Second Conference on Vitamin C: [papers] (Annals of the New York Academy of Sciences ; v. 258) 0890720118, 9780890720110

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if

ANNALS

OF THE

NEW

YORK

Volume

ACADEMY

OF SCIENCES

258

SECOND CONFERENCE ON VITAMIN C

Edited by C. G. King and: J.-J.

s6aed

Burns

The New York Academy of Sciences New York, New York 1975

Copyright, 1975, by The New York Academy of Sciences. All rights reserved. Except for brief quotations by reviewers, reproduction of this publication in whole or in part by any means whatever is strictly prohibited without written permission from the publisher.

Library of Congress Cataloging in Publication Data Conference on Vitamin C, 2d, New York, 1974.

Second Conference on Vitamin C. (Annals of the New York Academy of Sciences, v. 258)

1. Vitamin C—Congresses. 1920-— III. Series: New

JI. King, Charles Glen, 1896— Il. Burns, York Academy of Sciences. Annals; v. 258.

John J., Q11.N5

vol. 258 [QP772.A8] 508’.1s [641.1'8] ISBN

0-89072-011-8

75-26737

PGP Printed in the United States of America ISBN 0-89072-011-8

ANNALS

OF THE

NEW

YORK

ACADEMY

VOLUME September

SECOND

OF SCIENCES

258 30, 1975

CONFERENCE

ON

VITAMIN

C *

Editors and Conference Chairmen C. G. KING

AND

J. J. BURNS

ee

CONTENTS Part I. Metabolism of Ascorbic Acid

Introduction: Overview of Ascorbic Acid Metabolism. By J. J. BURNS ...... Biosynthesis and Metabolism of Ascorbic Acid in Plants. By FRANK A. LoEWuS, GEORGE WAGNER, AND JOAN C. YANG ...............-..--5Synthesis and Some Major Functions of Vitamin C in Animals. By I. B. CHATTERJEE, A. K. MAJUMDER, B. K. NANDI, AND N. SUBRAMANIAN .... Chemistry and Metabolism of Ascorbic Acid and Ascorbate Sulfate. By B. M. TOLBERT, M. DOWNING, R. W. CARLSON, M. K. KNIGHT, AND E. M. BAKER Liquid Chromatographic Analysis of Ascorbate and Ascorbate-2-Sulfate. By WITETTAM oNes BIGEERSAND DENNIS) UME UKE ILY seg eee ee Metabolism of Ascorbic Acid and Ascorbic-2-Sulfate in Man and the Subhuman Primate. By E. M. BAKER, J. E. HALVER, D. O. JOHNSEN, B. E. JOYCE) Ma Kak NIGHT. “AND .B aioe LOLBER Te cuee ert cee ie ean rte oa Utilization of Ascorbic Acid in Fish. By J. E. HALverR, R. R. Smitu, B. M. LOEBERT.

CAND Pe pM SsBAKER oe, iesre Aee creat a Oa

EN Nir

ies

ae

ce

5 7 24 48 70

72 81

Distribution of Ascorbic Acid, Metabolites and Analogues in Man and Animals. BYSDIETRICH

Part

II.

HORNIG:

o.4.c¢fa 406 ssa)

Interaction

with

Drugs

PEE

and

Mire

See lees eet eter caches oe

Environmental

103

Chemicals

Effects of Ascorbic Acid on Microsomal Drug Metabolism. By VINCENT G. LSNNONIGAND) PAUD CH AOA T Ove spaniels se ere tkca ewe: ie. -1 3sears Ascorbic Acid Requirements and Metabolism in Relation to Organochlorine Pesticidesse by Js GC, STREET AND) Ra We CHADWICK) | fone ase eae Protective Effects of Ascorbic Acid Against Toxicity of Heavy Metals. By IMAERERSPIVEY! EO Somer we ee i, ee ea ene caret eo aN ote cycoke Environmental Influences on Ascorbic Acid Requirements in Animals. By MIGTON: Le SCOTT Sane ta aetna CPL, te OE OTE a ae ee Vitamin) C and Cigarette Smokers, By OMER PELLETIER ...............-.. Effect of Ascorbic Acid on Amine-Nitrite Toxicity. By JEROME J. KAMM, T. IDASHIIAN Ai tis, CONNEY.cAND)Je) aBURNS ane an Seon: Se ee ae

119

132 144 151

169

* This series of papers is the result of the Second Conference on Vitamin C, sponsored by The New York Academy of Sciences and the Institute of Human Nutrition, College of Physicians and Surgeons, Columbia University, and held on October 9-12, 1974 in New York, New York.

Blocking the Formation of N-Nitroso Compounds with Ascorbic Acid in Vitro and in. Vivo.

By SIDNEY

Sa MURVISH

Gio

ctcers o> ereeie eenenei ea

ae

Reduction of Gastric Carcinogens with Ascorbic Acid. By R. RAINERI AND ROR AA, AG ics prtiguckame tetolerate naa eee Dene. J. HH: WEISBURGER ,.....+Part

III.

Ascorbic

Acid

and

Biological

KENNETH G. STROTHKAMP, AND KENNETH G. KRUL ......... Cytotoxic Aspects of the Interaction of Ascorbic Acid with Alloxan and Fd Hydroxydopamine. By RICHARD E. HEIKKILA AND GERALD COHEN . Some Properites of the Ascorbate Free Radical. By BENON H. J. BIELSKI, HELEN W. RICHTER, AND PHILLIP C. CHAN ... Dehydroascorbate Uptake and Reduction by Human Blood Neutrophils, Erythrocytes, and Lymphocytes. By LisusE STANKOVA, DEMETRIOS A. RIGAS: AND:.R: HS BIGLEY 2,005 epee es ie ee ee The Membrane Transport of Ascorbic Acid. By GEORGE V. MANN” AND PAMELA NEWTON@.*,..0o , A ee SO ee Le Ne ee eee Effect of Ascorbic Acid Deficiency on the Permeability and Collagen Biosynthesis of Oral Mucosal Epithelium. By MICHAEL C. ALFANO, SANFORD A. MILLER, AND JAMES F. DRUMMOND ................. : Function of Ascorbic Acid in Collagen Metabolism. By M. J. “BARNES = 3 Activation of Prolyl Hydroxylase in Fibroblasts by Ascorbic Acid. By GEORGE KUTTAN, ee

Ascorbic Acid and Collagen Synthesis in | Cultured Fibroblasts. AND. (@, JEVBATES:

30,8

arene

one

ce ee

ae

AND

Pharmacological

201 209 221 231

238 243

253 264

By C. I. LEVENE oe

Ascorbic Acid. By W. BYRON SMITH, STEPHEN B. SHOHET, ELLEN JESKI, AND BERTRAM H. LUBIN Effects of Ascorbic Acid on Health Parameters in Guinea Pigs. By VEEN-BAIGENT, A. R. TEN CATE, E. BRIGHT-SEE, AND A. V. RAo

IV.

190

SIDNEY

;

Ascorbic-2-Sulfate Metabolism by Human Fibroblasts. By A. D. BonD Instability and Function: Ascorbic Acid and Glutaminyl and Asparaginy] Residues. By ARTHUR B. ROBINSON AND STEVEN L. RICHHEIMER Tissue Changes Induced by Marginal Vitamin C ania By NorMAN M. SULKIN AND DorotHy F. SULKIN Alteration in Human Granulocyte Function After in ‘Vitro Incubation ‘with L-

Part

181

Systems

Ascorbic Acid and Electron Transport. By WILLI WEIS ..... Ascorbic Acid and Cytochromes. By EvA DEGKWITZ, SABINE WALSCH, Martin ICERaia eee [DUBBERSTEIN: “AND: J OR Gi WV.UIN ee Ascorbate Oxidase and Related Copper Proteins. By CHARLES R. DAWSON,

J. CARDINALE, FRANS L. H. STASSEN, RAMADASAN UDENFRIEND 5 38- (eUgL Se, tee er ee ie eeee

175

ZAGA-

M.

J.

Aspects

Clinical Pharmacological Aspects of Ascorbic Acid. By C. W. M. WILSON . Some Aspects of Current Vitamin C Usage: Diminished High-Altitude Resistance Following Overdosage. By G, N. SCHRAUZER, D. ISHMAEL, AND G, W. KIRRER AM, £12795 8S, eS er er ee oes ee Relationships of Protein and Mineral Intake to L- Ascorbic Acid Metabolism, Including Considerations of Some Directly Related Hormones. By G. C. CHATTERJEE, P. K. MAJUMDER, S. K. BANERJEE, R. K. Roy, B. RAY, AND DD IRODRABAL |o5 ies ink OC epee tacceta teen arta a Changes in Ascorbic Acid Metabolism of the Offspring Following High Maternal Intake of this Vitamin in the Pregnant Guinea Pig. By Epwarp P. INORKUS ANDOEEDRO}ROSSU see tenet en Ascorbic Acid in Cholesterol and Bile Acid Metabolism, By Emit GINTER |.

288 307

Effect of Steroidogenesis on Ascorbic Acid Content and Uptake in Isolated Adrenal Cells. By ABBAS E. KITABCHI AND WILLIAM H. WEST ..... : Proposed Uses of Ascorbic Acid in Prevention of Bladder Carcinoma. By ip U SCHLEGE Lae Sane EPC Se Gee APN ne, Meee ay eee Ee Part V.

Human

Requirements

and

422 432

Needs

Vitamin C Status: Methods and Findings. By H. E. SAUBERLICH ........... Biological Variation in Ascorbic Acid Needs. By MAN-LI S. YEW ..........

438 451

Ascorbic Acid and Athletic Performance. By H. Howa.p, B. SEGESSER, AND Wa EaRCOR NER aor cee eet, pete herent MR eee a yA SE Ant, cei css Relationships of Ascorbic Acid to Pregnancy, and Oral Contraceptive Steroids.

458

By JERRY M. RIVERS AND MARJORIE M. DEVINE ...................... Ascorbic Acid Deficiency in Clinical Disease Including Regional Enteritis. By GHARTESIE) SGERSONSE We te ee ee erg? ye Hie ae ee ee The Recommended Dietary Allowances for Ascorbic Acid. By ALFRED E ELAR PERE ie eae tree Oe a rae SOT ie nee EA eee rte ec eae, eeAe Part VI.

Ascorbic

Acid

and

Respiratory

GEORGE

W.

SHAFFER,

DENNIS

A.

GEORGE,

AND

THOMAS

491

498

C.

GTIATEMERS apy re teak Rr eee ee oe Re ep eee a Se oe Vitamin C and Upper Respiratory Illness in Navaho Children: Preliminary Observations (1974). By JOHN L. COULEHAN, LouIs KAPNER, SUSAN EBERHARD, FLoyp H. TAYLOR, AND KENNETH D. ROGERS ..................

Safety Considerations with High Ascorbic Acid Dosage. Ascorbic Acid Function and Metabolism During Colds.

483

Illness

Large-Scale Trials of Vitamin C. By TERENCE W. ANDERSON .............. A Controlled Clinical Trial of Ascorbic Acid for the Common Cold. By THOMAS L. LEwis, THOMAS R. KARLOWSKI, ALBERT Z. KAPIKIAN, JOHN M. LYNCH,

465

By LEwis A. BARNESS By C. W. M. WILSON

S05

ws

523 529

Se

Current Status of Vitamin C and Future Horizons. By C. G. KING PancieDiscussioni Ga Lies DRATONE GiGi 1G eae iter eee

......... eee

540 546

U

%

4

I~

Go,

@

on

&

tea

o=

=o?

“28

yout]

_

2

=

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i

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apne)

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ws

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allel? Gineuy 7

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ee

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tA

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wo

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Pars

a oa

ae

atm

ax

Part

I.

METABOLISM

OF

ASCORBIC

INTRODUCTION: ACID

ACID

OVERVIEW OF ASCORBIC METABOLISM J. J. Burns

Roche Research Center Hoffmann-La Roche Inc. Nutley, New Jersey 07110

Much new information has appeared since the first conference on vitamin C was held, October 7-10, 1960, under the auspices of The New York Academy of Sciences. In view of this, it is most appropriate to hold another conference

in order to review the present status of our knowledge on this important vitamin. Papers published in the monograph of the first conference indicated that

(6

oe ansa cle | rea |

p

t HOCH ¢h,0H

Wok cH 20H

of hell H0H

L-gulonolactone

2-keto-L-gulonolactone

L-ascorbic acid

J

HC

we?

HCOH

con

Hi Ay

HOCH

&

TPNH

Le

HCOH

OH

HCOH

Jing

OH

reas D-glucose

HOCH

HCOH |

eh

D-glucuronic acid

CH,0H L-gulonic acid

9

DPN 79

HC”

oly

HCOH HOCH

HOCH ¢-0

HCOH

HCOH

HCOH

HOCH

HOH

2

D-glucose

3-keto-L-gulonic acid

Pentose Phosphate Pothway

-C0,

CH.OH

NG CH,0H

ie

c-0

ms oc“

H

:

T

HCOH

CH,OH

ees —~ 2

D-xylulose-5-P

i

¢H0H

HOCH

0__ HCOH —

HOCH HOCHt HCOH

CH,0H D-xylulose

CH,0H L-xylulose

HCOH

D CH,0OH

Xylitol

Ficure 1. Glucuronic acid pathway of glucose metabolism.

5

6

Annals

New

York

Academy

of Sciences

COOH OXALIC

i l H—C

HO—C

0 |

HO-C—H

! CH20H

L-ASCORBIC ACID

)

o=c H—C

HO—C—H

! CH20H

( ——O2 ] H—C—OH l Ho—C—H o-c

CH20H

ACID

=n l H—C—OH 1 H—C—OH icBie-H

4 toh L-LYXONIC ACID

—_L-DEHYDRO ASCORBIC DIKETOACID L-GULONIC ACID

ance

AND

c7On HO-C-H i H-C-OH i

HO-C—H i

C HOH L-XYLONIC ACIO

C=0 | HO-C—H H-C—OH HO—C—H CH20H L-XYLOSE

FiGure 2. Metabolism of ascorbic acid.

considerable knowledge had been obtained prior to 1960 on the biosynthesis and metabolism of ascorbic acid. Radioactive tracer studies pointed out that ascorbic acid is synthesized from glucose through the intermediate formation of glucuronic acid and gulonic acid, as shown in FIGURE 1. Species such as man, monkey, and guinea pig lack the liver microsomal enzyme, L-gulonolactone oxidase, which is required for the last step in the formation of the vitamin. It is this missing step that necessitates the inclusion of vitamin C in their diet to prevent scurvy. The reactions in FIGURE 1 show that ascorbic acid is involved in a newly discovered pathway of glucose metabolism, which is referred to as the “glucuronic acid pathway.” This pathway also accounts for the synthesis of L-xylulose, the pentose excreted in urine of humans with the inborn error of metabolism, essential pentosuria. Studies presented in the first session of this conference will throw further light on the pathways of ascorbic acid biosynthesis and the genetic relationships involved. Research results presented at the first conference indicated that ascorbic acid was metabolized as shown in FiGuRE 2. Knowledge of these metabolic products was made possible primarily by studies with isotopically labeled forms of L-ascorbic acid. Further clarification of the metabolism of ascorbic acid will be given in this first session, especially the exciting information on a new metabolite, ascorbic acid-2-SO,. It is expected that papers presented in this session, as well as in the entire conference, will point out new directions for research on ascorbic acid.

BIOSYNTHESIS ASCORBIC Frank A. Loewus,+

AND METABOLISM ACID IN PLANTS *

George Wagner,t

OF

and Joan C. Yang

Division of Cell and Molecular Biology State University of New York at Buffalo Buffalo,

New

York

14214

INTRODUCTION

Formation, function, and fate of L-ascorbic acid in plants and microorganisms remain topics of great challenge for today’s biochemist. There has been some progress. Hexose is generally regarded as the ultimate source of the carbon chain, but neither intermediates nor enzymes operative in the conversion have been pinpointed and explored. Ascorbic acid appears to be ubiquitous in the plant world, yet a systematic survey of its presence and identity using modern methods of assay remains to be accomplished. The widespread occurrence of ascorbic acid implies a functional role or roles of broad significance, yet the nature of such role(s) are matters of speculation rather than of fact. Information regarding the fate of ascorbic acid is just beginning to appear, and its appearance offers promise of fresh new ways in which to approach the other topics, biosynthesis and activity. I wish to review here our recent work on the relationship between Lascorbic acid metabolism in plants and (+)-tartaric acid and oxalic acid formation. This relationship appears to hold the key to ascorbic acid biosynthesis, which I regard as an essential matter if the functional role of ascorbic acid in plants is to be fully understood. TARTARIC

ACID

BIOSYNTHESIS

FROM

ASCORBIC

ACID

When Herbert et al.! published the constitution of L-ascorbic acid in 1933, isolation of equivalent amounts of oxalate and tartrate following oxidation, as illustrated in FiGuRE 1, supplied evidence of structure. All three, ascorbic acid, oxalate,

and tartrate,

occur

in plants, often together.

The

latter two

are also

produced by certain bacteria. That L-ascorbic acid or a closely related substance might be a precursor of (+)-tartaric acid was suggested by Hough and Jones,? who relied on the configurational similarity between carbons 4 and 5 of L-ascorbic

acid and carbons

2 and

3 of (+)-tartaric

acid.

This possibility

was first tested by feeding L-ascorbic acid-6-1'C to a detached grape leaf.* Tartaric acid was isolated after 25 hours of metabolism and found to be virtually * This work was supported by National Institutes of Health research 12422 and a grant from Hoffmann-La Roche, Inc., Nutley, New Jersey.

+ Present address:

Department of Agricultural

versity, Pullman, Washington 99163. t Present address: Biology Department, New York 11973.

7

Chemistry, Washington

Brookhaven

National

grant GM-

State Uni-

Laboratory,

Upton,

8

Annals

mh 40

HO - C

New

York

Academy

34

ie

40

0

HO-C a —H- c HO-C-H CH20H L-Ascorbic Acid

of Sciences

C= 0

COOH

¢=0 |NoOl

Oxalic Acid

H- c

COOH

HO-C-H | CH20H

H-C-OH | HO-C-H

COOH HNO

H-C-OH HO-C-H

CH20H

COOH

DehydroL- Ascorbic

L- Threonic

(+)-Tartaric

Acid

Acid

Acid

FicureE 1. Chemical conversion of L-ascorbic acid to (-+)-tartaric acid and oxalic acid.*

devoid of label. Most of the carbon-14 was recovered as carbohydrate, mainly sucrose, glucose, and fructose. In 1969, Saito and Kasai* made the interesting discovery that young grape berries converted L-ascorbic acid-1-!*C to tartaric acid in high isotopic yield, 72% of the soluble label was recovered as tartaric acid after 24 hours of metabolism. Most of the label appeared in carboxyl carbon of the acid. pDGlucurono-6,3-lactone-6-14C was one-third to one-half as effective and p-glucuronate-6-'4C only one-tenth as effective as L-ascorbic acid-1-'*C. D-Glucurono-6,3-lactone is readily converted® to L-ascorbic acid by plants, but D-glucuronate is poorly converted, if at all.° Saito and Kasai’s observations has been confirmed in this laboratory with detached grape berries obtained from a closely related species of grape as well as detached leaves of a more distantly related species of the Vitaceae.* A portion of our results is presented in TABLE 1. Comparison of distribution of label among plant constituents after a feeding of L-ascorbic acid-1-!4C or -6-'4C revealed that only the former source of label

TABLE

1

DISTRIBUTION OF RADIOACTIVITY AMONG COMPONENTS IN THE WATER-SOLUBLE FRACTION OF DETACHED Vitis labrusca BERRIES AND Parthenocissus inserta LEAVES 24 Hours AFTER LABELING

Ascorbic Acid-1-“C V. lab. P. ins.

Tartaric acid Oxalic acid Other acids Sugars and neutrals

69.0 0.6 10.2 3.9

Other compounds

16.3

Ascorbic Acid-6-“C V. lab. Pans:

% of soluble radioactivity 38.4 1.4 0.6 0.4 30.2 11.4

33.8 45.3

0.6 0.5 50.8 38.4

19.4

YS)

oF,

Loewus et al.: Biosynthesis and Metabolism

9

resulted in significant incorporation of radioisotope into tartaric acid. Virtually all of the 14C in ascorbic acid-1-'*C-derived tartaric acid was in the carboxyl carbon. When ascorbic acid-6-!*C was supplied to these plants, about 40% of the soluble label appeared as sugar, mainly. sucrose, confirming the earlier report.* Labeled oxalic acid was not found in these experiments. Our findings, which support those of Saito and Kasai, indicate that carbon 1 of L-ascorbic acid becomes a carboxyl function in (+)-tartaric acid, and, by inference, carbons 1 through 4 of L-ascorbic acid provide the carbon chain of (+)-tartaric acid (FIGURE 2).

This grape study prompted investigation of another type of (+)-tartaric acid accumulator, the geranium. We chose Pelargonium crispum, a plant that accumulates about 10 mg of tartaric acid per g fresh weight of leaf. Experiments were designed in which detached vegetative tips (apices) imbibed the radioactive compound for a short period and then were left to metabolize the labeled material for a set period of time.* As before, comparison was made on distribution of label among plant constituents after feeding L-ascorbic acid-114C and -6-'4C. In addition, experiments were run with L-galactono-1,4-lactoneUL-"!C and -6-4C. The latter is a potent precursor of L-ascorbic acid in plants. 1° P. crispum apices that had been held in 1.7% L-galactono-1,4-lactone for 7 hours, then transferred to water, underwent a dramatic increase in ascorbic acid content, reaching nearly 10 times the normal level in 3 days then slowly declining over the next 3 days to a level still 6 times above the control (FIGURE 3). In the same period, tartaric acid increased only slightly. It should be noted that the endogenous level of tartaric acid was about 10 times than of ascorbic acid at the start of the experiment, and any metabolic contribution from ascorbic acid that had been generated from an exogenous source would be small compared to the endogenous tartaric acid pool.- There may be some significance in the fact that the rapid decline in tartaric acid content following detachment was arrested by ascorbic acid feeding. Tartaric acid was assayed by 2 methods, vanadate colorimetric procedure and gas-liquid chromatography of the trimethylsilyl derivative.

"COOH

a!) ¢

aC ¢

H-C-OH BA TEL

cae

i

COOH

10-¢ |Meco

>) oe

Sr

|

pie

HO-C-H _

“HOH «

(+)-Tartaric

acid

C

«fac|-

~——>

— Hexose

L-Ascorbic acid Ficure 2. Metabolism of L-ascorbic acid in the Vitaceae.

10

Annals

New

York

Academy

of Sciences

if

i

s

cx =

£

Pre, Ascorbic acid yd eae a ig ae

4

s

ra

=

5

p=4

rs]

Bir

/

2

rg me °

,

we

=

eS 2

= @

a S

2

B

yom L- Galactono-1, 4 - lactone

5

pee

\) {

cs

Be

ee

20 B

og

=

‘

10

@ i] 4

eee

25

end of

feeding

Figure

=>

(Sultt acid - vanadate

3.

Conversion

50

75

acid - GLC

100

|

125

150

TIME, hr of t-galactono-1,4-lactone

to

L-ascorbic

acid

and

(+)-

tartaric acid in detached Pelargonium crispum apices. Apices were held in 1.7% aqueous L-galactono-1,4-lactone for 7 hours and then transferred to distilled water. Ascorbic acid was assayed with 2,6-dichlorophenolindophenol and tartaric acid by reaction with vanadate or by gas-liquid chromatography as its trimethylsilyl derivative.”

Results from these P. crispum experiments are summarized in TABLE 2. Uniformly labeled L-galactono-1,4-lactone-''C-treated apices produced ascorbic, tartaric, and oxalic acids accounting for 20% of the label present in the soluble fraction. Another 50% was recovered as L-galactonic acid or its unchanged lactone. When L-galactono-1,4-lactone-6-''C was given, label appeared in ascorbic acid (90% located in carbon 6) and tartaric acid (96% located in TABLE

2

DISTRIBUTION OF RADIOACTIVITY AMONG COMPONENTS IN WATER-SOLUBLE FRACTION OF DETACHED Pelargonium crispum Apices 72 Hours AFTER LABELING Galactono-1,4-lactone

UL-“C

-6-%C

Ascorbic Acid

-6-4C

-1-“C

% of soluble radioactivity Ascorbic acid Tartaric acid Oxalic acid Other acids Sugars and neutrals Other compounds

10.0 8.5 2.0 55.4 11.8 3

2.6 15.4 0.2 40.8 15.8 Pees

PRIS! Bye) 0.03 20.0 1359 RUE:

18.2 0.4 ie 19.9 D5 3319

Loewus et al.: Biosynthesis and Metabolism

i

carboxyl carbon) but none was found in oxalic acid, findings which suggested that the carbon chain of tartaric acid originated from the bottom 4 carbons of L-ascorbic acid. This was confirmed by an L-ascorbic acid-6-''C experiment. Of the label present as soluble constituents in the latter, 32% was (+)-tartaric acid (99% located in carboxyl carbon). Oxalic acid was devoid of label. When L-ascorbic acid-1-'*C was supplied to P. crispum apices, appearance of label in (+)-tartaric acid and oxalic acid was reversed, now label appeared in oxalic acid but none in tartaric acid. In a more detailed study, incorporation of label from L-ascorbic acid-6-1#C into P. crispum was examined at intervals from 4 to 72 hours. Profiles of the soluble radioactivity eluted from Dowex 1 (formate) exchange columns are given in FIGURE 4. Radioactivity in ascorbic acid decreased with time while that in tartaric acid increased. A small radioactive peak II appeared in the eluate in the region characterized by threonic acid. Other peaks, I, III, and IV, were also examined by paper chromatography, but their identities remain tentative.

Oto O.1N acid

OA to 4N .

Formic

- I*Formic acid Tortaric

ao” L- Ascorbic acid

acid “a rr

Ww

{ 0 mn

2S x

10

24 hr

€ ™

ay ae oOo

>

40

10 hr

> a Ome =

ran)

4,5-Dienol of gluconic acid

00t

teal H-C-OH

‘=0

Pretaric acid Conversion

acid

|

COOH (+)-Tartaric acid

H-C—OH | CH,0H

10.

acid

HO-¢-H

0)

Figure

4-Ketogluconic

Bee eoU

oa ==

——*

5-Ketogluconic

CHO | er, CH,0H Glycoaldehyde

COOH CH20H Glycolic acid

of p-glucose to (+ )-tartaric acid via 5-ketogluconic

as proposed by Kotera et al."

acid

Loewus et al.: Biosynthesis and Metabolism

21

REFERENCES

.

HERBERT, R. W., E. L. Hirst, G. V. PERcIVAL, R. J. W. RENOLDs & F. SMITH. 1933. The constitution of ascorbic acid. J. Chem. Soc. London. 1270-1290.

Houcu,

L. & J. K. N. Jones.

1956.

The biosynthesis of the monosaccharides.

Advances Carbohydr. Chem. 11: 185—262 (see p. 240).

Loewus, F. A. & H. A. STAFFORD. 1958. Observations on the incorporation of C* into tartaric acid and the labeling pattern of p-glucose from an excised

grape leaf administered L-ascorbic-acid-6-C™. Plant Physiol. 33: 155—156. Sairo, K. & Z. Kasar. 1969. Tartaric acid synthesis from L-ascorbic-acid-1-“C in grape berries. Phytochemistry 8: 2177-2182. FINKLE, B. J., S. KELLY & F. A. LoEwus. 1960. Metabolism of D-[1-*C]- and D-[6-“C]glucuronolactone by the ripening strawberry. Biochim. Biophys. Acta 38: 332-339. Loewus, F. A., S. KELLY & E. F. NEUFELD. 1962. Metabolism of myo-inositol in plants: conversion to pectin, hemicellulose, p-xylose and sugar acids. Proc. Nat. Acad. Sci. U.S.A. 48: 421-425. WaGNER, G. & F. A. LoEwus. 1974. L-ascorbic acid metabolism in the Vitaceae.

Conversion

to (+ )-tartaric acid and hexoses.

787. WAGNER, G. & F. LoEwus. 1973. The biosynthesis Pelargonium crispum. Plant Physiol. 52: 651-654. Baic,

M.

M., S. KELLY

& F. LoEwus.

1970.

higher plants from L-gulono-1,4-lactone Physiol. 46: 277-280. .

and

Plant Physiol. 54: 784—

of (-+)-tartaric

L-ascorbic

acid

in

acid biosynthesis

in

tL-galactono-1,4-lactone.

Plant

Jackson, G. A. D., R. B. Woop & M. V. Prosser. 1961. Conversion of Lgalactono-y-lactone into L-ascorbic acid by plants. Nature 191: 282-283.

. WacGner, G. 1974. Carbohydrate interconversions involving the biosynthesis and metabolism of L-ascorbic acid and tartaric acid in plants. Ph.D. Thesis. State Univ. of New York at Buffalo. MILLERD, A., R. K. Morton & J. R. E. WELLS. 1963. Oxalic acid biosynthesis in shoots of Oxalis pes-caprae L. Biochem. J. 86: 57-62. .

CuHana, C-C. & H. BEEvERS.

1968.

Biogenesis of oxalate in plant tissues.

Physiol. 43: 1821-1828. Burns, J. J.. H. B. Burcn & C. G. Kinc. 1951. The metabolism ascorbic acid in guinea pigs. J. Biol. Chem. 191: 501-514. Burns, J. J., P. G. DAYTON

& S. SCHULENBURG.

the metabolism of L-ascorbic acid HELLMAN, L. & J. J. BuRNs. 1958. J. Biol. Chem. 230: 923-930. . DayTon, P. G., F. EISENBERG, Jr. labeled ascorbic, dehydroascorbic Biochem. Biophys. 81: 111-118.

Kina, C. G. & R. R. BECKER. .

1956.

Plant

of 1-C*-L-

Further observations

on

in guinea pigs. J. Biol. Chem. 218: 15-21. Metabolism of L-ascorbic acid-1-“C in man. & J. J. BuRNS.

1959.

Metabolism

of C*-

and diketogulonic acids in guinea pigs. Arch.

1959.

The biosynthesis of vitamin C (ascorbic

acid). Wld. Rev. Nutr. Diet. 1: 61-72. OxE, O. L. 1969. Oxalic acid in plants and in nutrition.

Wld. Rev. Nutr. Diet.

10: 262-303. .

Hacer,

L. & R. H. HERMAN.

1973.

Oxalate

metabolism.

I. Amer.

J. Clin.

Nutr. 26: 758-765. . Fassett, D. W. 1973. Oxalates. In Toxicants Occurring Naturally in Foods. 2nd edit. : 346—362. Nat. Acad. Sci., Washington, D.C. . Ranson, S. L. 1965. Plant acids. Jn Biosynthetic Pathways in Higher Plants. J. B. Pridham, Ed. : 179-198. Academic Press. New York, N.Y. Bucu,

M. L.

1960.

A Bibliography of Organic Acids in Higher Plants.

Handbook No. 164, U.S. Dept. Agric.

Washington, D.C.

Agric.

Annals

New

York

Academy

of Sciences

Hurrerraucu, R. & I. KEINER. 1968. Separation of ascorbic acid and dehydroascorbic acid on polyamide plates. Pharmazie 23: 157.

Dayton, P. G., M. McM. SNELL & J. M. Pere. 1966. Ascorbic and dehydroascorbic acids in guinea pigs and rats. J. Nutr. 88: 338-344.

Loewus,

F.

1971.

22: 337-364. Mapson, L. W.

Carbohydrate

1967.

Annu.

interconversions.

Rev.

Plant

Physiol.

Biogenesis of L-ascorbic acid in plants and animals.

Jn

The Vitamins. W. H. Sebrell, Jr. & R. S. Harris, Eds. 1: 369-385. Academic Press. New York, N.Y. Kina, C. G. 1973. The biological synthesis of ascorbic acid. Wld. Rev. Nutr. Diet. 18: 47-59.

Hassip, W. Z. 1970. Biosynthesis of sugars and polepaccharides. In The Carbohydrates. W. Pigman & D. Horton, Eds. 2nd edit., 2A: 301-373. LoEwus, F., M-S. CHEN & M. W. Loewus. 1973. The myo-inositol oxidation pathway to cell wall polysaccharides. Jn Biogenesis of Plant Cell Wall Polysaccharides. F. Loewus, Ed. : 1-27. Academic Press. New York, N.Y. Loewus, F. A. 1961. Aspects of ascorbic acid biosynthesis in plants. Ann. N.Y.

Acad. Sci. 92: Loewus, F. A. tochemistry 2: Logewus, F. A.

57-78. 1963. Tracer studies on ascorbic acid formation in plants. Phy109-128. 1965. Inositol metabolism and cell wall formation in plants.

Fed. Proc., FASEB. 24: 855-862. HANNINEN,

O., R.

RAUNIO

acid from myo-inositol.

& J. MARNIEMI.

Carbohydr.

1971.

Biosynthesis

of L-ascorbic

Res. 16: 343-351.

Loewus, F. 1974. The biochemistry of myo-inositol in plants. /n Metabolism and Regulation of Secondary Plant Products. V. C. Runeckles and E. E. Conn, Eds. Recent Advan. Phytochemistry 8: 179-207. Academic Press, New York, N.Y. LoEwus, F. A. & S. KELLY. 1962. Conversion of glucose to inositol in parsley leaves. Biochem. Biophys. Res. Commun. 7: 204—208. Loewus, F. A. & S. KELLY. 1963. Inositol metabolism in plants. I. Labeling patterns in cell wall polysaccharides from detached plants given myo-inositol2-t or -2-"C. Arch. Biochem. Biophys. 102: 96-105. IUPAC COMMISSION ON THE NOMENCLATURE OF ORGANIC CHEMISTRY AND IUPAC-IUB CoMMISSION ON BIOCHEMICAL NOMENCLATURE. 1968. J. Biol. Chem. 243: 5809-5819. Logewus, F. A., S. Ketty

42.

43.

& H. H. Hiatr.

1960.

Ascorbic

acid synthesis from

D-glucose-2-“C in the liver of the intact rat. J. Biol. Chem. 235: 937-939. Maroc-Gyr, J. 1965. The metabolism of glucose and gluconate in the leaves of Pelargonium zonale L, in relation to the formation of organic acids and in particular tartaric acid. Physiol. Veg. 3: 167-180. KopaMa, T., U. KoTera & K. YAMADA. 1972. Induction of mutants from the tartaric producing bacterium, Gluconobacter suboxydans and their properties. Agr. Biol. Chem. 36: 1299-1305. KoreraA, U., K. UMEHARA, T. KopAMA & K. YAMADA. 1972. Isolation method of highly tartaric acid producing mutants of Gluconobacter suboxydans, Agr. Biol. Chem. 36: 1307-1313. KorTera, U., T. KopAMaA, Y. Minopa & K. YAMADA. 1972. Isolation and chemi-

cal structure of new oxidation product of 5-ketogluconic acid, and a hypotheti-

44,

cal pathway from glucose to tartaric acid through this compound. Agr. Biol. Chem. 36: 1315-1325. BAKER, E. M., J. C. SAARI & B. M. TOLBERT. 1966. Ascorbic acid metabolism in man. Amer. J. Clin. Nutr. 19: 371-378.

Loewus et al.: Biosynthesis and Metabolism

ZS

DISCUSSION

Dr. J. J. BuRNs: In the data that you showed for the plants that form . oxalate, would you care to estimate how much oxalate may come via ascorbate in these plants or can it come from other sources? Dr. LoEwus: In the oxalate biosynthesis in plants, two other pathways have been proposed. One in which oxaloacetic acid is broken to form acetic acid and oxalic acid has been shown in vivo to occur in certain plants such as beet and spinach. The other pathway in which glycolate or glyoxalate is oxidized has been studied by Millard and others in Australia. Both of these proposals offer alternatives for oxalate biosynthesis, but neither has shown the dramatic conversion that we find now with ascorbic acid. Since ascorbic acid is being synthesized naturally at a substantial rate in plants, I think the best candidate for study at this particular time might be biosynthesis of ascorbic acid and its ultimate metabolism. Dr. JULIUS BERGER (Hoffmann-La Roche, Nutley, N.J.): Has there been any confirmed demonstration of the production of ascorbic acid by any microorganisms? Dr. Loewus: There are no definitive reports of ascorbic acid biosynthesis by bacteria. However, we find that the algae are very good at producing ascorbic acid. We have been examining one particular diatom that converts glucose to ascorbic acid but fragments it into such a redistribution that we are at a loss to explain the biosynthetic pathway at this particular time. Dr. KiNG: Have you found any organisms too far down the biological scale to have ascorbic acid present in them? Is there any indication at all of living organisms that do not require ascorbic acid either by synthesis or by intake? Dr. LoEwus:

Ascorbic acid seems to be ubiquitous in the plant world, but

it is not well established whether this extends to bacteria. Some species of fungi apparently have ascorbic acid. Modern methods of analysis such as gas chromatography should answer these questions. Up to now, for the most part, ascorbic acid has been identified by its oxidation-reduction properties, which is not a clear criterion.

SYNTHESIS

AND SOME MAJOR FUNCTIONS VITAMIN C IN ANIMALS *

OF

I. B. Chatterjee, A. K. Majumder, B, K. Nandi, and N. Subramanian Department of Biochemistry University College of Science Calcutta, India

The requirement of ascorbic acid (vitamin C) is a common property of living organisms, and it has long been considered that all animals except the guinea pig, monkey, and man can synthesize this vitamin. The classic method for determining the ability of an animal to synthesize ascorbic acid is to feed it a scorbutogenic diet for a prolonged period and to observe the appearance of the scurvy syndrome. Obviously, the method is laborious and time-consuming. Also, the onset of the scorbutic syndrome depends on the ascorbic-acid-retention capacity of the animal. For example, whereas the guinea pigs can be made scorbutic in about 3 weeks, it takes 3 to 4 months to produce scurvy in man. Since the discovery of the technique for studying ascorbic acid synthesis in vitro,.-= the task has become much simpler. In this technique, the tissue homogenates or the subcellular fractions are incubated with precursors of ascorbic acid and the amount of the vitamin formed is estimated. Using the in vitro method, we have examined the ascorbic acid synthesizing abilities of different species of animals in the phylogenetic tree, and the results are given below.

EVOLUTION

AND

BIOSYNTHESIS

OF

ASCORBIC

ACID

The ability to synthesize ascorbic acid is absent in the insects, invertebrates, and fishes. The biosynthetic capacity started in the kidney of amphibians, remained in that of reptiles, became transferred to the liver of mammals, and finally disappeared from the guinea pig, the flying mammals, the monkey, and man. A similar transition in the biosynthetic ability was observed in the branched evolution of birds.°-!’ TaBLe 1 shows ascorbic acid synthesis from L-gulono-1,4-lactone

animals.

FIGURE

in microsomal fraction from tissues of different species of | shows how the overall pattern of ascorbic acid synthesis by

different species of animals is correlated to their phylogeny. Evolution of the Biosynthetic Capacity

The incapability of insects, invertebrates, and fishes to synthesize ascorbic acid apparently raises the question whether ascorbic acid is an essential requirement

for these species.

It has been

reported

that salmon,

desert locusts #® are dependent on dietary ascorbic acid.

trout,!*:15

and

the

However, the need of

* This work was supported, in part, by United States PL-480 grant No. FG- In-416.

24

Chatterjee et al.: Synthesis and Some Major Functions TABLE ASCORBIC

aS

1

ACID SYNTHESIS FROM L-GULONO-1,4-LACTONE IN MICROSOMAL FROM TISSUES OF DIFFERENT SPECIES OF ANIMALS *

FRACTIONS

Ascorbic Acid Synthesized (xg/mg Protein/Hr) Kidney Liver

Animals Insects + ¢ Invertebrates + = Fishes +

= —_— =

— — —

144+10 itelisiet= lO)

— —

Amphibians Toad (Bufo melanostictus) Frog (Rana tigrina)

Reptiles Turtle (Lissemys punctata) Bloodsucker (Caloter versicolor)

98+8 SOs}

— —

House lizard (Hemidactylus flaviviridis)

46+6

—

Common Indian Monitor (Varanus monitor)

3244

—

Angani (Mabuya carinata) Snake (Natrix piscator) Tortoise (Testudo elegans)

254 18+2 4a)

— — —

Mammals Goat Cow

Sheep Rat Mouse

— ——_ ——

68+6 50+6 43+4 39+4 3524

Squirrel Gerbil Rabbit

on — —

3044 26+4 easy)

Cat Dog Guinea pig

— — —

Sista peel} —

Indian fruit bat (Pteropus medius)

=

—

Indian pipistrel (Vesperugo abramus)

=

_—

Monkey (Macaca mulatta)

—

ies

Man

—

—

Flying mammals

Primates

* For incubation and other detail conditions see Reference 13. Each datum represents an average from a minimum of 8 animals + S.D. In case of house lizards,

kidneys from 12 lizards were pooled for 1 determination, and 4 such determinations were made. + Accounts of insects, invertebrates and fishes have been given elsewhere.’ *

t In cases of insects and invertebrates, homogenates of the fat body or the hepatopancreas

and the malpighian

tubules were

used in place of liver and kidney micro-

somes.

insects, invertebrates, and fishes for ascorbic acid may be very small, and they may obtain enough vitamin through food. Naturally, there was no necessity to synthesize the vitamin, and the biosynthetic mechanism did not evolve in these earlier species of the phylogenetic tree. The emergence of the biosynthetic ability in the amphibians suggests that a

26

2 &

Annals

New

York

Academy

of Sciences

PRIMATES

.S)

= >

“

S

MAM

.\

MALS

B

BIRDS |

ee

REP ULES

he z $

AMPHIBIANS

uw

ae

ORDER)

E

FicuRE

1. Schematic representa-

tion of ascorbic-acid-synthesizing abilities of various species of animals in relation to their phylogeny. (From Chatterjee.’* By permission of Science.)

- INSECT:

oO

=< a


Additionally,

-OH

‘OH + OH

(16)

may form from the iron catalyzed breakdown

H.O., + Fe? ——>

‘OH + OH

It is not surprising, in light of the above been observed during the autoxidation of There is evidence that O.-”° as well H.O,, O.- and -OH may all be involved (and its quinones)

+0,

and dialurate

of H.O.:

+ Fe**

(17)

reactions, that formation of -OH has 6-OHDA and dialuric acid.** as ‘OH °°: ** is cytotoxic. Therefore, in the cytotoxic actions of 6-OHDA

(and alloxan).

In a recent study, we observed

that ethanol, an agent that can function both to catalase) and -OH (by direct reaction), protected action of alloxan.?8 Tissue-reducing agents like an important role in the degenerative process by these above species.

remove H.O. (substrate for mice against the diabetogenic ascorbic acid, may well play controlling the generation of

REFERENCES

1.

2.

3.

MALMrors, T. & H. THOENEN, Eds. 1971. 6-Hydroxydopamine and Catecholamine Neurons. North Holland Publishing Company. Amsterdam, The Netherlands. HerkkiLa, R. E., C. MyTILINEOU, L. CoTE & G. COHEN. 1973. Evidence for degeneration of sympathetic nerve terminals caused by the ortho- and paraquinones of 6-hydroxydopamine. J. Neurochem. 20: 1345-1350. TRANZER, J. P. & H. THOENEN. 1973. Selective destruction of adrenergic nerve terminals by chemical analogues of 6-hydroxydopamine. Experientia 15: 314—

Sly, 4.

LuNpsTRoM,

J., H. ONG, J. Daty

& C. R. CREVELING.

1973.

Isomers

of 2,4,5-

trihydroxyphenylethylamine (6-hydroxydopamine): Long term effects of the accumulation of [*H]-norepinephrine in mouse heart in vivo. Molec. Pharma-

col. 9: 505-513. 5. 6.

7. 8.

9.

10. 11.

12.

Rose, C. S. & P. Gyorcy. 1950. Hemolysis with alloxan and alloxan-like compounds, and the protective action of tocopherol. Blood 5: 1062-1074. HEIKKILA, R. E., J. A. Mezick & D. G. CORNWELL. 1971. Destruction of specific membrane phospholipids during peroxidative hemolysis of vitamin E deficient erythrocytes. Physiol. Chem. Physics 3: 93-97.

BRUCKMANN, G. & E. WERTHEIMER. 1947. Alloxan studies: The action of alloxan homologues and related compounds. J. Biol. Chem. 168: 241-256. HEeIKKILA, R. E. & G. ConEN. 1972. Further studies on the generation of hydrogen peroxide by 6-hydroxydopamine: Potentiation by ascorbic acid. Molec. Pharmacol. 8: 241-248. HEIKKILA, R. E. & G. CoHEN. 1973. 6-Hydroxydopamine: Evidence for the superoxide radical as an oxidative intermediate. Science 181: 456-457. Conen, G., D. DemMsBiec & J. Marcus. 1970. Measurement of catalase activity in tissue extracts. Anal. Biochem. 34: 30-38. HEeIKKILA, R. E. & G. CoHEN. 1972. In vivo generation of hydrogen peroxide from 6-hydroxydopamine. Experientia 28: 1197-1198.

DEAMER, D. W., R. E. HEIKKILA, R. V. PANGANAMALA, G. COHEN & D. G. CorN-

WELL. 1971. The alloxan-dialuric acid cycle and the generation of hydrogen peroxide. Physiol. Chem. Physics 3: 426-430.

Heikkila & Cohen:

Alloxan 1964.

and 6-Hydroxydopamine

229

13.

CoHEN, G. & P. HocHsTEIN.

14.

cytes by hemolytic agents. Biochemistry 3: 895-900. COHEN, G. & P. HOCHSTEIN. 1965. In vivo generation of H»O. in mouse erythrocytes by hemolytic agents. J. Pharmacol. Exp. Ther. 147: 139-143.

15.

Sanger, A. & H. THOENEN.

1971.

Generation of hydrogen peroxide in erythro-

Model experiments on the molecular mecha-

nism of action of 6-hydroxydopamine.

Molec. Pharmacol.

7: 147-154.

16.

BREESE, G. R. & T. D. TrRayitor. 1970. Effect of 6-hydroxydopamine on brain norepinephrine and dopamine: Evidence for selective degeneration of catecholamine neurons. J. Pharmacol. Exp. Ther. 174: 413-420.

17.

Uretsxy, N. J. & L. L. Iversen.

18.

19.

Misra, H. P. & I. FripovicH.

dation of epinephrine 20.

21.

1970.

Effects of 6-hydroxydopamine on cate-

cholamine containing neurones in the rat brain. J. Neurochem. 17: 269-278. HoxreLtt, T. & U. UNGERsTEDT. 1973. Specificity of 6-hydroxydopamine induced degeneration of central monoamine neurones: An electron and fluorescence microscopic study with special reference to intracerebral injection on the nigro-striatal dopamine system. Brain Res. 60: 269-297. 1972.

The role of superoxide anion in the autoxi-

and a simple assay for superoxide dismutase.

J. Biol.

Chem. 247: 3170-3175. McCreery, R. L., R. DREILING & R. N. ApaMs. 1974. Voltammetry in brain tissue: The fate of injected 6-hydroxydopamine. Brain Res. 73: 15-21. HeErkkILA, R. E. & G. COHEN. 1971. Inhibition of biogenic amine uptake by hy-

drogen peroxide:

A mechanism

for toxic effects of 6-hydroxydopamine.

Sci-

ence 172: 1257-1258.

22. 23. 24. 25.

26.

CHARALAMPOOS, F. C. & D. M. HEGSTED. 1949. Susceptibility of the guinea pig to action of alloxan as compared with the rat. Proc. Soc. Exp. Biol. Med. 70: 207-210. COHEN, G. & R. E. HEIkkiLA. 1974. The generation of hydrogen peroxide, superoxide radical, and hydroxyl radical by 6-hydroxydopamine, dialuric acid, and related cytotoxic agents. J. Biol. Chem. 249: 2447-2452. Haser, F. & J. Weiss. 1934. The catalytic decomposition of hydrogen peroxide by iron salts. Proc. Roy. Soc. Ser. A. 147: 332-351. McCorp, J. M., B. B. KEELE & I. FRipovicH. 1971. An enzyme based theory of obligate anaerobiosis: The physiological role of superoxide dismutase. Proc. Nat. Acad. Sci. U.S.A. 68: 1024-1027. Myers,

L. S., Jr.

1973.

Free

ponents by ionizing radiation.

radical

damage

of nucleic

acids and their com-

Fed. Proc. 32: 1882-1894.

27.

Pryor, W. A. 1973. Free radical reactions and their importance cal systems. Fed. Proc. 32: 1862-1869.

in biochemi-

28.

HEIKKILA, R. E., H. BARDEN & G. COHEN. 1974. Prevention of alloxan induced diabetes by ethanol administration. J. Pharmacol. Exp. Ther. 190: 501-506.

DISCUSSION

Dr. A. E. Kitascut: Could you prevent the hyperglycemic and other effects by the in vivo addition of well-known antioxidants or free radical trappers, such as santoquin, vitamin E, DPPD, and others? Dr. HEIKKILA: We have not tried to prevent the diabetogenic action of alloxan with vitamin E or the other free radical trappers you mention. However, we have given several other alcohols in addition to ethyl alcohol. We've given

methanol, ethanol, propanol, and butanol, and all four of these alcohols prevent the alloxan diabetes. In addition, we’ve performed some experiments with thiourea, which is a very good radical trapping agent, and the results are some-

230

Annals

New

York

Academy

of Sciences

what equivocal. With thiourea, we seem to get an all-or-none situation; although many of the animals are protected, others are not. Dr. H. SpRINCE (Veterans Administration Hospital, Coatsville, Pa.): One other point, in the living animal, where one obtains a destruction of free nerve endings with 6-hydroxydopamine, pretreating the animal with ascorbic acid resulted in greater damage. Recently, in Biochemical Pharmacology, Jonsson and Sachs from Sweden reported giving large doses of ascorbic acid. By some of the classic criteria for degeneration, they obtained no more degeneration. It has also been that individuals given large doses of ascorbic acid have decreased norepinephrine in the heart. This by itself is one criterion for nerve terminal degeneration. Does this mean that large doses of ascorbic acid might potentially have toxic effects on the nervous system and the heart in people who are taking massive doses? Dr. HEIKKILA: If an individual were taking a quinone-type compound that could react with ascorbic acid, large doses of ascorbic acid could cause the production of large amounts of peroxide, superoxide radical, and hydroxyl radical. There might be deleterious effects. Whether compounds that can react with ascorbic acid are present endogenously is not known. Dr. SPRINCE: Are the effects you obtained a function of the concentration of ascorbic acid? Dr. HEIKKILA: The uptake experiments (see Reference 8) were performed with increasing concentrations of ascorbic acid, up to 10°? M. There was an increasing inhibition of uptake, so the effect was a function of concentration. Dr. M. L. Scott: Do you have any ideas about the relationship between your work and the fact that dialuric acid is known to cause erythrocyte hemolysis in vitamin E-deficient animals? Ascorbic acid is such a good inducer for oxidation in microsomes and mitochondria of such animals but doesn’t have any effect in chicks that are receiving adequate vitamin E and selenium. Dr. HEIKKILA: There are probably several lines of defense against hemolysis, depending on how you stress. Ascorbic acid is a relatively weak peroxide generator compared to dialuric acid. Dr. W. WEIS: With respect to dehydroascorbic acid, in 1952 and 1956, Patterson demonstrated that dehydroascorbic acid itself, in the doses reported, produces alloxan diabetes, with histopathologic destruction of the pancreas.

SOME

PROPERTIES OF THE ASCORBATE FREE RADICAL *

Benon

H.

J. Bielski

and

Helen

W.

Richter

Department of Chemistry Brookhaven National Laboratory Upton, New York 11973

Phillip C. Chan Department of Biochemistry State University of New York Downstate Medical Center Brooklyn, New York 11203

Ascorbic acid (AH.,) has been known to be an essential biological component for a long time, but its specific role in the living cell is still not clearly understood. When ascorbic acid is oxidized it generally follows the Michaelis ! concept of a two-step oxidation involving a free radical intermediate. Therefore, it is of interest to understand not only the reactivity of ascorbic acid toward other biological compounds but also the reactivity of the ascorbate radical (A=). Since the discovery of the production of ascorbic acid radicals during the enzymatic oxidation of ascorbic acid,”: * several research groups have put great effort into the study of this transient species.'!" In most of these studies the ascorbic acid radical was generated by high-energy ionizing radiation.’ * 1°. 12-16 The advantage of this method is that the radical can be generated rapidly (in microseconds) from the parent compounds (ascorbic acid or dehydroascorbic acid) in the absence of interfering chemicals. The purpose of our investigation is to determine the reactivity of the ascorbate radical with a number of biologically significant compounds. The results with the compounds that have been tested are reported herein. When dilute aqueous solutions are exposed to ionizing radiation, essentially all of the energy is absorbed by the water: 1* 1°

H,O —w>OH(2.74) + ey,(2.76) + H(0.55) + H.O,(0.72) + H,(0.45)

@

The numerical value given in parentheses for each product in Reaction I is the G value, which is defined as the number of product molecules (or particles) formed per 100 eV of energy absorbed by the aqueous solution.'* The primary radicals produced by the water decomposition can undergo further reactions with added solutes. It has been found experimentally that in neutral and alkaline solutions the primary reducing radical, e-,,, is readily converted to OH radical by reaction with nitrous oxide:

N.O + e,, + H.O —— N, + OH + OH* Research carried out at Brookhaven National Laboratory under the United States Energy Research and Development Administration.

Pa |

(1) contract

with

Annals

B32

New

Academy

York

of Sciences

Hence, when an ascorbic acid solution saturated with N.O is exposed to highenergy ionizing radiation, the ascorbate radical is generated with a G(A=) = Gon + G.-

:

aq

AH, AH

AH

+ H*

(2,-2)

+ OH ——> AH: + H,O

(3)

Spectrophotometric studies of ascorbic acid radicals generated by pulse radiolysis * have shown that the species has two absorption maxima: one at 360 nm

(principal)

and

one

between

285

and

310

nm

(the latter maximum

undergoes a red shift with decreasing acidity). The radical disappears by second-order kinetics and is most stable in the pH range between 8 and 10, where the decay constant is k-—=7>10* M"? sec}. Detailed studies of the electronic structure of the ascorbate radical and model compounds by electron paramagnetic resonance spectroscopy 1°11: 14-16 suggest that the predominant form present over the entire pH range (0 to 13) is:

agPaNarn TS nee

ie

A survey of the literature shows that, despite the fact that the ascorbate radical has been known and studied for over a decade, almost no information is available about its chemistry of interaction with other compounds (QH.,,Q) of biological importance, e.g.:

A: + QH, ——> QH: + AH Ar + Q——>Q-+A

(4) (5)

The reason for the lack of such information is that it is difficult to generate the ascorbate radical chemically without introducing high concentrations of interfering chemicals as in the Ti-H.O. system, the Fenton reagent system, and the ceric sulfate system. Similarly, great difficulties have been encountered in attempts to study secondary radical reactions like (4) and (5) by pulse radiolysis. Because of interfering, competitive reactions, studies of these relatively slow reactions require high concentrations of the scavenger QH,. However, the OH radical required for the generation of A> reacts equally fast with the scavenger:

QH,, + OH

——> QH: + H,O

(6)

Thus, the reaction system after the pulse consists of AH., As, QH., and QH-. To overcome some of the above difficulties, a system consisting of a modified Durrum fast kinetics spectrophotometer on line with a Van de Graaff generator was developed in which one solution passes through a 2 MeV electron

Bielski beam

where

the radical

et al.: Ascorbate of interest

Free

is generated

Radical

in isolation,

233 while

the other,

nonirradiated, solution carries the scavenger and/or the buffer of a desired pH. The resolution time of the system is in the millisecond range and adequate for the study of a considerable number of radicals that decay at a relatively slow rate, e.g., ascorbate radicals, superoxide radicals, and catecholamine radicals. In the present study the rate of interaction of A> with a given compound (Reactions 4 and 5) is followed spectrophotometrically at 360 nm (when possible), where the ascorbate radical absorbs with an extinction of 4.9 « 103 M-? cm. Since the decay of A> is affected in neutral and alkaline solutions by the buffer concentration, the second-order rate constant, k., has to be deter-

mined in each case under the given experimental conditions (e.g., pH, concentration of buffer, slit setting on the spectrophotometer, temperature, etc.): A> + A= + H+

Figure

1.

Second-order

——>A+

AH-

(7)

decay

of ascorbate radicals. A nitrous oxide-saturated solution of ascorbate was irradiated and mixed with a nitrogen-saturated phosphate buffer at pH 8.7. The solution after mixing contained 2.65 uM _ ascorbate radicals, 47.35 uM ascorbic acid, 50 mM sodium phosphate, 11 mM nitrous oxide, and 0.32 mM nitrogen. The change in absorbance (inset) at 360 nm (0.005 absorbance units/cm) with time (0.5 sec/cm) was determined with an optical light path of 2 cm. The measurements were taken at 23° C.

tN [e)[e)

nm 366 (ABSORBANCE) 1/

0

1.0

2.0 SECONDS

3.0

4.0

An example of such a determination is shown in FiGuRE 1, where the ascorbic in which A> had been formed by Reactions I, 1, 2, and 3 was mixed rapidly in a jet mixing chamber with an equal volume of a buffer solution and the radical decay was monitored in the 2-cm optical cell. The decay is strictly second order, with a rate constant k, = 2.8 x 10° M~! sec+, where k is defined by 2kt= (1/C — 1/C,). Once k, is known for a given set of experimental conditions, varying amounts of scavenger (QH.,) are added to the buffer solution and the rate of Reaction 4 or 5 is determined under pseudo-first-order conditions. As an example, the reaction of A> with dopamine (QH.) is illustrated in FicurE 2. The pseudoacid solution

first-order rate constants that were determined when the dopamine concentration

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was varied by a factor of five show good agreement and give a corresponding second-order rate constant k = 360 + 40 M*! sec’?. The reactivity of ascorbate radicals with molecular oxygen was investigated because in the physiological pH range oxygen easily oxidizes ascorbic acid. Contrary to expectations, the second-order decay of A~ was not affected by the presence of oxygen, indicating that there was no interaction between the radical and O.. Methanol was selected for the study because it was used as a solvent for water-insoluble compounds. There is general agreement at present that the reduction of ferricytochrome c (cyt c+) by ascorbic acid is correctly described by the overall Reaction 8, in which two molecules of ferricytochrome c are reduced for every molecule of ascorbate oxidized to dehydroascorbate: 2 cyt c?+ + AH, ——>

2 cytc?+ + H++A

(8)

Based on kinetic 2? as well as on electron paramagnetic resonance ** studies, it had been suggested that Reaction 8 proceeds in two steps, Reactions 9 and 10, in which two electrons from one molecule of ascorbic acid are transferred to two molecules of cyt c**: cyt c?+ + AH- ——>

cyt c*+ + A= + H+

cyt c+ + Ay ——>scytc?*+A

(9) (10)

The results from our studies indicate that the upper limit for k,, is 6.6 * 10° M-? sec-!. This estimated value was obtained from a number of experiments in which the final reaction mixture contained 7 »M A-, 43 uM AH, and variable amounts of cyt c+ (from 38 to 147 1M). Under the most favorable conditions, that is in presence of 147 uM cyt c*+, less than 15% of the ascorbate radical

disappeared by Reaction 10, while the rest disproportionated by Reaction 7. In view of these observations it is suggested that the main pathway of the reduction of cytochrome c by ascorbic acid is via Reactions 9 and 7. The contribution of Reaction 10, although significant at pH 7.4, becomes negligible

0,010

FicurRE 2. Pseudo-first-order disappearance of ascorbate free radicals observed at 360 nm in the presence of dopamine (A:

0.001

20 hmMss Beee20) mM. Gero mM). The rates were deter-

(ABSORBANCE) 369 nm

mined in nitrogen-saturated solutions at pH 8.4 and 23°C.

0

L 0.1

al 0.2

al Ie 0.3 0.4 SECONDS

1 0.5

al 0.65»

(O57,

Bielski

et al.:

Ascorbate TABLE

Free

Radical

235

1

INTERACTION OF ASCORBATE RADICALS (A‘) WITH VARIOUS BIOCHEMICAL COMPOUNDS *

(Q) or (A*) ons (uM)

(QH:) (mM )

pH

Rate Constant (M™* sect)

A -+A Age one A’ + cytochrome c A* + Oz A* + methanol A’ + lactate A’ + pyruvate

Del 5.8 6.4 4.7 Sal 6.6 6.2

10-50 0.20 0.63 7.40 50.00 50.00

8.7 8.4 7.4 8.6 8.8 8.6 8.6

(2.822022) 52 10° (3562510:4)) 41107 6.6x 10? ASSENO p-fructose = L-sorbose, which resembles the relationship described here for DHA transport. That similarity suggests that these saccharides use a common transport mechanism. However, we have seen two differences. The DHA system is much less sensitive to PCMB and to mercury than is the glucose system, and the ability of human cells to transport DHA diminishes

FiGuRE 7. The effect of aging of erythrocytes at several pH levels on the transport of dehydroascorbate.

with aging of the cells in vitro. Previous transport studies with sugars have often been done with overaged blood bank cells. The mechanism of this impairment of DHA transport with cell age is unknown. It is not changed by converting the hemoglobin to carboxyhemoglobin. The effects of copper suggest that the limiting reaction in transport of DHA may be the disposition of intracellular DHA rather than the membrane translocation. Whatever the mechanism, these findings have implications for the function of vitamin C. Species that require dietary vitamin C must also have an appropriate mechanism for transporting this into cells. The present data suggest that hyperglycemia, of whatever cause, may compromise the intracellular supply of vitamin C. While several sugars have such an effect, only glucose impairs at levels that are often found in the blood. The histopathologic manifestations of deficiency produced by locally-impaired transport may be different from those produced by a deficient extracellular supply of vitamin C. Thus, the lesions of impaired transport may not be those of classic scurvy. This would be a more probable consequence if it were shown that different cell types have differing transport facility for DHA.

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The human red cell is known to have a unique persistence of fast, “fetal-type” monosaccharide transport.'* Since the renal threshold for AA is low,'® correction of impaired transport by raising plasma levels of AA will be ineffectual. The effect of increasing the level of DHA in overcoming the inhibition of glucose was small in these trials because DHA is itself inhibitory at high concentrations. Furthermore, DHA is a potent pharmacologic substance !* and cannot safely be given in quantity. The observation that copper ion will accelerate transport in vitro offers another approach to facilitating the transport of DHA since some drugs, e.g., phenylhydantoin, will markedly increase the level of serum copper in human beings.*°

Summary

A system for measuring the rate of transport of dehydroascorbate into human red blood cells shows Michaelis-Menten type kinetics with substrate inhibition at levels above 150 «»M DHA. The addition of sugars impairs this transport in the diminishing hierarchy p-glucose, D-mannose, D-xylose, D-galactose, L-lyxose, D-araboascorbate, L-sorbose and 2-deoxy-p-ribose. The effect of glucose on transport of ascorbate is marked at physiological levels. Transport of DHA is accelerated by copper ion and allows dehydroascorbate to move against a concentration gradient. The evidence supports the hypotheses proposing that hyperglycemia will impair the intracellular availability of vitamin C.

References

I. RALLI, E. P. & S. SHERRY. 1939. Effect of insulin on plasma level and excretion of vitamin C. Proc. Soc. Exp. Biol. Med. 43: 669-672. 2. SHERRY, S. & E. P. RAL. 1948. Further studies of the effects of insulin on the metabolism of vitamin C. J. Clin. Invest. 27: 217. 3. Hat, H. 1941. Behavior of vitamin C content of blood during insulin shock.

4.

Z. Med. 139: 485-488. Cox, B. D., M. J. WHICHELOW, W. J. H. BUTTERFIELD & P. NICHOLAS. 1974. Peripheral vitamin C metabolism in diabetics and nondiabetics: Effect of intraarterial insulin. Clin. Sci. Mol. Med. 47. In press.

5.

PAuLING,

6.

Sci. U.S.A. 67: 1643-1648. MANN, G. V. 1974. Hypothesis: The role of vitamin C in diabetic angiopathy. Perspect. Biol. Med. 17: 210-212.

L.

1970.

7.

ROSENBLOOM,

A.

Evolution

L.

1973.

and the need for ascorbic

The

natural

history

acid.

of diabetes

Proc. Nat.

mellitus.

Acad.

Public

Health Rey. 2: 115-154. 8. Gore, IL, M. Wapa & L. GOODMAN. 1968. Capillary hemorrhage in ascorbicacid-deficient guinea pigs. Arch. Path. 85: 493-502. 9. SIPERSTEIN, M. D. 1971. The role of microangiopathy in the diabetic syndrome.

Acta Diabetica 8: 249-273. 10. 11.

12.

PATTERSON, J. W. 1950. The diabetogenic effect of dehydroascorbic and dehydroisoascorbic acids. J. Biol. Chem. 183: 81-88. Levine, M. & W. D. STEIN. 1966. The kinetic parameters of the monosaccharide transfer system of the human erythrocyte. Biochem. Biophys. Acta 127:

179-193. Baitiie, L. A. 1960. Determination of liquid scintillation counting efficiency by pulse height shift. Int. J. Appl. Radiat. 8: 1-12.

Mann 13.

14. 15.

16.

17. 18. 19. 20.

& Newton:

Membrane

Transport

2

HuGues, R. E. & S.C. MATon. 1968. The passage of vitamin C across the erythrocyte membrane. Brit. J. Haemat. 14: 247-253. Martin, J. R. & C. E. Mecca. 1961. Studies in distribution of L-ascorbic acid in the rat. Arch. Biochem. Biophys. 93: 110-114. MILLER, D. M. 1969. Monosaccharide transport in human erythrocytes. Jn Red Cell Membrane-Structure and Function. G. A. Jamieson & T. J. Greenwalt, Eds. J. B. Lippincott. Philadelphia, Pa. LeFevre, P. G. 1961. Sugar transport in the red blood cell: Structure activity relationships in substrates and antagonists. Pharmacol. Rev. 13: 39-70. Wippas, W. F. 1955. Hexose permeability of foetal erythrocytes. J. Physiol. 127: 318-327. TERuucHI, J. & H. Mocuizuxr. 1960. Metabolism of dehydroascorbic acid in human subjects. J. Vitamin. 6: 163-170. SgosTRAND, S. E. 1970. Pharmacologic properties of dehydroascorbic acid and ascorbic acid. Acta Physiol. Scand. (suppl.) 356: 1-79. Taytor,

J. D., P. M. Kroun

diphenylhydantoin.

& T. N. Hiccins.

1974.

Serum

copper levels and

Amer. J. Clin. Path. 61: 577-578.

DISCUSSION

Dr. WAGNER: Have you looked at the effect of sucrose? Dr. MANN: No, but we have looked at fructose. Dr. H. SprINCE: It is surprising that alcohol has a hypoglycemic effect. Dr. MANN: It does have a hypoglycemic effect, but I suppose that was for different reasons than transport. Dr. W. B. SMITH: How did you preserve the red cells when they were 48 hours old? Dr. MANN: The cells were drawn, washed, and stored in a phosphate-saline buffer. Dr. SMITH: You can store them, but they leak within 24 hours of storage. Dr. MANN: Maybe this is the reason for this aging effect. I think it is unfortunate that much of the glucose transport work has been performed with overaged cells, which surely must be leaky. But, we see no real effect in the first 3—4 days of storage of washed cells at 5° C. Dr. N. R. STEVENSON (Rutgers Medical School, Piscataway, N.J.): 1 agree with you completely on your questioning of your third hypothesis, and I question whether you can even speak of transport of dehydroascorbic acid per se, considering that conversion does occur within the cell. Second, there is a difference between the membranes of various tissue organs in their translocation capacity for L-ascorbic acid vs. dehydroascorbic acid. The gut, for instance, does not move dehydroascorbic acid to any degree, whereas red blood cells and polymorphonuclear leukocytes take it up. Dr. MANN: We are talking about the movement of the material from the extra-

The

to the intracellular

compartment.

point is that I’m not sure

ascorbate

inside

So, we

are

that the conversion

the cell is, indeed,

the limiting

talking

about

transport.

of dehydroascorbate

factor

to

in this phenomenon.

Other tissues, for example, the retina, have an energy-dependent transport of dehydroascorbic. I am speaking here of human red blood cells. Dr. C. W. M. WILSON: We looked at this in the white cells and also in

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the red cells, but in the white cells; we have shown that as people age, the saturation limit for ascorbic acid absorption diminishes. Dr. A. E Kitascui: What percentage of total ascorbic acid in human plasma and in white blood cells is dehydroascorbic acid? Dr. MANN: It’s perhaps of the order of 10%. If the total ascorbate level is of the order of 0.8-1.2 mg%, there will be less than 0.1 mg% of dehydroascorbic acid. Dr. Kitascnti: Do you have data to support the thesis that dehydroascorbic acid is transported rather than stuck on the membrane? Dr. MANN: These cells are drawn and washed, and then hemolyzed, deproteinized, and the supernatant discarded, so I would concede that the dehydroascorbate may be in or on the membrane.

EFFECT OF ASCORBIC ACID DEFICIENCY ON THE PERMEABILITY AND COLLAGEN BIOSYNTHESIS OF ORAL MUCOSAL EPITHELIUM * Michael

C. Alfano,+ Sanford A. Miller, and James F. Drummond ¢

Department of Nutrition and Food Science Massachusetts Institute of Technology Cambridge, Massachusetts 02139

INTRODUCTION

One of the primary mechanisms of host defense against infectious disease is the maintenance of an effective epithelial barrier, which minimizes the penetration of bacteria and toxic bacterial by-products into the underlying connective tissue. The importance of this barrier function is clearly demonstrated by the epithelium that lines the crevice between the gingiva and the tooth. This nonkeratinized stratified squamous epithelium is constantly challenged by dense concentrations of bacteria in the subgingival dental plaque.? The penetration of toxic or antigenic macromolecular by-products of this bacterial plaque through the epithelial lining of the gingival crevice may initiate inflammatory periodontal disease, which ultimately results in the destruction of supporting bone and consequent tooth loss.*:4 Obviously, the effectiveness of the epithelial “barrier function” is an important determinant in the maintenance of periodontal health. Several qualitative reports suggest that the barrier function of mucosal epithelium may be seriously impaired by malnutrition.*-!° Unfortunately, neither the mechanism by which malnutrition alters epithelial permeability nor the degree to which this alteration occurs has been determined. Recent studies in our laboratories have indicated that the basement membrane represents the ratelimiting barrier to the penetration of bacterial endotoxin through nonkeratinized oral mucosal epithelium.'!» 1” Therefore, any nutritionally modulated increase in the permeability of oral mucosal epithelium to endotoxin must be mediated by some alteration in the structural integrity of the basement membrane. Ascorbic acid deficiency has been qualitatively related to an altered mucosal barrier function,” decreased resistance to infectious disease,! increased incidence of perio-

dontal disease,'* and altered synthesis of basement membrane collagen.1* 1 Therefore, the purpose of this investigation was to quantitate the effect of ascorbic acid deficiency on both the epithelial barrier to endotoxin penetration and basement membrane collagen biosynthesis.

* Supported by United States Public Health Service Grant No. D.E. 105-9. This is contribution No. 2606 from the Department of Nutrition and Food Science, Massachusetts Institute of Technology. + Reprint requests should be addressed to Dr. Alfano, Department of Periodontics and Oral Medicine, Fairleigh Dickinson University, School of Dentistry, Hackensack, New Jersey 07601. ¢ Present address: Department of Oral Pathology, University of Kentucky, College of Dentistry, Lexington, Kentucky 40506.

253

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MATERIALS

AND

Preparation

of Sciences

METHODS

of Endotoxin

Endotoxin was selected as a model for penetrating macromolecules because of its large molecular size, potent biological activity, and implication in periodontal disease. FE. coli endotoxin type 055:B5 (Difco Laboratories, Detroit, Mich.) was gas-tritiated (New England Nuclear Corp., Boston, Mass.) according to the method described by Schwartz and coworkers.' Free tritium was removed from the endotoxin by exhaustive dialysis against distilled water, as described by Schrader and Woolfrey,'® resulting in a final specific activity of 94.5 »Ci/mg. Animal

Model

System

The nonkeratinized epithelium of the ventral surface of the base of the guinea-pig tongue has been shown to be histologically analogous to the epithelium that lines the human gingival crevice.!* This finding, in addition to the fact that guinea pigs may be rapidly depleted of ascorbic acid, makes the guinea pig a useful model for studying the effects of ascorbate deficiency on mucosal barrier function. Eighty-two young adult male Hartley guinea pigs weighing about 350 grams each were randomly divided into three groups. The animals in all three groups were fed the same pelleted semisynthetic ascorbic-acid-deficient diet (Nutritional Biochemical Co., Cleveland, Ohio) and water ad libitum. The “deficient” group consisted of 32 animals that were fed only the deficient diet and water. The “pair-fed” group consisted of 32 animals that were paired by weight to the animals in the deficient group. Each animal in this pair-fed group was given the same amount of diet as consumed daily by the corresponding animal in the deficient group, except that, in addition, the pair-fed animals received oral supplementation with 10.0 mg of L-ascorbic acid daily as described by Ginter et al.17 The last group (“ad libitum”) consisted of 18 animals that were maintained on an identical dietary and supplementation regime as the pair-fed group, except that the amount of diet consumed was unrestricted. The food intake of each animal was recorded daily, and the animals were weighed every three days. Eight animals from each of the deficient and pair-fed groups were sacrificed after one, two, three, or five weeks on the diets. The deficient animals in the five-week group were rehabilitated with 10.0 mg of L-ascorbic acid daily for two weeks after three weeks on the ascorbate-restricted diet. Six animals from the ad libitum group were sacrificed after 0, three, or five weeks on the diet. At sacrifice, ascorbate levels were determined in the tongue, liver, and whole blood.'* In addition, the mucosa from the ventral surface of the base of the tongue was assayed for permeability to tritiated bacterial endotoxin using an in vitro system outlined in the next section. Finally, the biosynthesis of basement membrane collagen by mucosal epithelial explants from each of the animals was assayed in vitro as described below.

Alfano

et al.: Ascorbate Assay

Deficiency and Mucosal

for Mucosal

Epithelium

255

Permeability

The method for quantitating mucosal permeability is based on the skinperfusion chamber of Ainsworth !° and has been described in detail in a previous report.*° Briefly, the tongues of the guinea pigs were excised at sacrifice and a mucosal specimen approximately 6 X 6 mm in size was dissected from the ventral surface of the base of each tongue. The mucosal tissue was then clamped in an adapter and mounted in a permeability cell in which the connective tissue surface of the tissue was perfused with phosphate-buffered saline solution at a flow-rate of 3.0 ml/hr (FicurE 1). Fifty microliters (47 wCi) of the tritiated endotoxin solution was then placed on the epithelial surface of the tissue. The perfusion was regulated by a multispeed peristaltic pump (Harvard Apparatus, Millis, Mass.) and conducted in an incubator at 37° C to ensure physiological temperature during the 3.0-hour perfusion period (FIGURE 2). One-milliliter

PENETRATION

SOLUTION

MUCOSAL TISSUE ~ ACRYLIC i

ADAPTER

jt

TE=*5— perFusioN SOLUTION INLET - CE pERFUSION SOLUTION OUTLET

—

=

FIGURE tion).

1. Permeability

(From

Alfano

cell with mucosal

et al.°

By permission

tissue and adapter of the Journal

in place

of Dental

(cross sec-

Research.)

aliquots of the perfusate were collected at 20-minute intervals for analysis of radioactivity. A “penetration coefficient” was then calculated based on the ratio of the penetration rate at steady state to the concentration of the endotoxin applied.°°: 74 Penetration coefficients were calculated for each group of tissues,?° and the data were statistically evaluated using a two-way analysis of variance. This analysis was accomplished by use of the UCLA Biomedical computer program No. BMD 10V for multiple linear regression.** Collagen

Biosynthesis

by Epithelial Explants

After the mucosal specimen was removed from the ventral base of the tongue for permeability measurements as described above, two additional mucosal samples (2 X 3 mm each) were dissected from the tongue immediately

Annals

250

FIGURE

2.

Permeability

phosphate-buffered (une (3H (C)).

New

York

Academy

cells, collecting

saline solution assembled

of Sciences

vials, peristaltic

pump,

and

reservoir

in incubator to insure constant

of

tempera-

anterior to the initial sample site. The epithelium was separated from the underlying connective tissue in these samples by incubation in 20 mM Na, EDTA in phosphate-buffered saline solution at 37° C for one hour.?* Each of the isolated epithelial sheets was placed on a sterilized regenerated cellulose substrate (Cole Parmer, Chicago, Ill.) suspended over basal medium.

One of the paired samples

was placed on basal medium supplemented with 50.0 ug L-ascorbic acid per ml; the other sample was cultured on unsupplemented basal medium. The basal medium consisted of: (1) Dulbecco’s modified Eagle’s medium; (2) 5.0 yg L-proline/ml; (3) 100 units penicillin/ml; (4) 100 ug streptomycin/ ml. After six hours in culture at 37° C in a humidified atmosphere of 95% air and 5% CO.,, the explants were pulsed for three hours with 'C-.-proline. This was accomplished by switching the explants and their substrates to ascorbatesupplemented or unsupplemented basal medium, as indicated, which was modified to contain 2.0 »Ci/ml of *C-L-proline (specific activity 0.4 uCi/ug proline). After incubation the explants were removed from culture, weighed, and frozen at —20°C. The explants were then homogenized in 500 volumes of distilled water and a 200-yl aliquot was removed for DNA analysis.24 The remaining homogenate was heat-inactivated for 10.0 minutes at 100° C, dialyzed against two changes of cold distilled water for 24 hours, evaporated to dryness under nitrogen, and then hydrolyzed. The radio-labeled proline and hydroxyproline in the hydrolysate were separated in the presence of carrier proline and hydroxyproline by thin-layer chromatography,'* and then analyzed for radioactivity by liquid scintillation spectrometry. The results were expressed as dpm/hydroxyproline/ug DNA, and reflect the amount of [''C]hydroxyproline incorporated per cell into the nondialyzable

Alfano et al.: Ascorbate

Deficiency and Mucosal

Epithelium

257

collagen fraction. The radio-labeled hydroxyproline in this fraction represents the basement membrane collagen synthesized during the labeling period. The effects of dietary regime, time on the diet, and ascorbic acid in the medium on basement membrane collagen biosynthesis were analyzed using a three-way analysis of variance. The BMD-10V computer program for multiple linear regression was used to generate the analysis of variance table.?2

RESULTS

The data on the food intake and body weight (FIGURE 3) as well as ascorbate levels in the tissues and blood (FiGureE 4) during the course of the investigation are consistent with several other reports.'’: °°-2* The fact that rehabilitation of the deficient group only with ascorbic acid resulted in both a rapid increase in food intake and reversal of weight loss, and that the ad libitum group grew well during the course of the study (FIGURE 3) indicates that the diet was deficient in ascorbic acid but adequate in other nutrients. As illustrated in Ficure 4, the ascorbic acid content of the liver and tongue rapidly fell to low levels in the deficient animals, while remaining relatively constant for the other two groups. In contrast, the blood levels of ascorbic acid in the deficient group remained near normal values for three weeks, at which time a moderate decrease occurred. The tissue levels of ascorbic acid rose markedly in the deficient animals that were rehabilitated for two weeks with 10.0 mg of L-ascorbate daily. The effect of progressive ascorbic acid deficiency on the permeability of the

4—-4 ©—o @--@

600

AD LIBITUM+!OmgASCORBIC ACID DAILY PAIR FED +10mg ASCORBIC ACID DAILY DEFICIENT

560

520

§ 480

s

FicurE

3.

Food

intake

and

weight gain (in grams) of guinea pigs maintained on an_ascorbic-

acid-deficient diet or pair-fed- and ad libitum-fed ascorbate-deficient diets that were supplemented with 10.0 mg L-ascorbic acid daily.

=gao g 400

360

3 2 (e)

Nip sre

iE

30 BA

en

ASCORBIC ACID SUPPLEMENTATION INITIATED (grams) CONSUMPTION FOOD DAILY 0 3

6

9

2°15

8 2) 24 27 DAYS ON DIET

30

33

Annals

258

Pa oo°o

xa wo @

New

of Sciences

Academy

York

[_] Ab LIBITUM + 10mg ASCORBIC ACID DAILY g PAIR FED + 10mg ASCORBIC ACID DAIL AILY YZ DEFICIENT

JI STANDARD

ERROR

a ie)

+ ie)

awOo °

Ficure

weight) (4g/gm LIVER wet

60/—

Ascorbic

blood,

of guinea asconnle ACD

acid levels

tongue,

and

pigs maintained

liver

on

an

ascorbic-acid-deficient diet or pair-

AAT Daa en

fed- and ad-libitum-fed

sof-

ascorbate-

deficient diets that were supplemented with 10.0 mg _ L-ascorbic acid daily. (The five-week deficient group was rehabilitated by supplementation with 10.0 mg L-ascorbic

fs) |

oe Si

LEVELS ACID ASCORBIC

weight) (449/gm. TONGUE wet

ASCORBIC ACID SUPPLEMENTATION aT eee

ath eT S2 a |

o

4,

in whole

ND. =Oo oO

on

acid per day for two weeks after three weeks on an unsupplemented ascorbate-deficient diet).

VA

Oo WEEKS

ASCORBIC ACID SUPPLEMENTATION INITIATED

AFTER

3 WEEKS

mucosal tissue is illustrated in FiGuRE 5, in which an increase in the penetration coefficient directly reflects an increase in tissue permeability. The increase in permeability for both deficient and pair-fed groups after two or three weeks on the diet is noteworthy; and the analysis of variance indicated that the main effects of both diet and time were significant (p < 0.001). Mucosal tissues from animals with severe ascorbate deficiency (two- or three-week groups) demonstrated markedly greater permeability when contrasted to tissues from normal animals (0 time) (p < 0.05); however, when the permeability of the tissues from deficient animals was contrasted to that of the pair-fed control animals at each time point, the increase in permeability was less marked, and no longer statistically significant (p > 0.05). These findings indicate that, although ascorbate-

Figure 5, Mucosal penetration coefficients for mucosal tissue from guinea pigs maintained on an ascorbic-acid-deficient diet, or pairfed- and

ad libitum-fed

AD PAIR 200}-

LIBITUM FED

+10 mg

+ !Omg

ASCORBIC

ASCORBIC

ACID

ACID

DAILY

DAILY

DEFICIENT

] STANDARD

ERROR

ascorbate-

deficient diets that were supplemented with 10.0 mg_ L-ascorbic acid daily. (*The five week deficient group was rehabilitated by supplementation with 10.0 mg Lascorbic acid per day for two weeks after three weeks on an unsupplemented diet).

.

ascorbate-deficient

') min (PENETRATION COEFFICIENT xem WEEKS

neh na INITIATED AFTER 3 WEEKS

Alfano

et al.: Ascorbate

Deficiency and Mucosal

Epithelium

259

deficient animals demonstrated consistently greater permeability than either control group, much of this large increase in permeability must be attributed to the inanition that accompanies ascorbate deficiency. Therefore, in addition to ascorbic acid, other nutritional factors must be important in modulating oral mucosal permeability.

Two effects of ascorbic acid on the biosynthesis of basement membrane collagen were studied: first, the direct effect of the presence or absence of ascorbic acid in the culture medium

was noted; second, the conditioning effect

of various stages of in vivo ascorbic acid deficiency on the in vitro synthesis of basement membrane collagen was evaluated. Analysis of the data as summarized in TABLE | indicated that the addition of 50.0 ug/ml of L-ascorbic acid significantly increased (p < 0.0001) the ability of mucosal epithelial explants to incor-

TABLE

|

DPM HyYDROXYPROLINE/uG DNA+S.D. FoR MucosAL EPITHELIAL EXPLANTS FROM ASCORBATE-DEFICIENT, PAIR-FED, OR AD LIBITUM-FED GUINEA PIGS GROWN ON BASAL MEDIUM * EITHER SUPPLEMENTED (+) oR Nor (—) WITH 50 uwG/ML OF L-ASCORBIC ACID

Days

Pair-Fed

on

Diet 0 Ul 14 P| 35

bis =

Deficient

i

be

ad Libitum

in

aes

a

= 27.8+18.5

hia 15.4+7.7

38.0+10.4 18.0+9.6 ROSES — Pip 2a —I ee ire Wests) s) 22.9+14.5 14.6+8.6 — — 25° 2== [e2e eel) lel See OF2 == 2e) oil D204 (Oe ail 2: ees Seas 25 .9)2= 102 1 1L6==2°8 1D.6== OO 20 1SSr Saie 2 eee 328 14.5+4.0

* The basal medium consisted of: Dulbecco’s modified Eagle’s medium + 2 uCi/ ml ["C]proline (S.A. 0.4 wCi/ug proline), 100 units penicillin, and 100 ug streptomycin per ml. + This group was switched to an ascorbate-supplemented diet (10 mg/day) after 21 days on the deficient diet.

porate hydroxyproline into basement membrane collagen. In contrast, the effect of dietary conditioning on the ability of the explants to synthesize basement membrane collagen was not significant (p > 0.05). DISCUSSION

The marked decrease in ascorbic acid tissue levels in the deficient group and the considerable increase in these levels after administration of ascorbic acid (FIGURE 4) are consistent with the anticipated effects of an ascorbic-aciddeficient diet. The slight increase in ascorbic acid levels in the tissues of the pair-fed group at two weeks (FiGURE 4) may reflect the reported relationship between ascorbic acid levels and protein status.**: °° The fact that the tongue lost its ascorbic acid stores in the deficient group as rapidly as the liver demonstrates that, unlike the lens, brain, and adrenal,”* it does not have the capacity

to concentrate

and store ascorbic acid.

This finding insures that the mucosal

260

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tissues of the ascorbate-restricted animals in this study were progressively deficient in their ascorbic acid content. Several effects of ascorbate deficiency on the permeability of oral mucosal tissues are worthy of discussion. First, it is important to note that both in a pilot study 2° and in the present investigation, oral mucosal tissues from scorbutic animals demonstrated significantly greater permeability to [*H]endotoxin than did tissues from normal healthy animals (0 time, FiGURE 5).

However, the fact

that the permeability of the mucosal tissues of the pair-fed group also increased, although not significant statistically, merits discussion. Examination of the dietary intake of the pair-fed group indicated that the amount of food supplied became progressively deficient until these animals ultimately received less than 40% of the food that the ad libitum animals were consuming. This markedly deficient food intake was reflected in the altered growth curve for this group (FIGURE 3), and suggests that the animals were probably deficient in total calories, and probably protein and other nutrients as well. Considering that the basement membrane, the rate-limiting barrier to endotoxin penetration of these tissues,!! is a complex structure composed of glycoproteins in addition to collagen," it is reasonable to assume that nutritional deficiencies other than ascorbic acid may adversely affect the integrity of this structure. Therefore, the increase in the mucosal permeability of the pair-fed group may actually be a reflection of some other nutrient deficiency resulting from the decreased food intake. This finding indicates that mucosal barrier function is probably a multinutritional-dependent parameter. In view of the apparent sensitivity of this parameter, it is reasonable to suggest that it may prove to be a useful tool in establishing optimal levels of nutrient intake. The data in FiGurRE 5 also demonstrate that, although a mild deficiency in ascorbic acid (one week) does not alter permeability, a moderate deficiency (two weeks) results in a statistically significant increase in permeability which is almost as severe as that in the scorbutic animals. Although the increase in mucosal permeability during the deficiency paralleled the decline in tissue levels of ascorbate, it occurred sooner and to a more marked extent than one could predict in light of the blood levels of ascorbate (FIGURE 4). This fact illustrates the unreliability of blood levels of ascorbate in predicting ascorbic acid status in the tissues. In addition, the finding that the permeability of the tissues declines only slightly after two weeks of ascorbic acid supplementation indicates that the effects of ascorbate deficiency on mucosal barrier function are not rapidly reversible. Considering the notable changes that were observed in both tissue levels of ascorbic acid and mucosal permeability during ascorbate deficiency, it was interesting to note that this conditioning did not appear to alter the ability of the explants to synthesize collagen in vitro (TABLE 1). One explanation for this finding is the possibility that ascorbic acid may not be stored to any significant extent in the mucosal epithelium. Thus, the marked differences in ascorbic acid levels in the tongues from the various groups may be a reflection only of differences in the connective tissue stores of the vitamin. Therefore, it is possible that epithelial explants from deficient or control animals contained relatively equal quantities of ascorbic acid when they were placed in culture. This would explain the failure of control tissues to synthesize more collagen than depleted tissues when cultured on ascorbate-free medium. Two additional explanations for the apparent inability of dietary conditioning

Alfano

et al.: Ascorbate

Deficiency and Mucosal

Epithelium

261

to alter in vitro collagen biosynthesis are possible. First, the variability in assaying these small tissue samples is large, and this factor may mask moderate inherent differences in the tissues. Also, since the primary effect of ascorbic acid on collagen biosynthesis is a local one, that is, occurring at the site of biosynthesis, the marked differences in ascorbate levels in the two culture media may have negated the effect of in vivo conditioning. The results of this investigation did indicate that ascorbic acid significantly increases the ability of primary mucosal epithelial explants to incorporate hydroxyproline into basement membrane collagen in vitro (TABLE 1). Although this finding is consistent with numerous reports on the in vitro effect of ascorbic acid on the biosynthesis both of interstitial collagen by fibroblasts and basement membrane

collagen by a cancerous

epithelial cell line, #1: *? it is significant

because it represents the first time that ascorbic acid has been shown to be important in the biosynthesis of basement membrane collagen by primary adult epithelial cells in culture. Furthermore, this finding strongly suggests that an alteration in basement membrane collagen biosynthesis constitutes the mechanism by which ascorbic acid deficiency compromises the barrier function of the basement membrane. This suggestion is consistent with the observation of Robert and Godeau ** that collagenase significantly alters the permeability of the basement membrane. Finally, it is conceivable that this altered capacity to synthesize basement membrane collagen during ascorbate restriction, and the concomitant compromise of basement membrane barrier integrity may represent the elusive mechanism by which capillary fragility is induced in scurvy. The implications of these findings in terms of host resistance to infectious disease are significant since they represent the first time that the deleterious effects of malnutrition on mucosal barrier function have been quantitatively documented. In addition, the data demonstrate that although two weeks of nutritional rehabilitation result in a rapid increase in food consumption, weight gain and tissue levels of ascorbic acid, there is not a rapid return of mucosal permeability to baseline levels. This finding suggests that acute episodes of malnutrition may potentially compromise host defenses for prolonged periods of time. The importance of this study in terms of host resistance to periodontal disease is particularly interesting. The initiation of periodontal disease is generally thought to require the penetration of some quantity of toxic or antigenic macromolecules from the bacterial plaque through the gingival crevicular epithelium and into the connective tissue.* As calculated from the data in FIGURE 5, the nutritional stress imposed in this investigation almost doubled the permeability of the mucosal epithelium to endotoxin. Assuming that a comparable effect occurs in the histologically analogous epithelium of the gingival crevice, it is clear that the increase in the toxic macromolecular load to the connective tissue may predispose the host to periodontal disease. In fact, if prior to the introduction of nutritional stress, the host defenses balance the virulence of the bacterial plaque, it as appropriate to suggest that the nutritional alteration in the epithelial barrier may actually initiate the disease process. The fact that plaque bacteria are capable of releasing potent collagenolytic enzymes **: *° capable of destroying basement membrane collagen *° further accentuates the importance of the nutritionally altered capacity of the host to synthesize basement membrane

collagen,

and thereby maintain

an effective epithelial barrier.

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ACKNOWLEDGMENTS

We thank Professors Nevin S. Scrimshaw, Bernard S. Gould, and Paul M. Newberne of the Massachusetts Institute of Technology for their perceptive comments and helpful suggestions during this research.

REFERENCES

1968. Interactions of NutriScriMsHaw, N. S., C. E. Taytor & J. R. Gordon. tion and Infection. World Health Organization. Geneva, Switzerland. Socransky, S. S., R. J. Gippons, A. C. DALE, L. BoRTNICK, E. ROSENTHAL & 1963. The microbiota of the gingival crevice of man. J. B. MacDonaLp. I. Total microscopic and viable counts and counts of specific organisms.

Arch. Oral Biol. 8: 275—280. SocRANSKY, S. S. 1970. Relationship of bacteria to the etiology of periodontal disease. J. Dent. Res. 49: 203-221. ScHwartz, J., F. L. Stinson & R. B. PARKER. 1972. The passage of tritiated

bacterial endotoxin across intact crevicular epithelium. 276. GRANT,

A. H.

1926.

Effect of the calcium,

vitamin

J. Periodont. 43: 270-

C, vitamin

diet on the permeability of the intestinal wall to bacteria. 502-508. Hongo,

S., M. TAKASAKA,

T. Fucrwara,

K. IMaizumr

D ratio in the

J. Infect. Dis. 39:

& H. OGAwa.

1969.

Shi-

gellosis in cynomolgus monkeys. VII. Experimental production of dysentery with a relatively small dose of Shigella flexneri 2a in ascorbic acid deficient monkeys. Japan. J. Med. Sci. Biol. 22: 149-162. Lyncu,

J. E.

1957.

Histological

used in experimental 813-819. WEAVER,

H. M.

affected 14-21.

1946.

by intake

WORTHINGTON,

amebiasis Resistance

of vitamin

observations

on the influence

in guinea-pigs. of cotton

of a special

J. Trop.

Med.

Hyg.

rats to the virus of poliomyelitis

A, partial

B. S., E. S. BOATMAN

Amer.

inanition,

and

& G. E. KENNEY.

sex.

1974.

J. Pediat.

diet

6: as

28:

Intestinal absorp-

tion of intact proteins in normal and protein deficient rats. Amer. J. Clin. Nutr. 27; 276-286. WORTHINGTON, B. S. & E. S. BOATMAN. 1974. The influence of protein malnutrition on ileal permeability to macromolecules in the rat. Amer. J. Digest. Dis. 19; 43-55. ALFANO, M. C., J. F. DRUMMOND & S. A. MILLER. Localization of the ratelimiting barrier to the penetration of endotoxin through non-keratinized oral mucosa Jn Vitro.

ALFANO,

M. C.

J, Dent. Res.

1974.

The

In press.

Effect of Ascorbic

Acid

Deficiency

Function of Guinea-pig Oral Mucosal Epithelium. Archives, Massachusetts Institute of Technology. CLARK, J. W., E. CHERASKIN & W. RINGSDORF. 1970.

Patient. Priest,

: 127. Charles

R. E.

1970.

C Thomas.

Formation

Doctoral

on

the Barrier

thesis,

Institute

Diet and the Periodontal

Springfield, Ill.

of epithelial

basement

membrane

is restricted

by scurvy in vitro and is stimulated by vitamin C, Nature 225: 744-745. ALFANO, M. C., J. F. DRUMMOND & S. A. MILLER. 1975. Effect of ascorbic acid on the biosynthesis of basement membrane collagen. J. Dent. Res, 54 (special issue A): L107.

SCHRADER,

W. H. & B. F. Woorrrey.

1963.

Studies with tritiated

I, Preparation and analysis of tritiated endotoxin.

endotoxin.

J. Clin. Invest. 42: 225-235.

GINTER, E., P. BoBEK & M. Ovecka. 1968. Model of chronic hypovitaminosis C in guinea-pigs. Internat. Z. Vit. Forsch. 38: 104-113.

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Rog, J. H. & C. A. KUETHER. 1943. The determination of ascorbic acid in whole blood and urine through the 2,4-dinitrophenylhydrazine derivative of dehydroascorbic acid. J. Biol. Chem. 147: 399-407. AINSWORTH, M. 1960. Methods for measuring percutaneous absorption. J. Soc. Cosm. Chem. 9: 69-78. ALFANO, M. C., J. F.DRUMMOND & S. A. MILLER. 1975. Technique for studying the dynamics of oral mucosal permeability in vitro. J. Dent. Res. 54: 194. TREGEAR, R. G. 1966. Physical Functions of Skin. Academic Press. New York,

INBY: Dixon, W. J. 1973.

Biomedical Computer Programs.

fornia Press. Los Angeles, Calif. ALFANO, M. C., J. F.DRUMMoND & S. A. MILLER. sal epithelium from connective tissue by EDTA, Dent. Res. 53 (special issue): 247. PrasaD, A. S., E. Du

MouUCHELLE,

D. Konruck

: 719.

University of Cali-

1974. Separation of mucoelastase or collagenase. J.

& D. OGERLEAS.

1972.

A sim-

ple fluorometric method for the determination of RNA and DNA in tissues. J. Lab. Clin. Med. 80: 598-602. RICHMOND, V. & E. L. R. Stoxstap. 1969. Effect of ascorbic acid on guineapig skin collagen synthesis: I. Total collagen. J. Dent. Res. 48: 863-871. Hucues, R. E., R. J. Hurtey & P. R. Jones. 1971. The retention acid by guinea-pig tissues. Brit. J. Nutr. 26: 433-438.

of ascorbic

Opumosu, A. & C. W. M. WILson. 1973. Metabolic availability of vitamin C in the guinea-pig. Nature 242: 519-521. CHATTERJEE, G. C. 1967. Effects of ascorbic acid deficiency in animals. In The Vitamins.

W.

Sebrell

and

R. S. Harris,

Eds.

: 407.

Academic

Press.

New

York, N.Y. WILLIAMS, R. S. & R. E. HuGHes. 1972. Dietary protein, growth and retention of ascorbic acid in guinea-pigs. Brit. J. Nutr. 28: 167-174. KEFALIDES,

N. A.

1973.

Structure

and

biosynthesis

of basement

membranes.

Int. Rev. Conn. Tiss. Res. 6: 63-104. Bates, C. J., A. J. BArLEy, C. J. PRYNNE & C. I. LEVENE. 1972. The effect of ascorbic acid on the synthesis of collagen precursor secreted by 3T6 mouse fibroblasts in culture. Biochem, Biophys. Acta 278: 372-390. DELL’ORCco, R. T. & J. H. NAsH. 1973. Effects of ascorbic acid on collagen synthesis in non-mitotic human diploid fibroblasts. Proc. Soc. Exp. Biol. Med.

144: 621-622.

34.

BD:

Rosert, A. M. & G. GopEAu. 1974. Action of proteolytic and glycolytic enzymes on the permeability of the blood brain barrier. Biomedicine 21: 36-39. Grppons, R. J. & J. B. MACDONALD. 1961. Degradation of collagenous substrates by Bacteroides melaninogenicus. J. Bact. 81: 614-621. ALFANO, M. C., R. E. MoruHart, G. METCALF & J. F. DRUMMOND. 1974. Presence of collagenase from Clostridium histolyticum in gingival sulcal debris of a primitive population. J. Dent. Res. 53: 142.

FUNCTION OF ASCORBIC ACID COLLAGEN METABOLISM M.

IN

J. Barnes

Medical Research Council and Department University of Cambridge Cambridge, England

of Pathology

Direct involvement of ascorbic acid in collagen synthesis is well known and represents perhaps the most clearly defined biochemical role of the vitamin. The absence of wound healing and occurrence of fractures that fail to repair are classically-recognized features of scurvy that can be attributed to impaired collagen formation arising from lack of vitamin C. The presence of hemorrhage in this disease may conceivably be regarded as another.’ In this paper it is my intention to outline the progress that has been made in the understanding of the mode of action of the vitamin in collagen formation since the time this subject was considered at the first conference on vitamin C organized by The New York Academy of Sciences in 1960.*;* It was then mooted 2 that its most likely role was in the formation of collagen hydroxyproline through hydroxylation of proline before the latter was incorporated into peptide linkage. It is now known from studies utilizing isolated collagensynthesizing systems that ascorbic acid does, as thought possible, participate in this reaction and also in the analogous reaction leading to the formation of collagen hydroxylysine, but that the hydroxylation of both the proline and the lysine occurs only after the pertinent amino acid has been incorporated within the collagen polypeptide chain formed during ribosomal collagen protein synthesis. This paper will briefly summarize the relevant evidence that has accumulated over the past decade leading to this conclusion and will consider whether the collagen lesion as it occurs in vivo in scurvy is fully accountable in terms of impaired hydroxylation (of either peptidyl proline and/or peptidyl lysine). The function of ascorbic acid in collagen synthesis has been reviewed recently by a number of authors.‘~7 FORMATION OF COLLAGEN EVIDENCE FOR THE

HYDROXYPROLINE PARTICIPATION OF

AND HYDROXYLYSINE: ASCORBIC ACID

It was demonstrated some years ago that free hydroxyproline and hydroxylysine were not incorporated into collagen. The presence of these two unusual amino acids in collagen arises in fact through the hydroxylation of particular prolyl and lysyl residues previously incorporated into peptide linkage during the process of ribosomal collagen polypeptide synthesis. Hydroxylation appears to occur primarily while the polypeptide chain is being formulated and is therefore still attached to the ribosome.**?®: 88, 89 Evidence that hydroxylation does not precede translation arose initially from isotopic studies utilizing isolated collagen-synthesizing systems that demonstrated that formation of collagen hydroxyproline could be markedly inhibited without, at the same time, preventing collagen protein synthesis. Thus, under appropriate conditions, incorporation of labeled proline into collagen hydroxy-

264

Barnes:

Collagen Metabolism

265

proline could be inhibited while still achieving incorporation of radioactivity into peptide-bound proline contained in material that, like collagen, was extractable with hot trichloroacetic acid and was collagenase-susceptible. These studies implied the formation of a proline-enriched hydroxyproline-deficient collagen, termed protocollagen, when hydroxylation was impaired.1! Formation of protocollagen has been demonstrated by isotopic means when hydroxylation is impaired either by inclusion of the chelating agent «,a’-dipyridyl in the incubation medium or through exclusion of oxygen by incubation of tissues under nitrogen.* The constituent unhydroxylated polypeptide chains of protocollagen have now been separated by chromatography by procedures similar to those for the separation of the equivalent hydroxylated polypeptide chains of collagen.!2-?1

TABLE

1

INCORPORATION OF ["“'C]PROLINE INTO THE COLLAGEN PROLINE AND HYDROXYPROLINE OF GRANULOMAS FROM NORMAL AND SCORBUTIC GUINEA

PIGS

Specific Activity (dpm/umole)

*

Normal

Scorbutic

Hydroxy-

Proline/ Hydroxy-

Proline

proline

proline

790 745

697 680

heal) 1.10

644 8994 4860

564 3888 1870

(cpm/flask)

tl 23 2.6

Hydroxy-

Proline/ Hydroxy-

Proline

proline

proline

787 530

ROH + X+ H,O

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where R is the substrate undergoing hydroxylation (peptidyl proline or lysine), XH,, the cosubstrate, and ROH the hydroxylated product (peptidyl hydroxyproline or hydroxylysine). With the establishment of ascorbic acid as a cofactor, it was naturally assumed that the vitamin was required as the cosubstrate in the mixed function oxidation, as occurs for example in the hydroxylation of dopamine by the copper-dependent enzyme dopamine-f-hydroxylase.°° It was thought that a-ketoglutarate might act as an allosteric activator.*! However, it then became apparent the role of cosubstrate was undertaken by a-ketoglutarate itself, which, during the course of hydroxylation, underwent a stoichiometric decarboxylation to succinate.*!:°2 It was shown that one-half of the oxygen molecule was incorporated into the substrate undergoing hydroxylation while the other half, in contrast to the reaction depicted in the above equation, appeared in succinate rather than water.°? These enzymes have therefore been classified as dioxygenases.** It became clear that a number of enzymes could be grouped together as belonging to a newly recognized class of hydroxylase, all requiring as cofactors molecular oxygen, ferrous ion, a 2-keto acid, and a reducing agent, the most effective being ascorbic acid, and all being stimulated by catalase.*° Thus, in addition to prolyl and lysyl hydroxylases, the enzyme y-butyrobetaine hydroxylase, from both animal and bacterial origins, that catalyzes the hydroxylation of y-butyrobetaine to carnitine, oxygenases identified in Neurospora crassa that catalyze the oxygenation of thymine to 5-carboxyuracil and the conversion of thymidine to thymine riboside and deoxyuridine to uridine, and the enzyme p-hydroxyphenylpyruvic acid oxidase that catalyzes the conversion of p-hydroxyphenylpyruvic acid to homogentisic acid have all been shown to display in common those cofactor activity requirements listed above. With the establishment of a-ketoglutarate as cosubstrate, the role of ascorbic acid as reductant in the reactions catalyzed by prolyl and lysyl hydroxylases was left unexplained. In contrast to the absolute requirement for a-ketoglutarate and ferrous ion, that for ascorbate has been found to be not highly specific.34 #5, 56,57 The vitamin, in the isolated cell-free system at any rate, can be replaced by any of its stereoisomers (but not by dehydroascorbic acid), by various reduced pteridines, and by a number of thiol compounds including dithiothreitol and cysteine. The same lack of specificity appears to hold true for the other enzymes in this group. Generally, however, ascorbic acid appears to be the most effective reductant. Besides its ability to replace ascorbic acid as a reductant, dithiothreitol appears to exert an additional effect since at low concentrations it causes stimulation of enzyme activity even in the presence of saturating levels of ascorbic acid.*® At the same time, however, dithiothreitol can also cause loss of enzyme activity, probably by disrupting essential disulphide linkages and causing thereby disaggregation of the active enzyme.**: #4) 97 » 58 A reaction mechanism for prolyl and lysyl hydroxylases and other a-ketoglutarate-dependent dioxygenases is proposed ®® in which oxygen is activated by ferrous ion Lound to the enzyme, followed by the formation of an intermediate peroxy compound between substrate and a-ketoglutarate (the initial oxidative attack probably being towards a-ketoglutarate) and then cleavage of the peroxy compound to yield hydroxylated substrate, succinate, and carbon dioxide. The role of the reductant in this scheme is not clear. As proposed by Holme et al.,°®: °° Fe+* remains reduced during the reaction and the reductant may either serve to maintain Fe*+ in the reduced state or to protect essential

Barnes:

Collagen Metabolism

269

-SH groups. In the scheme proposed by Hurych et al.,*!:6? iron undergoes a cyclic oxidation and reduction. They have presented evidence for the oxidation of Fe** when added to enzyme and «-ketoglutarate. Oxidation, it is suggested, occurs during activation of oxygen, and reduction is accomplished by the reducing cofactor. In this case ascorbic acid should be utilized in stoichiometric proportions. This has yet to be established. Bhatnagar and Liu“ also suggest that iron may undergo a cyclic oxidation and reduction and that ascorbate is utilized in the generation of the superoxide radical, which serves to reduce ferric to ferrous ion. Recent studies in cell culture **:°* have introduced a new aspect to the problem of the mode of action of vitamin C in the mechanism of hydroxylation of collagen proline. This subject is dealt with in other contributions to this monograph *°: °' and will not therefore be described in detail here. It appears that the lack of hydroxylation in cell cultures when ascorbate is absent is due to an actual lack of active enzyme (rather than to a failure of active enzyme to hydroxylate because of the absence of an essential reducing cofactor). Ascorbic acid may be involved in the conversion of an inactive precursor to active enzyme. The question that arises is whether the two activities of ascorbic acid (its activation of enzyme and participation in hydroxylation by active enzyme) are perhaps really two aspects of the same phenomenon. Conceivably, ascorbic acid serves in the cell-free system to maintain enzyme in an active state in a manner related to its ability to cause activation of the inactive enzyme in the whole cell. Possibly reduction of some specific site on the enzyme (Fet++?) not only permits hydroxylation but also permits the retention of an active conformation of enzyme. Dithiothreitol causes inactivation of enzyme both in whole cells °* and in vitro *°.°" and has been shown to cause disaggregation of the molecule.*® This can be reversed by ascorbic acid in cell culture.** Ascorbic acid also affords protection against the effects of dithiothreitol in vitro.°*

THE

NATURE

OF

IMPAIRED COLLAGEN SYNTHESIS ASCORBIC ACID DEFICIENCY

In Vivo

IN

Studies by the author and his colleagues were undertaken in an attempt to establish if impaired collagen synthesis in vivo in ascorbic acid deficiency could be explained satisfactorily in terms of a functioning of vitamin C in the hydroxylation of collagen proline and lysine in accord with the behavior of the vitamin in vitro. There were certain grounds for believing that the lesion in vivo might not simply be impaired hydroxylation. Thus, early attempts to separate and identify by amino acid analysis a proline-enriched, hydroxyproline-deficient collagen from the tissues of scorbutic guinea pigs were unsuccessful °°" and thereby lent weight to the contention that hydroxylation of proline in the formation of collagen hydroxyproline must precede translation. Isotopic studies by Gould et al.“* and Robertson et al."° also led to the conclusion that the rapid synthesis of collagen in scorbutic guinea pigs following the administration of vitamin C was not due to the hydroxylation of a hydroxyproline-deficient precursor accumulated in scurvy but must involve de novo synthesis. Impaired hydroxylation would be expected to give rise to a fall in hydroxyproline excretion. In scorbutic guinea pigs, however, it was found that hydroxyproline excretion remained normal or even slightly elevated despite impaired

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collagen synthesis in the tissues.’:°* Similarly the level of diffusible hydroxyproline and hydroxylysine in the skin showed no significant change.” ° A fall in hydroxyproline excretion did not occur until after two weeks of ascorbic acid deprivation, some days after the effect on tissue collagen was first noted. This fall occurred in control animals also and was considered attributable to inanition. An increased excretion of hydroxyproline has been reported in cases of human scurvy.” @ Although studies with the purified enzyme prolyl hydroxylase have indicated a requirement for ascorbic acid in the hydroxylation of collagen proline, the requirement is not an absolute one and the vitamin can be replaced by other reductants. This raises the possibility that in vivo the requirement for a reductant could be met by compounds other than vitamin C and that the impairment of collagen synthesis in scurvy reflects a role of ascorbic acid in collagen metabolism other than one in hydroxylation. We decided therefore to seek evidence of impaired hydroxylation in vivo by isotopic methods, studying the incorporation of labeled proiine into collagen proline and hydroxyproline. Since it seemed possible, in view of the failure to detect its gross accumulation in tissues, that formation of protocollagen might be transitory or that its existence was short-lived, we conducted these incorporation studies at varying periods of ascorbic acid deprivation, measuring the incorporation at varying times after administration of the isotope. Incorporation into elastin was studied as well, since this extracellular polymer also contains some hydroxyproline, although much less than collagen. It was anticipated that increasing ascorbic acid deprivation would lead to increasing hydroxylation impairment and therefore incorporation of radioactivity into hydroxyproline relative to that into proline would decrease. Precisely this situation was found in elastin.®*® Incorporation of radioactivity into elastin hydroxyproline became negligible while that into elastin proline was little affected (TABLE 4). The elastin proline/hydroxyproline specific radioactivity ratio thus became increasingly large in contrast to the value of unity in controls (the value of unity, of course, indicating that the degree of hydroxylation in the newly synthesized labeled material was the same as that in the preformed unlabeled protein).

The

results thus indicated the formation in the scorbutic animals of a polymer increasingly deficient in hydroxyproline. However, in the case of skin collagen,®® incorporation into both proline and hydroxyproline showed a rapid fall occurring around the 8th—-10th day of ascorbic acid deprivation. The proline/hydroxyproline specific activity ratio showed only a very slight (but nevertheless highly significant: p < 0.001) rise (TABLE 4). The results implied the formation, but in rapidly diminishing amounts, of a slightly underhydroxylated collagen in which the level of hydroxylation was reduced by 5-10%. This collagen behaved as normal collagen as regards its distribution between fractions of different solubility and its rate of turnover, and it was concluded therefore that hydroxylation was not sufficiently reduced to impair its function.*:° A similarly, slightly underhydroxylated collagen has since been detected in the scorbutic catfish.*2 The question that arose was whether the drastic fall in incorporation of radioactivity into skin collagen arose from an actual reduction in collagen protein synthesis or from the formation of a more substantially underhydroxylated collagen that failed to accumulate in the tissues because it was entirely degraded. Degradation would have to be rapid since we could not detect such

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New

Annals

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Academy

of Sciences

a moiety in the tissues even when they were examined as soon as 2 hours after administration of isotope.'? An increase in proline radioactivity (measured after hydrolysis) in the diffusible fraction of skin concomitant with the fall in incorporation of radioactivity into collagen suggested degradation of underhydroxylated collagen (protocollagen) might be occurring.** We decided to explore this problem further by urinary excretion studies in guinea pigs administered labeled proline following ascorbic acid deprivation for varying periods of time. It was anticipated that if underhydroxylated collagen with an increasing deficiency of hydroxyproline was being formed and degraded in increasing amounts then the excretion of hydroxyproline-containing peptides should be replaced, to an increasing extent, by the excretion of the equivalent hydroxyprolinefree peptides.

TABLE

5

EXCRETION OF FREE GLYCINE AND OF RADIOACTIVITY IN FREE PROLINE AND THE PEPTIDES PROLYLHYDROXYPROLINE AND PROLYLPROLINE IN THE URINE OF SCORBUTIC GUINEA PIGS RELATIVE TO CONTROLS *

Day of

Prolylhy-

Experiment

Glycine

6 8 10 12 14

0.48 3.29 6.19 Om 0.84

* Animals

(six in each

Proline

droxyproline

Prolylproline

1.34 ft7 1.43 0.87 1.30

i°7 0.85 — tao 1.28

3.83 2.36 eles 0.84 0.79

group)

received

L-[G-*H]proline

Urine was collected for 24 hours after administration

on

the days

of the isotope.

indicated.

Urines were

combined within each group. The urinary constituents listed were separated by ionexchange chromatography. Free glycine was estimated with ninhydrin. Radioactivity in free proline, prolylproline, and prolylhydroxyproline was measured by liquid scintillation counting. Results are expressed as a ratio of the excretion in the ascorbic— acid-deficient group relative to that in the appropriate control group for each period of the experiment.

These studies revealed first that approximately “% of the total hydroxyproline excretion occurred in the form of relatively large, nondiffusible peptides. A similar situation is known to occur in humans."® Examination of these peptides by collagenase digestion revealed that there was only a slight underhydroxylation of proline in this fraction from scorbutic animals, similar to that observed in skin collagen. This implied that if a substantially underhydroxylated collagen were

formed,

its degradation

must

be different

from

that of normal

collagen,

i.e., it must be degraded entirely to a diffusible form. It was found that the major diffusible hydroxyproline-containing peptide in urine was prolylhydroxyproline. Excretion of radioactivity in this and the peptide prolylproline, however, showed little change in scorbutic animals relative to controls (TABLE 5). This was in accord with the observation that total hydroxyproline excretion and the amount of radioactivity in the total hydroxy-

Barnes:

Collagen Metabolism

Pagfs)

proline excreted showed little if any change in scorbutic animals. Radioactivity appearing in free proline, however, showed a temporary increase, and at about the same time a very sharp but transitory increase in free glycine excretion was noted; these changes occurring at a time when incorporation of radioactivity into skin collagen was rapidly declining (TaBLe 5). These changes were reflected in the plasma concentration of free glycine and proline. Hornig et al.” also found a transitory rise in the concentration of free proline in the plasma of scorbutic guinea pigs. It may be argued that these increases could arise from the formation and immediate degradation of protocollagen. However, since there was no indication of an increasing prolylproline excretion relative to that of prolylhydroxyproline, excretion of the latter remaining relatively constant during the above changes and since there was no evidence of substantial and increasing underhydroxylation of collagen proline in the non-diffusible fraction of urine, we conclude that severe underhydroxylation of collagen does not occur in the scorbutic guinea pig. It seems probable that there is initially in ascorbic acid deficiency a slight fall in hydroxylation of proline, insufficient to impair the functioning of the collagen molecule, with the result that a collagen can be detected in the tissues with a 5-10% reduction in hydroxylation. There then occurs a somewhat greater reduction, causing instability in the molecule, which, as a consequence, undergoes degradation. Degradation of such a molecule may maintain the excretion of prolylhydroxyproline and prolylproline more or less within the normal range and at a normal ratio to each other. It has recently been emphatically demonstrated that the hydroxyproline content of collagen is critical in regard to the stability of the triple helical structure of the molecule and that reduction in the hydroxyproline content gives rise to a molecule in which the triple helix is unstable at body temperature.1®: 2° We consider therefore that in the scorbutic guinea pig a slight additional reduction in hydroxylation beyond the 5-10% detected in the tissues gives rise to a molecule that remains in the form of unassociated a-chains and, lacking the protection of the triple helical structure, is rapidly subjected to the action of preteolytic enzymes.*® We also conclude that further reduction in hydroxylation beyond this stage does not occur because of an inhibition of collagen protein synthesis. The increased free glycine excretion and increased amount of radioactivity in free proline in urine is thought to arise from impaired synthesis rather than degradation of protocollagen. Impaired collagen protein synthesis seems the most reasonable explanation for the continuing lack of accumulation of protocollagen in scurvy rather than the continuous formation and degradation of protocollagen, especially in view of the lack of evidence for the occurrence of appreciable underhydroxylation. Impaired hydroxylation by means of anaerobic conditions, use of a,a’-dipyridyl, or ascorbate deficiency in isolated systems in vitro appears to cause,

at least in short-term

incubations,

an

intracellular

accumulation

of

the un- or underhydroxylated material.1* *1) 27.7678 Similarly, in vivo intracellular accumulation of slightly-underhydroxylated a-chains may occur, and this may lead to a feedback inhibition of further synthesis.*" °° The electronmicroscopical studies of Ross and Benditt*® and the centrifugation studies of Fernandiz-Madrid and Pita *° and Harwood et al.*! demonstrating disaggregation of polyribosomes in the fibroblasts of scorbutic tissues all imply disrupted collagen protein synthesis in scurvy. There is no situation so far known to occur in vivo where impaired hydroxylation of collagen proline can be demonstrated without at the same time impaired collagen synthesis or accumulation. Inter-

274

Annals

New

York

Academy

of Sciences

ference with hydroxylation by means of «,a’-dipyridyl or the use of proline analogues both lead to reduced collagen synthesis or accumulation.*?*’ Studies in guinea pigs with chronic hypovitaminosis C failed to reveal impaired hydroxylation without impaired synthesis. As in acute deficiency, a slightly-underhydroxylated collagen was produced and incorporation of labeled proline into collagen was at the same time markedly reduced.*” All of these data point to impaired synthesis as a consequence of impaired hydroxylation in vivo. The results with collagen and especially with elastin described above indicate that hydroxylation of peptidyl proline is in fact impaired in vivo in scurvy. It has yet to be established, however, whether the hydroxylation of peptidyl lysine is impaired. Studies in cell culture suggest hydroxylation of collagen lysine may be less affected by ascorbate deficiency ‘® and preliminary results suggest this may also be so in vivo.°° It is not yet known whether the hydroxylation of peptidyl proline in vivo is impaired through the absence of the vitamin as an essential cofactor participating directly in the hydroxylation mechanism or through lack of active enzyme. As already discussed, these two aspects of the vitamin’s action may in any event prove to be different facets of a single mechanism. The studies of Mussini et al.8° suggest there may be a lack of active enzyme in vivo in scorbutic tissues.

CONCLUSIONS

Studies in isolated collagen-synthesizing systems have demonstrated that ascorbic acid participates in the synthesis of collagen hydroxyproline and hydroxylysine, both of which are formed by the hydroxylation of particular prolyl and lysyl residues previously incorporated into peptide linkage during the process of ribosomal collagen protein synthesis. The precise mode of action of the vitamin in these hydroxylations has yet to be elucidated. The need for ascorbic acid as a reductant in vitro in these reactions is not highly specific. Because of this lack of specificity, the possibility existed that compounds other than vitamin C might perform this function in vivo and that impaired collagen synthesis in scurvy might imply a role for ascorbic acid in collagen metabolism other than one in hydroxylation. There appears to be no evidence, however, for the participation of other reductants in the hydroxylation mechanism in vivo. Studies in scorbutic guinea pigs have indicated that hydroxylation at least of peptidyl proline is impaired in vivo in ascorbic acid deficiency, and we conclude that this is the primary lesion in collagen synthesis in scurvy. Studies in folic-acid-deficient rats have lent support to the contention that ascorbic acid participates directly in this reaction in vivo.S? Nevertheless, hydroxylation of collagen proline in vivo is only slightly impaired in scorbutic animals. There is no evidence for the occurrence of a substantially underhydroxylated moiety. It is believed that a slight additional reduction beyond an initial S-10%, which does not appear to impair collagen function, causes a lack of formation of the triple helical structure of collagen, with consequent degradation of the unassociated a-chains. Accumulation of the latter within the cell may also cause a feedback inhibition of further collagen protein synthesis. The continuous absence of accumulation of unhydroxylated material in the tissues of scorbutic guinea pigs is considered attributable to reduced collagen protein synthesis, which is regarded as a secondary feature of impaired hydroxylation.

Barnes:

Collagen Metabolism

219

ACKNOWLEDGMENTS

The author wishes to thank Mr. B. J. Constable

skilled technical advice.

assistance

and

Dr.

E. Kodicek

and Mr. L. F. Morton

for his encouragement

for

and

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Bera, R. A. & D. J. Procxop. 1973. Biochemistry 12: 3395-3401. JIMENEZ, S. A., P. DEHM, B. R. OLSEN & D. J. Prockop. 1973. J. Biol. Chem. 248: 720-729. Pontz, B. F., P. K. MULLER & W. N. MEIGEL. 1973. J. Biol. Chem. 248: 75587564. RAMALEY,

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Uirto, J. & D. J. Prockop. 1974. Europ. J. Biochem. 43: 221-230. STonE, N. & A. MEISTER. 1962. Nature (London) 194: 555-557. Gotr.ies, A. A., A.KAPLAN & S. UDENFRIEND. 1966. J. Biol. Chem. 241: 1551-

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Hutton, J. J., A. L. TApPpEL & S. UDENFRIEND. 1967. Arch. Biochem. Biophys. 118: 23 1-240. Berc, R. A. & D. J. Prockop. 1973. J. Biol. Chem. 248: 1175-1182. HAuSMANN, E. 1967. Biochim. Biophys. Acta 133: 591-593. KivirIkko, K. I. & D. J. Prockop. 1967. Arch. Biochem. Biophys. 118: 611-618. Kivirikko, K. I. & D. J. Prockop. 1967. Proc. Nat. Acad. Sci. U.S.A. 57: 782— 789. MILLER, R. L. 1971. Arch. Biochem. Biophys. 147: 339-342. KrvirIkko, K. I. & D. J. Prockop. 1972. Biochim. Biophys. Acta 258: 366-379. PopeNog,

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& S. UDENEFRIEND.

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& M. Torrr.

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: 365-372.

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Hosza, P., J. HurycH & R. ZAHRADNIK. 1973. Biochim. Biophys. Acta 304: 466— 472. BHATNAGAR, R. S. & T. Z. Lru. 1972. FEBS Lett. 26: 32-34; 1973. Abstract 9th Int. Cong. Biochem. : 335.

64.

242: 4007-4012. 1969. Arch. Biochem.

& E. M. BAKER.

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Fibroblast. E. Kulonen New York, N.Y.

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HayalsuHt, O. 1974. In Molecular Mechanisms of Oxygen Activation. O. Hayaishi, Ed. : 1-28. Academic Press. New York, N.Y. AsBoTT, M. T. & S. UDENFRIEND. 1974. In Molecular Mechanisms of Oxygen Activation. O. Hayaishi, Ed. : 167-214. Academic Press. New York, N.Y. KutTnick, M. A., B. M. ToLBert, V. L. RICHMOND

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Biophys. 133: 286-292. Ruoaps, R. E. & S. UDENFRIEND. 1970. Arch. Biochem. Biophys. 139: 329-339. Bates, C. J., C. J. PRYNNE & C. I. LEVENE. 1972. Biochim. Biophys. Acta 278: 610-616. BARNES, M. J., B. J. CONSTABLE, L. F. MorToN & E. Kopicex. 1973. Biochim. Biophys. Acta 328: 373-382. BarngEs, M. J., B. J. CONSTABLE, L. F. Morton & P. M. Royce. 1974. Biochem. J. 139: 461-468. BARNES, M. J., B. J. CONSTABLE, L. F. Morton & E. Kopicex. 1971. Biochem. at25 6b 17 Re FRIEDMAN, S. & S. KAUFMAN. 1966. J. Biol. Chem. 241: 2256-2259. Ruoaps, R. E. & S. UDENFRIEND. 1968. Proc. Nat. Acad. Sci. U.S.A. 60: 14731478. Kivirikko, K. I., K. SHupo, S. SAKAKIBARA & D. J. PRocxop. 1972. Biochemistry 11: 122-129. Res. Commun.

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Collagen Metabolism

24 |

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GouLp, B. S., G. MANNER, H. M. GoLpMaNn & J. STOLMAN. 1960. Ann. N.Y. Acad. Sci. 85: 385-398. Gross, J. 1959. J. Exp. Med. 109: 557-569. Barnes, M. J., B. J. ConstaBte & E. Kopicex. 1969. Biochim. Biophys. Acta 184: 358-365. Barnes, M. J., B. J. CoNsTaBLe, L. F. Morton & E. KopiceK. 1970. Biochem. J. 119: 575-585. BuRKLEY, K. 1968. M.S. Thesis, University of Iowa. Iowa City, Iowa.

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1968. Biochim. Biophys. Acta 165: 238-250. WILSON, R. P. & W. E. PoE. 1973. J. Nutr. 103: 1359-1364. KRANE, S. M., A. J. Munoz & E. D. Harris. 1970. J. Clin. Invest. 49: 716-729. Hornic, D., F. WEBER & O. Wiss. 1971. Int. J. Vitam. Nutr. Res. 41: 86-89. Hurycu, J.. M. CHvapiL, M. TicHy & F. BENrAc. 1967. Europ. J. Biochem. 3: 242-247.

67. 68. 69.

M. L., E. M. Bixsy, T. D. R. Hockapay,

P. B. & J. ROSENBLOOM.

1971.

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Marco.is, R. L. & L. N. Lukens. 1971. Arch. Biochem. Biophys. 147: 612618. Switzer, B. R. & G. K. SUMMER. 1973. In Vitro 9: 160-166. Ross, R. & E. P. BENpDITY. 1964. J. Cell Biol. 22: 365-389. FERNANDEZ-Maprip, F. & J. Pita. 1970. Jn Chemistry and Molecular Biology

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81.

of the Intercellular Matrix. E. A. Balazs, Ed. 1: 439-448. Academic Press. New York, N.Y. HArwoop, R., M. E. Grant & D. S. Jackson. 1973. Biochem. Soc. Trans. 1:

85. 86.

IZA —=12 19" CuHvaPIL, M., J. HurycH & E. EHRLICHOVA. 1968. Hoppe-Seyler’s Z. Physiol. Chem. 349: 218-222. t Lane, J. M., P. DEHM & D. J. Procxop. 1971. Biochim. Biophys. Acta 236: SSW Lane, J. M., L. J. Parkes & D. J. Prockop. 1971. Biochim. Biophys. Acta 236: §28—541. JIMENEZ, S. A. & D. J. PRockop. 1971. J. Clin. Invest. 50: 49a. MussinI, E., J. J. Hutron & S. UDENFRIEND. 1967. Science 157: 927-929.

87.

Hautrvast, T. G. A. J.

88. 89.

Harwoop, R., M. E. GRANT & D. S. JACKSON. 1974. Biochem. J. 144: 123-130. Urtto, J. & D. J. Prockop. 1974. Arch. Biochem. Biophys. 164: 210-217.

90.

BARNES,

82. 83.

84.

& M. J. BARNES.

M. J., B. J. ConsTaBLe

1974.

Brit. J. Nutr. 32: 457-469.

& L. F. Morton.

1974.

Unpublished

observa-

tion.

DISCUSSION

Dr. BARNES: Ascorbate deficiency has no effect on the preformed collagen, that is, the collagen that has been formed before the vitamin deficiency occurred. There is no evidence of increased degradation of this collagen. The amount of soluble collagen is reduced in scorbutic guinea pigs. This fraction of collagen consists of the recently synthesized material and its amount is determined, on the one hand, by the rate of synthesis and, on the other, by the rate of removal from the pool by degradation or conversion to insoluble collagen. The amount is reduced in scurvy because of the lack of synthesis (or retention) of new collagen whilst loss from the pool is occurring as normally. The concentration (but not the amount) of insoluble collagen seems to increase in the scorbutic state, probably because of dehydration of tissues through a reduced intake of water during vitamin deprivation.

ACTIVATION OF PROLYL HYDROXYLASE FIBROBLASTS BY ASCORBIC ACID George J. Cardinale, Frans L. H. Stassen, Ramadasan

IN

Kuttan,

and Sidney Udenfriend Roche

Institute of Molecular Nutley,

New

Jersey

Biology

07110

Green and Goldberg! first reported that in cultured fibroblasts significant amounts of peptidyl hydroxyproline first appeared in late log-phase. They also demonstrated that the addition of lactate to the culture medium in early logphase resulted in a large increase in peptidyl hydroxyproline formation.* Subsequently, Gribble et al.* showed that even though L-929 mouse skin fibroblasts synthesized primary collagen chains in early log-phase, prolyl hydroxylase activity did not increase until late log-phase and that it was at this stage that peptidyl hydroxyproline was formed. Other studies by these authors *° revealed that the activity of prolyl hydroxylase in early log-phase could be stimulated severalfold if the cells were concentrated to a higher density or if lactate were added to the medium. Since the activation was independent of protein and RNA synthesis, the results indicated the existence of an inactive precursor of prolyl hydroxylase in early log-phase cells that is activated during normal cell growth or by cell crowding or lactate treatment. Further evidence for this hypothesis was obtained by McGee et al.,° who demonstrated that during cell crowding or lactate treatment, the amount of protein that cross-reacted with a monospecific antibody to prolyl hydroxylase remained virtually constant while the enzyme activity increased severalfold. In succeeding work* they were able to isolate an enzymatically inactive protein from early log-phase L-929 fibroblasts that cross-reacted with antibody to prolyl hydroxylase. Furthermore, they also demonstrated that this material had a molecular weight about one-third that of the active enzyme and therefore suggested that the cross-reacting protein may be a subunit precursor of the active enzyme. There have been many reports‘ '* that the amount of hydroxyproline formed by cultured fibroblasts and osteoblasts is increased by treatment with ascorbate. Peterkofsky*®° reported that ascorbate rapidly deteriorates under tissue culture conditions and that maintenance of adequate ascorbate levels results in a marked increase in peptidyl hydroxyproline in early log-phase cells. To determine whether the increased hydroxyproline formation is due to enzyme activation we investigated the effect of ascorbate on prolyl hydroxylase activity in early log-phase L-929 fibroblasts.

Materials

and

Methods

L-929 fibroblasts were grown as previously reported,® except that no ascorbate was added to the medium, The compounds to be tested were added to the medium of early log-phase cultures between 38 and 46 hours after inoculation. Cells were then harvested and washed and sonicates were assayed as previously

278

Cardinale

et al.: Activation TABLE

of Prolyl Hydroxylase

29

|

ACTIVATION OF PROLYL HyYDROXYLASE IN EARLY LOG-PHASE MONOLAYER CULTURES BY ASCORBATE AND LACTATE *

L-929

Prolyl Hydroxylase Activity (cpm/mg protein)

Additions

None

Eco

Ascorbate (2.5 10-* M) Lactate (8 x 10° M)

aeee

76,000 72,800

* Prior to harvesting 48 hours after inoculation, replicate flasks were treated for 3 hours with ascorbate or for 6 hours with lactate.”

described.1® Prolyl hydroxylase activity was measured by the tritium release assay of Hutton et al.® ‘7 Both total antigenic protein and the amount of enzymatically inactive, cross-reacting protein were determined by the enzyme immunoassay of Stassen et al.1* Results

The effect of ascorbate on prolyl hydroxylase activity in early log-phase L-929 fibroblasts was determined by adding ascorbate to the culture medium. As can be seen in TABLE |, ascorbate caused the same fivefold activation as lactate but in a shorter time and at a much lower concentration. TABLE 2 shows that this activation by ascorbate was also independent of RNA and protein synthesis. The concentrations of inhibitors used were the same as those used by Comstock and Udenfriend® when they demonstrated that the lactate activation was not due to de novo synthesis of the enzyme. In addition, by using the enzyme immunoassay,!> it was shown that the increase in prolyl hydroxylase activity was not accompanied by an increase in enzyme-related antigen.

TABLE ACTIVATION OF

2

OF PROLYL HYDROXYLASE BY ASCORBATE IN THE PRESENCE INHIBITORS OF RNA AND PROTEIN SYNTHESIS *

Additions

Prolyl Hydroxylase Activity (cpm/mg protein)

None Ascorbate

27,700 58,800

Actinomycin D + ascorbate Puromycin -++ ascorbate Cycloheximide + ascorbate

58,700 62,900 65,100

* Actinomycin D (5 ug/ml), puromycin (10 ug/ml) and cycloheximide (25 ug/ml) were added to replicate flasks 15 minutes prior to the addition of 2.5 10 M ascorbate. After an additional incubation for 1 hour, the cells were harvested (41 hours after inoculation).

Annals

280 50FT

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New

York

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Academy

of Sciences

F

e

7

> oO

=r

is} 3/mg protein) x10 (cpm

|

ar

ACTIVITY HYDROXYLASE PROLYL

FicuRE 1. Stimulation of prolyl hydroxylase activity in early log-phase L-929 monolayer cultures as a function of the ascorbate concentration. The cells were harvested 1 hour after the addition of ascorbate. (From Stassen et al.)

Slacre eh een ie io°

107

10° (2x0 |O-°

1o*

10°

M ASCORBATE IN CULTURE MEDIUM

The effect of ascorbate concentration on prolyl hydroxylase activity in early log-phase cells is shown in FIGURE 1. Activation began when the ascorbate concentration in the medium reached 5 * 10-* M and increased until a concentration of 2.5 X 10° M was reached. In this 1-hour incubation, 50% of the maximal activity was reached at an ascorbate concentration of 2 x 10° M, whereas a lactate concentration of 4 10° M was required to achieve the same effect.° The time course of activation at a 5 x 10°° M ascorbate concentration is shown in FIGURE 2. Here the activation was complete in 2 hours while lactate required 6 hours for maximal activation.” The relationship between active enzyme and cross-reacting protein was further studied in stationary L-929 cells. On addition of 10 mM dithiothreitol to the culture medium, hydroxylase activity was almost completely lost while the total amount of antigen (enzyme plus cross-reacting protein) did not change (TABLE 3). Enzyme activity could be partially restored by treatment of the cells with ascorbate for 3 hours in the presence of an inhibitor of protein synthesis. After incubation of the cells for 24 hours in fresh medium with cycloheximide, enzyme activity was restored to about 40% of control values. However, there was evidence of marked cell damage under these conditions. When the cells were incubated for 24 hours in fresh medium without cycloheximide, enzyme activity returned to almost normal levels. In this case, the increase was accompanied by significant de novo synthesis of antigen. Interest-

.

-

;

= =e Os as »~

FiGuRE

2.

Prolyl

hydroxylase

ac-

tivity of early log-phase L-929 mono-

vier

layer cultures as a function of time after administration of ascorbate. Ascorbate was added to the culture medium at a concentration of 5 « 107° M. (From Stassen et al."’)

x = So =n = 5 =

= HOURS

AFTER

ASCORBATE

——s2 2 Fo ADMINISTRATION

Cardinale

et al.: Activation

of Prolyl Hydroxylase

281

ingly, treatment with dithiothreitol alone does not appear to damage the cells. To characterize the enzyme and cross-reacting protein, cell extracts were prepared as described in the footnote to TaBLE 3 and chromatographed on DEAE-Sephadex®. The fractions were assayed for enzyme activity and for antigen. The enzyme and cross-reacting protein were found to elute in two distinct regions. Controls contained both enzyme and cross-reacting protein (FicureE 3A). After treatment with dithiothreitol, the peak of enzyme activity essentially disappeared and cross-reacting protein increased (FIGURE 3B). No new antigen peak was found, nor was there any antigen left in the region of enzyme activity. The enzyme activity that was restored by treatment with ascorbate or by prolonged incubation in fresh medium eluted at the same salt concentration as the control enzyme (FIGURE 3C and 3D).

TABLE

3

PROLYL HyDROXYLASE AND CROSS-REACTING PROTEIN IN STATIONARY L-929 CULTURES AFTER INACTIVATION BY DITHIOTHREITOL AND REACTIVATION BY ASCORBATE OR BY PROLONGED INCUBATION IN FRESH MEDIUM * Prolyl

Additions

Total

Hydroxylase Antigen Cross-Reacting Activity (A) (B) Protein (B—A) (cpm x 10° per mg protein)

None Dithiothreitol

90.2 0.8

236.4 258.6

146.2 257.8

Dithiothreitol, followed by ascorbate and cycloheximide

18.4

223.6

DOS2

37.0

222

1922

76.9

309.8

232.9

Dithiothreitol, followed by fresh medium and cycloheximide for 24 hours Dithiothreitol, followed by fresh medium

for 24 hours

* The cells were grown in monolayer cultures for 6 days. Incubation with dithiothreitol (10° M) was carried out for 1 hour, with ascorbate (5 x 10° M) for 3 hours. The cycloheximide concentration was 25 ywg/ml. Antigen is expressed in terms of enzyme activity.”

The enzyme immunoassay'* was then used to examine rat and mouse tissues for cross-reacting protein. TABLE 4 shows the amount of prolyl hydroxylase activity and cross-reacting protein in 27,000 g supernatants from various rat and mouse tissues. In each case there were significantly higher levels of cross-reacting protein than active enzyme, especially in the liver. When enzyme and cross-reacting protein from mouse skin were purified by DEAESephadex (FicuRE 4) or molecular-sieve chromatography (FIGURE 5), the same relationship as to molecular weight and charge were found between these proteins as between enzyme and the cross-reacting protein obtained from fibroblasts.” Recently we have been able to achieve a severalfold activation of prolyl hydroxylase activity in sonicates of early log-phase cells by incubation with ascorbate and the other cofactors required for hydroxylation. The activation

282

Annals

Figure

3.

New

York

Academy

of Sciences

DEAE-Sephadex

chromatography of prolyl hydroxy-

lase extracted from stationary L929 cultures after inactivation by dithiothreitol and reactivation by ascorbate or by prolonged incuba-

)

tion in fresh medium. A, control; B, after incubation for 1 hour in

medium

containing

threitol;

C, after incubation

10° M dithiofor

1

(

hour with 10° M dithiothreitol, followed by a 3 hour incubation with 5 x 10° M ascorbate and cyclo-

heximide (25 ug/ml); and D, after incubation for 1 hour with 10° M dithiothreitol,

followed

by incuba-

tion for 24 hours in fresh medium. (From Stassen et al.*°)

(CPMx10-3 CROSS-RE PROTEIN (CPMx10-3) PROLYL HYDROXYLASE ACTIVITY

FRACTION

500

a Asa tea

NUMBER

“ah

> — 400 = =

Figure 4. DEAE-Sephadex chromatography of prolyl hy-




a

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=

>=

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120

NUMBER

Figure 5. Molecular sieve chromatography of prolyl hydroxylase and crossreacting protein from mouse skin. (From Stassen et al.* By permission of Archives of Biochemistry and Biophysics.)

is carried out at 30° for 3 hours in the presence

of 50 mM

Tris HCl

pH 7.2,

0.1 mM ferrous ion, 0.1 mM a-ketoglutarate, and 1.0 mM ascorbate. The data given in TABLE 5 demonstrate that all the cofactors are required for this activation. Discussion

In fibroblast cultures most of the hydroxyproline is formed in late log-phase and stationary growth stages.'!:* During the same growth period the activity

TABLE REQUIREMENTS

Additions to Sonicate

None Complete Complete Complete Complete

5

FOR THE ACTIVATION OF PROLYL HYDROXYLASE OF EARLY LOG-PHASE L-929 FIBROBLASTS *

system system—ascorbate system—Fe** ssytem—a-ketoglutarate

IN

SONICATES

Prolyl Hydroxylase Activity (%)

100 320 120 165 100

* Complete system contains 1.0 mM ascorbate, 0.1 mM ferrous ion, and 0.1 mM a-ketoglutarate. To determine the requirement for «-ketoglutarate, the sonicate was first dialyzed overnight against phosphate-buffered saline.

Cardinale

et al.: Activation

of Prolyl Hydroxylase

285

of prolyl hydroxylase is greatly increased.*: ° However, although the hydroxylase activity in early log-phase fibroblasts is low, these cells contain high concentrations of enzymatically inactive protein that is immunologically indistinguishable from prolyl hydroxylase.* This cross-reacting protein is more acidic and also has a lower molecular weight than the active enzyme.’ During growth the cellular level of the cross-reacting protein bears an inverse relationship to active prolyl hydroxylase.®°.* The low level of prolyl hydroxylase activity in early log-phase cells can be elevated by cell crowding or lactate treatment in the presence of inhibitors of protein and RNA synthesis. This body of evidence led to the postulate that the cross-reacting protein may be a precursor of prolyl hydroxylase. The molecular weight relationship between cross-reacting protein and active enzyme suggests that the cross-reacting protein may be a subunit of prolyl hydroxylase and that activation may entail some type of subunit aggregation.* Berg and Prockop have reported that prolyl hydroxylase from chick embryo is composed of 4 inactive subunits that can be obtained from the active enzyme by treatment with high levels of dithiothreitol.1® Several laboratories have reported that ascorbate markedly stimulates hydroxyproline formation in fibroblast cultures.*:!° With purified enzyme the requirement of a reducing agent for the hydroxylation of peptidyl proline is best fulfilled by ascorbate.*°: *! The obvious assumption is that ascorbate in vivo serves as a cofactor in a manner analogous to its in vitro function. This has been used to explain the ascorbate-mediated increase of hydroxyproline formation. However, in the present report it has been shown that ascorbate treatment greatly increases the amount of active enzyme. This activation takes place in the presence of inhibitors of protein synthesis and without any change in the amount of prolyl-hydroxylase-related antigen. Ascorbate, in some way, brings about conversion of an enzymatically inactive, putative precursor to active enzyme. A similar effect of ascorbate on prolyl hydroxylase activity has also been observed by Levene et al. in 3T6 mouse fibroblast cultures.” The reversible inactivation of prolyl hydroxylase on treatment of cells with dithiothreitol provides a potent tool for studying the mechanism of enzyme activation. Dithiothreitol-inactivated enzyme retains its ability to compete with active enzyme for antibody binding sites. On DEAE-Sephadex the inactivated enzyme cochromatographs with cross-reacting protein and not with active enzyme. Thus dithiothreitol converts enzyme to cross-reacting protein. The enzyme activity that reappears on treatment with ascorbate or on prolonged incubation in fresh medium is eluted in the same position as the original enzyme. The present data demonstrate that normal rat and mouse tissues also contain a protein that cross-reacts with antibody to prolyl hydroxylase. As in cultured fibroblasts, this cross-reacting protein is enzymatically inactive and can be separated from prolyl hydroxylase by ion-exchange and molecular-sieve chromatography. In all tissues investigated, the molecular weight relationships of cross-reacting protein and prolyl hydroxylase have been found to be the same. The cross-reacting protein is generally “3 to % the size of the active enzyme. By analogy with the findings in fibroblast cultures, therefore, cross-reacting protein may also play a role in the regulation of prolyl hydroxylase activity in vivo. The large amounts of antigen compared to active enzyme are rather surprising. They may represent an intracellular pool of enzyme precursor that can be activated rapidly in circumstances requiring rapid collagen biosynthesis. The recently observed activation of prolyl hydroxylase in sonicates of early log-phase fibroblasts requires the presence of all the cofactors necessary for

286

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York

Academy

of Sciences

hydroxylation. At present we do not know if this activation is the same as that observed in whole cells by cell crowding or by lactate or ascorbate treatment. Since the hydroxylation cofactors are required, it is possible that activation of the inactive form of the enzyme involves hydroxylation of proline residues. References 1. GREEN, H. & B. GoLpBERG. 1963. Nature 200: 1097. 2. GREEN, H. & B. GOLDBERG. 1964. Nature 204: 347. 3. GRIBLE, T. J., J. P. Comstock & S. UDENFRIEND. 1969. phys. 129: 308.

4. 5. 6.

Arch.

Biochem.

Bio-

Comstock, J. P., T. J. GrispLe & S. UDENFRIEND. 1970. Arch. Biochem. Biophys. 137: 115. Comstock, J. P. & S. UDENFRIEND. 1970. Proc. Nat. Acad. Sci. U.S.A. 66: 552. McGEE,

J. O’D., U. LANGNEss

& S. UDENFRIEND.

1971.

Proc. Nat. Acad.

7.

U.S.A. 68: 1585. McGee, J. O’D. & S. UDENFRIEND.

8. 9.

GREEN, H. & B. GoLpBEeRG. 1964. Proc. Soc. Exp. Biol. Med. 117: 258. Peck, W. A., S. J. BirGE, Jr. & J. BRANDT. 1967. Biochim. Biophys. Acta

1972.

Sci.

Arch. Biochem. Biophys. 152: 216. 142:

St 10.

Priest, R. E. & C. Busiitz.

11.

Castor, C. W.

1967.

Lab. Invest. 17: 371.

12. 13.

Levene, C. I. & C. J. BATES. 1970. J. Cell Sci. 7: 671. Bates, C. J., C. J. PRYNNE & C. I. LEVENE. 1972. Biochim.

1970. J. Lab. Clin. Med. 75: 798. Biophys. Acta 263:

397. 14.

Switzer, B. R. & G. K. SUMMER.

15. 16.

PETERKOFSKY, B. 1972. Arch. Biochem. Biophys. 152: 318. STASSEN, F. L. H., G. J. CARDINALE & S. UDENFRIEND. 1973. Proc. Nat. Acad. Sci. U.S.A. 70: 1090. Hurron, J. J., Jr., A. L. TAPPEL & S. UDENFRIEND. 1966. Anal. Biochem. 16: 384. STASSEN, F. L. H., G. J. CaRDINALE, J. O'D. MCGEE & S. UDENFRIEND. 1974. Arch. Biochem. Biophys. 160: 340.

17. 18. 19. 20.

21. 22.

1972.

Berc, R. A. & D. J. Procxor. 1973. PETERKOFSKY, B. & S. UDENFRIEND.

J. Nutr. 102: 721.

J. Biol. Chem. 248: 1175. 1965. Proc. Nat. Acad. Sci.

U.S.A.

53:

B35) Hutton, J. J. Jr., A. L. TAppeEL & S. UDENFRIEND. 1967. Arch. Biochem. Biophys. 118: 231. Levene, C. I., J. J. ALEO, C. J. PRYNNE & C. J. BATES, 1974. Biochim. Biophys. Acta 338: 29.

DISCUSSION

QUESTION: As far as you know, does the precursor to the hydroxylase reside in the cytoplasm or in some other cell fraction? Dr. CARDINALE: The precursor is soluble and is located in the 100,000 g supernatant.

Dr. H. SprincE: What would have happened if you had used dehydroascorbic acid instead of ascorbic acid? Dr, CaRDINALE: We have conflicting evidence. Often, the same dehydro-

Cardinale

et al.: Activation

of Prolyl Hydroxylase

287

ascorbic preparation that would not serve as a cofactor in the in vitro assay did activate our L-929 cells. I know that Dr. Levene cannot obtain activation with dehydroascorbate in his 3T6 cells. It is possible that there was something wrong with our dehydroascorbate, although it did not work as a cofactor. Dr. E. Deckwitz: What are the iron concentrations that are needed for collagen biosynthesis? Dr. CaRDINALE: The K,, for the in vitro reaction for the prolyl hydroxylase is known. We have not studied the iron concentration. In culture, as long as ascorbate is present, there is enough iron in the cells to naturally hydroxylate the collagen. Dr. V. ZANNONI: Did you look at the kinetics of the activated enzymes? Dr. CARDINALE: We have always assumed the activated enzyme to be identical

to the

isolated

enzyme,

where

all the

kinetics

have

already

been

worked out. There might be more than one active enzyme. Dr. ZANNONI: How do you view the activation with ascorbic acid and lactate? Dr. CARDINALE: Since ascorbate was effective at a much lower concentration than lactate, perhaps it was the primary step. Lactate does produce an effect similar to the ascorbic activation. Dr. W. WEIS: What is the point of attack of DIT? The enzyme, the substrate, or the cofactors? Dr. CARDINALE: Probably the enzyme. Dr. J. Gross: Do you think this occurs by breaking of disulfide bonds? Dr. CARDINALE: It seems obvious that a reduction of disulfide bonds occurs.

ASCORBIC ACID AND COLLAGEN SYNTHESIS IN CULTURED FIBROBLASTS CL,

Levenes

Department University

of Pathology of Cambridge

Cambridge, CJ Medical Dunn

England Bates

Research Council Nutrition Unit

Cambridge,

England

The choice of mammalian models available for in vivo studies on the mode of action of ascorbic acid is limited by the small number of mammalian species that need an exogenous dietary source of this vitamin. Moreover, studies in whole animals are complicated by a multiplicity of cell types that may respond in different ways to variations in the supply of ascorbic acid, by multiple ascorbic acid compartments and stores, capable of undergoing redistribution in response to physiological stimuli, and, of course, by interactions between different cells and organs that preserve the status quo under adverse conditions.

These

studies are also bedeviled by the problems of anorexia and inanition, which can produce diverse secondary effects not attributable to the primary action of ascorbic acid. When Green et al.’ recently succeeded in isolating and stabilizing a number of mammalian fibroblast lines in tissue culture and demonstrated a response, in terms of increased hydroxyproline synthesis, to ascorbic acid supplements, a powerful new tool was made available for the study of ascorbic acid in relation to connective tissue metabolism. The response to, and thus the requirement for, added ascorbic acid is observed in fibroblasts derived from mice, which are independent of a dietary source of the vitamin, as well as in those from humans, who are totally dependent on dietary ascorbic acid. This reinforces the supposition that fibroblasts in connective tissue are normally dependent on liver or kidney parenchymal cells ° for their ascorbic acid supply. The cultured fibroblast, a prototype connective-tissue-synthesizing cell, provides a model of growth and cell-specific function in a chemically controlled growth medium under conditions of minimum variables. Our own studies,*~'4 which have been confined to the 316 line of mouse fibroblasts originally isolated by Green,' have attempted to clarify the role of ascorbic acid in connective-tissue biosynthesis, and to understand how the failure of an early step in collagen biosynthesis can lead to several different functional abnormalities that may be

responsible for some of the clinical symptoms of scurvy. EXPERIMENTAL

DESIGN

The cells were plated at 25,000/60 mm Falcon petri dish and were fed every 2 days with 4 ml Dulbecco and Vogt’s modification of Minimal Eagle’s * Member

of the external staff of the Medical.Research

288

Council.

Levene

& Bates:

Collagen in Cultured

Fibroblasts

289

Medium, supplemented with 10% fetal calf serum.1 The cultures were grown at 37° in Fildes jars in a gas phase of 20% oxygen, 75% nitrogen, and 5% carbon dioxide. They grew logarithmically for 6-8 days, reaching about 8 X 10° cells per dish, as a multilayered sheet attached to the base. During the next 6-8 days, referred to as “stationary phase,” they grew much more slowly, to a final yield of about 12 x 10° cells per dish, after which the sheet detached and curled up, and its metabolism, as measured by glucose utilization, lactate production, and amino acid incorporation, ceased abruptly. The experiments were therefore terminated before curling commenced. Electron micrographs (FIGURE 1) of stationary phase cultures revealed collagen fibrils with characteristic 640-A periodicity in the pericellular region, adjacent to the cell membrane. A potential source of ascorbic acid in the unsupplemented growth medium is the fetal calf serum,

but in practice the contribution

from

this source

was

less than 0.1 x 10°* M. Initial attachment of the cells to the plastic was inhibited by ascorbic acid; therefore the supplements were not included until at least day 3. The optimal supplementation level appeared to be 0.5—2.8 x 10-4 M,°: '° which is similar to the level required by other fibroblast strains.1°-?! In our experience, about 80% of the ascorbic acid disappeared from the culture medium during 24 hours at 37° at pH 7.2: most of the radioactivity from 1-14C-labeled ascorbic acid was converted during this period to more acidic products. Optimal hydroxylation of proline in collagen could, however, be achieved during a 24-hour period with a single initial dose of ascorbic acid at 0.5 x 10°* M or even lower; therefore it seems unlikely that ascorbic acid, supplied at 2.8 & 10-4 M, falls to a rate-limiting concentration within 24 hours. In addition, the presence of growing cultures probably helps to preserve the ascorbic acid, first by lowering the pH through lactic acid production, and second by reducing, intracellularly, some of the dehydroascorbic acid produced by aerial oxidation. The selection of isotopic precursors, which were usually added 24 hours before harvesting, was designed to provide specific information about the various biosynthetic processes operative in connective tissue synthesis. Proline was the obvious choice as a precursor of collagen proline and hydroxyproline; lysine as the precursor of collagen lysine, hydroxylysine and glycosylated hydroxylysine; and glucosamine was found to be a useful precursor of both glucosamine and galactosamine in glycosamino glycans. Tryptophan (which is absent from collagen) was used as an index of noncollagenous protein synthesis. Highly purified bacterial collagenase facilitated the resolution of collagen (including procollagen and the underhydroxylated forms) from noncollagenous proteins.22,° Standard chromatographic procedures were used to separate the different forms of collagen from each other and to separate the individual labeled components after acid or alkaline hydrolysis.®:

THE

SPECIFICITY

OF

ASCORBATE

ACTION

IN

COLLAGEN

BIOSYNTHESIS

Initially we studied the effect of ascorbic acid on stationary phase cultures, beginning supplementation at day 8 and harvesting at day 14.7 From TABLE 1 it is evident that cell proliferation, measured initially by hemocytometer or Coulter counting and later monitored by DNA determination, was slightly but significantly stimulated by ascorbic acid. This marginal stimulatory effect was also observed in logarithmically growing cultures.

290

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Incorporation of labeled tryptophan or proline per cell was unchanged by ascorbate supplementation (TABLE 1); thus, general protein synthesis is probably unaffected by ascorbate status under these conditions. It is particularly inter-

Ficure 1. Electron micrograph of collagen fibril lying at edge of 3T6 fibroblast; stained with phosphotungstic acid. > 144,000.

esting that collagen polypeptide synthesis was also unaffected. This contrasts with the situation in guinea pig skin,?* where acute scurvy appears to result in a marked reduction in synthesis of the unhydroxylated precursor of collagen. Hydroxylation of proline in collagen, on the other hand, clearly is extremely

29 Collagen in Cultured Fibroblasts & Bates Levene

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292

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Academy

of Sciences

sensitive to ascorbate status in 3T6 cultures. This is illustrated (TABLE 1) by the change in proline:hydroxyproline ratio in the fragments obtained after collagenase digestion. It is the specific failure of hydroxylation, therefore, rather than a reduction of polypeptide synthesis that is responsible for the reduced hydroxyproline content of cultures grown in the absence of ascorbate. Glycosaminoglycan synthesis, as measured by ['*C]glucosamine incorporation, either into total high-molecular-weight material or into a specific cetylpyridinium-precipitable fraction, was unaffected by ascorbate status (TABLE 1). Confirmation of this was obtained by [*°S]sulfate incorporation and uronic acid synthesis measurements.*:!!_ The synthesis of galactosamine, which had previously been shown to be depressed in healing Achilles tendonectomy wounds in scorbutic guinea pigs,?' 2° was not affected by ascorbate in 3T6 cultures** (TABLE 1). Small amounts of chondroitin sulfates and heparan sulfate were identified in 3T6 cultures,!4 and these, like the major hyaluronic acid component, were unaffected by ascorbate status. The action of ascorbate in logarithmically growing cultures was qualitatively similar to that in stationary phase cultures. In logarithmic phase, however, the unsupplemented cultures produced collagen that was much more severely underhydroxylated, with a proline:hydroxyproline ratio of 20:1 or even higher. This ratio fell to about 2.5:1 in stationary phase in the deficient cultures. Hydroxyproline synthesis per cell rose to a peak as the cells became more crowded and then declined, presumably owing to reduced availability of substrate.° This phenomenon has been reported for L-929 cultures,*° and it explains the observation 27, °5 that hydroxyproline synthesis declines less rapidly than total protein synthesis as cultures become crowded and enter stationary phase.

SEQUELAE

OF ASCORBATE

DEPRIVATION:

UNDERHYDROXYLATED

THE

PROPERTIES

OF

COLLAGEN

Recent studies °°: °°. 42 have established the following events (not necessarily in sequence) during the synthesis and maturation of collagen: (a) synthesis of the polypeptide chain, by the processes common to general protein synthesis; (b) hydroxylation of specific proline and lysine residues to give the collagencharacteristic amino acids, hydroxyproline and hydroxylysine, in peptide linkage; (c) association of 3 chains, possibly under the influence of an “extra” N-terminal segment, the “registration peptide,” to form a triple helical unit known as “procollagen;” (d) glycosylation of certain hydroxylysine residues to galactosylhydroxylysine and glucosyl-galactosyl-hydroxylysine; (e) extrusion of the procollagen through the cell membrane probably via the Golgi apparatus, to the pericellular, connective-tissue deposition zone; (f) excision of the “registration peptide,” by procollagen peptidase, to give a less soluble product—tropocollagen; (g) precipitation of tropocollagen units as collagen fibrils; (h) oxidation of certain e-amino groups of lysine and hydroxylysine to the corresponding aldehydes by lysyl oxidase; (i) formation of reducible Schiff base or aldol condensation products between adjacent lysines, hydroxylysines, and their aldehyde derivatives; and (j) stabilization of the reducible cross-links, by processes not yet elucidated, to give mature, insoluble, and cross-linked collagen fibers. All of these events do, apparently, occur during the development of the connective tissue matrix of 3T6 fibroblast cultures. As we have seen, the first step, synthesis of the polypeptide chain, is unaffected by ascorbate status, but

Levene

& Bates:

Collagen in Cultured

Fibroblasts

295

all the subsequent steps are potentially dependent on the second step, hydroxylation, which is severely inhibited by ascorbate deficiency. In 3T6 cultures, lysine hydroxylation seems not to be as sensitive to ascorbate deprivation as is proline hydroxylation.!2 Indeed, glycosylated hydroxylysine content is unchanged, or even slightly increased, by ascorbate deprivation.!” Total hydroxylysine synthesis is only slightly diminished in unsupplemented cultures (TABLE 2), but this conceals a fairly severe deficiency of nonglycosylated hydroxylysine. Recent evidence from several laboratories *1~*° has indicated that hydroxyproline-deficient collagen has a lower melting, or denaturation, temperature than fully hydroxylated collagen. At 37°, the temperature at which 3T6 cultures are grown, the triple helix of unhydroxylated collagen would become unstable, and would dissociate into constituent chains, whereas that of fully hydroxylated

TABLE

2

EFFECT OF ASCORBATE DEFICIENCY ON PROLINE HYDROXYLATION HYDROXYLATION IN COLLAGEN SYNTHESIZED BY 376 FIBROBLASTS

Plus Ascorbate

AND LYSINE IN CULTURE *

Minus Ascorbate

% Hydroxylation of proline

Cell layer Medium % Hydroxylation of lysine Cell layer Medium Free hydroxylysine % Cell layer Medium

37 41

15 7

26 33

24 23

19

12

23

12

* Cultures in late logarithmic phase received 5-[*H]proline, 4,5-[*H]lysine, or U['C]lysine for 24 hours, with or without a 24-hour supplement of ascorbic acid, 2.8 10%M. Peptides obtained specifically from collagen by digestion with bacterial collagenase were acid-hydrolysed and analyzed, by standard techniques, for the percentage hydroxylation of proline and lysine. Free hydroxylysine was measured after alkaline hydrolysis, which leaves the glycosyl linkages in glycosylated hydroxylysine intact.

collagen is stable. Our observations '! on the physical properties of the soluble collagen liberated into the growth medium are consistent with a reduction of triple-helix stability in the absence of ascorbate. Whereas ascorbate-supplemented cultures yield a product that can, at least in part, be coprecipitated with rat-tail tendon collagen during trichloroacetic acid-ethanol fractionation, that from deprived cultures is coprecipitated to a much smaller extent (TABLE 3). Different chromatographic patterns on Biogel® under mildly denaturing conditions and different susceptibilities to pepsin digestion also bear out the idea of a change in physical properties consistent with reduced triple-helix stability. Clearly this factor is likely to have an important bearing on the subsequent fate of the newly synthesized collagen. Towards the end of logarithmic phase, about half the collagen which is synthesized but not immediately degraded ‘* during a 24-hour labeling period

Annals

294

TABLE

TRICHLOROACETIC OF

of Sciences

York Academy

New

3

ACID-ETHANOL

PROLINE-LABELED

FRACTIONATION

GROWTH

MEDIUM

*

Percentage of Total Radioactivity Occurring in Ethanol-Insoluble Fraction Experiment

1

Minus Ascorbate

Plus Ascorbate

63

24.2

6.5

21.4

4.1 ps,

16.0 123

,

”

3 4 Guinea pig skin collagen control * Growth medium 5-[*H]proline

83.5 from late logarithmic phase cultures labeled for 24 hours with

in the presence

or absence

of ascorbic

acid.

2.8

10°M

were

mixed

with unlabeled rat tail collagen carrier and fractionated by the trichloroacetic acidethanol procedure.’ The figures given indicate the percentage of the total nondiffusible (i.e., incorporated) label that was soluble in trichloroacetic acid, pH 3.5, and insoluble in 14% ethanol. The recovery of labeled guinea pig skin collagen is shown as a control value.

appears as a soluble, high-molecular-weight form in the growth medium, and the other half remains associated with the cell layer. At present we have no reliable

means

of distinguishing

the intracellular,

unsecreted

chains

from

the

pericellular fibrillar material. Several other workers have reported that the inhibition of hydroxylation by various means produces an inhibition of collagen extrusion,”°: 79 36-41 whereas we have consistently observed a higher proportion of the total collagenous material (of a-chain size or larger) in the medium in ascorbate-deprived cultures than in supplemented ones (TABLE 4). One possible explanation of this is that underhydroxylation inhibits fibrillogenesis, under our conditions, to an even greater extent than extrusion, so that a larger proportion of the secreted, underhydroxylated procollagen tends to escape from the peri- © cellular, fibril-forming zone, and appears to escape peptide excision by procollagen peptidase. It is worth noting that the collagen that appears in the growth medium of unsupplemented cultures is generally less fully hydroxylated than that remaining in the cell layer (TABLE 2) and that this material apparently contains a higher proportion of pro-« chains and a correspondingly lower proportion of a-chains than the fully hydroxylated material from supplemented cultures. These observations tend to support the theory that the cell-associated collagen mostly consists of material that has been deposited as fibrils in the pericellular mat, while that in the growth medium has been rejected by, or has escaped from, the fibril-forming process. Of the collagen in the cell layer, a small proportion can be extracted by salt solutions, while the majority is insoluble in both salt solutions and dilute acetic

acid.* In ascorbate-supplemented cultures, the salt-soluble fraction is smaller than in deficient cultures (TABLE 5); cross-linking may therefore be more efficient when the level of hydroxylation (presumably of lysine) is high. Treatment of whole cell layers with tritiated borohydride followed by analysis of the labeled reducible cross-links in acid hydrolysates revealed that deficient cultures

Levene

& Bates:

Collagen

in Cultured Fibroblasts

295

produced relatively more lysine-derived reducible cross-links whereas supplemented cultures produced relatively more hydroxylysine-derived ones 1° (FIGURE 2). This could be ascribed to the deficiency of hydroxylysine, particularly of nonglycosylated hydroxylysine, in the deficient cultures.12 It may be significant that hydroxylysine-derived cross-links are, in some instances, chemically more stable than the corresponding lysine-derived ones.*” The probable sequelae of deficient collagen hydroxylation that we have observed in 3T6 cultures are summarized in FiGuRE 3. Although some of the newly synthesized collagen in 3T6 cultures undoubtedly is rapidly degraded,’ ** the underhydroxylated collagen that has escaped into the growth medium seems relatively stable. This may be due to the inhibitory effect of serum «,-macroglobulins on collagenolytic proteases.'' This type of protection might be absent from connective tissues in the whole animal, possibly resulting in rapid degradation of underhydroxylated collagen to small fragments and subsequent rapid elimination.*® THE

ROLE

oF ASCORBIC ACID IN STIMULATION OF COLLAGEN PROLINE HYDROXYLASE ACTIVITY

Since the primary site of ascorbate action in 3T6 cultures seemed to be in controlling the activity of the enzymes responsible for hydroxylation of collagen, we decided to examine, in more detail, its effect on one of these enzymes,

TABLE EFFECT

oF

AscorBIC ACID ON BETWEEN THE CELL

colla-

4

THE DISTRIBUTION OF COLLAGENOUS MATERIAL LAYER AND THE GROWTH MEDIUM *

Labeled Proline-Incorporation into Collagenase-Sensitive Material (% in Cell Layer) Experiment

1

Minus Ascorbate

Plus Ascorbate

irs

2 3

21.7

PaleS) 41.9

44.8 56.1

Labeled Lysine Incorporation into Collagenase-Sensitive Material (% in Cell Layer) Minus Ascorbate

4

21.9

5 6

DY 29.9

Plus Ascorbate

30.0 pial 42.3

* Cultures were grown in the absence of ascorbate to late logarithmic phase, and were then incubated with 5-[*H]proline or 4,5-[*H]lysine for 24 hours in the presence

or absence

of ascorbic acid, 2.8x10'M.

Cell layers and growth

media

were

sepa-

rately harvested, and collagen-derived fragments were obtained by collagenase digestion. The percentage of total labeled collagenous material that appeared in the cell layer was calculated from these figures.

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Annals

[e}0],

LOasIy AO

296

Levene

& Bates:

Collagen in Cultured

Fibroblasts

297]

gen proline hydroxylase.!* Ascorbate has, for some time, been thought to act as a cofactor in the transfer of a hydroxyl group to peptidyl proline in collagen.*° The enzyme responsible for this is a mixed-function oxidase, and the cosubstrate,

or second oxygen-acceptor, is now known to be a-ketoglutarate.**: 48 Ascorbate does not, therefore, fulfill an oxygen-acceptor role as it appears to for dopamine 8-hydroxylase.*® Nevertheless, the in vitro activity of collagen proline hydroxylase is negligible in the absence of ascorbic acid; on this our own observations with the 3T6 enzyme 1° are in complete accord with those of other workers.**: °° Ascorbic acid can, however, be replaced in vitro by a wide variety of other

CONTROL SYNDESINE

------

MINUS

VIT. Cc

REDUCED LYSINONORLEUCINE OH-

LYSINONORLEUCINE

OH-NORLEUCINE DiNORLEUCINE OH-

102 ACTIVITY TRITIUM 4X (c.p.m)

PHE 00

sale HYLYS 300

TYR ELUTION

VOLUME

LYS (ml)

Ficure 2. Effect of ascorbic acid on KB*H,-reducible cross-links and cross-link precursors of collagen in the cell layer; --- -- -; minus ascorbic acid; ——, plus ascorbic acid. Cultures were grown without ascorbic acid for 6 days and were then divided into 2 sets. One set was fed every 2 days with medium minus ascorbate and the other was

fed similarly

with

medium

plus ascorbate,

2.8x10‘*M.

The

cultures

were harvested on day 12, the cell layers were treated with KB*H,, and the labeled, reduced products were analyzed by ion-exchange chromatography. Hatched areas

denote the position of normal amino Biochimica et Biophysica Acta.)

acids.

(From Levene et al.’ By permission of

reducing substances 1*: 47-°° such as thiol compounds, reduced pteridines, and even to some extent by inorganic reducing agents such as dithionite or sulfite, if these are present at very high concentrations 1° (TABLE 6). Although ascorbic acid is undoubtedly active at a lower concentration than any other reducing agent so far tested, the comparatively nonspecific nature of its in vitro action made us speculate whether ascorbate might have another additional role in enhancing the activity of this enzyme. Intrigued by the observation *' that tissues from scorbutic guinea pigs yield subnormal amounts of prolyl hydroxylase even when all the essential cofactors, including ascorbic acid, are added to the in vitro assay system, we measured the

Annals

298

New Lack

of

collagen

York Academy ascorbate

proline

cofactor

and

lysine

of Sciences

of

hydroxylases

gid? Reduced

Reduced

hydroxylation

|

Reduced

hydroxylation

(no change

of proline

of lysine

in glycosylation

of

hydroxylysine)

stability

collagen

triple

dissociation

into

!

Reduced

of

single

content

’

chains

Reduced

'Escape'from

liberation

as

molecular

region;

free,

underhydroxylated

of procollagen

number

of reducible

hydroxylysine-derived

pericellular

fibril-forming

of non-glycosylated

hydroxylysine

helix;

soluble,

inter-

cross-links

2

chains

Reduced

in growth

cross-link

increased

medium

cellular

stability;

solubility fibres

of peri-

in salt

solutions

Ficure 3. Summary of the observed results of reduced proline and lysine hydroxylation in the collagen synthesized by ascorbate-deficient cultures.

TABLE

REPLACEMENT

OF

ASCORBIC

ACID

IN THE

6

in

Vitro

HYDROXYLATION

ASSAY *

Relative Activity Compound L-Ascorbate D-Isoascorbate Dehydroascorbate

10°*M

10°°M

10°M

10°M

15 = —

87 ~—

100 108 1

81 —— 112 235

Dithiothreitol

—

16

210

Mercaptoethanol L-Cysteine L-Homocysteine Thioglycollic acid 6,7-Dimethyltetrahydro-

— — — —_—

—

1 6 10

8 99 26 135

pteridine Tetrahydrofolic acid Dithionite Sulfite

2 — — —-

9 — — —-

108 5 15 4

* Sonic

extracts

of 316

cultures

were

incubated

with

216 384 —

a — — 37

4-[*H]proline-labeled

chick

embryo substrate, a-ketogluterate, ferrous iron, and a variety of reducing agents. Proline hydroxylase activity, measured in terms of radioactivity released as tritiated water, is expressed relative to that obtained with 10°M ascorbic acid. Negligible activity was observed

when

no reducing compound

By permission of Biochimica et Biophysica Acta.)

was

added.

(From

Levene

et al.”

Levene

& Bates:

Collagen in Cultured Fibroblasts

299

activity of the enzyme in extracts of 3T6 cultures grown under various conditions.** Cultures growing logarithmically in the absence of ascorbate yielded very little active enzyme, even when ascorbate was present in high concentration in the in vitro assay. As the cultures approached confluence, still in the absence of ascorbate, however, the enzyme activity in extracts increased sharply (FIGURE 4). This phenomenon could presumably help to account for the increase in hydroxylation observed when cultures approach confluence in the absence of ascorbate; an increase in the amount of active enzyme may partly counteract the deficiency of ascorbate as cofactor, especially if other reducing substances are present intracellularly.

ratio

Minus

ascorbate

Plus ascorbate

100}

8.0

6.0

x 4.0

ox

x

Proline / hydroxyproline fatio eo ee

*

6

8

10

12

4

6

8

10

12

soo

14

FicurE 4. Changes in collagen proline hydroxylase and in proline : hydroxyproline ratios during growth. Cultures were grown for various periods without ascorbic acid; half then received ascorbic acid, 2.8 10*M

for 24 hours.

Some

of each group

also received 5-[*H]proline. Proline hydroxylase activity was assayed in sonic extracts of the unlabeled cultures, and proline : hydroxyproline ratios were measured in collagenase digests of the labeled cell layers. X ------- X Proline : hydroxyproline

ratios. @------- @ Collagen proline hydroxylase activity, expressed as cpm released into tritiated water, per 10° cells. (From Levene et al.”* By permission of Biochimica et Biophysica Acta.)

When ascorbate was added to logarithmic cultures (FIGURE 5) it produced a rapid, dose-related increase in prolyl hydroxylase activity in extracts that soon reached a level approaching that found in confluent cultures. Correspondingly, the proline:hydroxyproline ratio falls towards unity (FIGURE 4). Whereas an increase

in enzyme

activity in the absence

of ascorbate

(caused,

for instance,

by high cell density) never quite produces optimal hydroxylation, a similar increase produced by ascorbate usually is associated with a proline: hydroxyproline ratio of unity. The activation of prolyl hydroxylase by ascorbate that we have observed in 3T6 mouse fibroblast cultures 1’: °? has been observed in L-929 fibroblast cultures by Stassen et al.°*:°+ and appears to resemble the

a)

Dose response

Relative prone h

curve

b)

CO

Time

course

I

DNA

omaniey

FS oe at

/ plate

=

ee

oe ee ete o 1150

°/o

Proline hydroxylase plus ascorbate

lore}

ine hydroxylase

minus ascorbate

°

1.0

20 30 40 Ascorbate conc. x10°4M

° 10 20 30 40 so Hrs, Incubation with 2.8 x 10 “M ascorbate

°

Ficure 5. Activation of collagen proline hydroxylase by ascorbic acid: doseresponse curve and time course. Cultures were grown to late logarithmic phase in the absence of ascorbic acid. Half of them were then supplemented with ascorbic acid and they were harvested after a further incubation period. Collagen proline hydroxylase activity was assayed in sonic extracts and expressed on a per Cell basis relative to the control (no added ascorbic acid): a) Dose-response curve for a 24 hr incubation with ascorbate and b) Time course of response with ascorbate at 2.8 x 10% M. Changes in total DNA per culture, which is the index of cell proliferation, are also shown. (From Levene et al.“ By permission of Biochimica et Biophysica Acta.)

TABLE

FAcTors THAT INCREASE COLLAGEN AND THE HYDROXYPROLINE CONTENT

7

PROLINE HYDROXYLASE ACTIVITY OF COLLAGEN IN WHOLE CELLS * Relative Proline

Addition or Modification None Ascorbate, 2.8

Number of Experiments

Hydroxylase Activity as % of Control Culture (Average )

6

100

Proline: |Hydroxyproline Ratio (Average) 10.0

10M

(50 ug/ml) L-Lactate, 4 10°M Reduced O, tension

2

198

1.4

4 1

222 360

4.8 6.0

received ascorbate, 2.8 10M, or L-lactate, 4 10°°M, or were transferred to a gas phase of 95% nitrogen—5S% carbon dioxide, for 24 hours before harvesting. Some

were labeled with 5-[*H]proline for the measurement

of proline : hydroxyproline ra-

tios in collagenase-sensitive peptides in the cell layer; others (unlabeled) were used for extraction and measurement of collagen proline hydroxylase activity, which was

expressed relative to a control culture that had received no modification tion to the growth medium.

300

of or addi-

Levene

& Bates:

Collagen in Cultured

Fibroblasts

301

activation produced by high concentrations of lactate ion,’: !*: °° and by protein synthesis inhibitors such as cycloheximide.'*: °° Low oxygen tensions also seem capable of causing activation.'* Conditions that produce an increase in amount of extractable hydroxylase stimulate proline hydroxylation in the newly synthesized collagen (TABLE 7). The common factor between the various activating conditions is not yet understood, but may be related to a reducing intracellular environment. Recent investigations ** °° favor the idea that activation is produced by the association of inactive subunits of the enzyme, probably by a process involving formation or exchange of disulfide bonds. It is significant that, whereas moderate concentrations of free thiols can replace the in vitro cofactor activity of ascorbate, high concentrations can cause irreversible inactivation, probably by subunit dissociation.!3: *!: 6. 61 Thus ascorbic acid seems to have 2 roles in maintaining the level of proline hydroxylation in collagen, neither of them specific for the vitamin, both probably related to its electron-donating properties. Certain other naturally occurring substances or conditions can mimic ascorbic acid in one or another of these roles, but none has yet been found that can replace both roles entirely.

CONCLUSION

The primary effect of ascorbate on connective tissue synthesis by 3T6 fibroblast cultures appears to be located, rather specifically, at the level of proline hydroxylation, and to a lesser extent lysine hydroxylation, in collagen. When ascorbate is absent, the cultures produce an underhydroxylated form of collagen, which shows some abnormal physical properties. The soluble material secreted into the growth medium dissociates more readily than is normal into single, pro-a-sized chains, is not coprecipitated with normal tropocollagen, and can readily be degraded by pepsin. Less of the synthesized collagenous material is found in the cell layer, possibly because of reduced pericellular fibril formation. The material that is found associated with the cell layer is abnormally soluble in salt solutions and possesses an abnormal pattern of reducible cross-links, Ascorbate seems to have two roles in controlling collagen proline hydroxylase activity. One, demonstrable in vitro, can be mimicked by high concentrations of thiol compounds and by other reducing agents. The other, an activation effect that occurs only in living cells, is mimicked by such conditions as high cell density, high lactate concentration, and low oxygen tension—all of which are likely to occur in stationary phase cultures. Each of these effects can increase the level of proline hydroxylation in nascent collagen. It is not yet known which is functionally the more important, but both are probably needed to achieve complete hydroxylation. This dual action of ascorbate may help to explain its specificity as the antiscorbutic substance in vivo. Whereas several unrelated and naturally occurring compounds can replace it in one of its roles, none is yet known that can replace it entirely in both.

ACKNOWLEDGMENT

The authors are indebted to Dr. D. C. Barker for the electronmicroscopy.

Annals

302

New

York Academy

of Sciences

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& H. GREEN.

1963.

Quantitative studies of the growth of mouse

embryo cells in culture and their development Biol. 17: 299-313.

into established lines.

J. Cell

GOLbDBERG, B., H. GREEN & G. J. TopAro. 1963. Collagen formation in vitro by established mammalian cell lines. Exp. Cell Res. 31: 444-447. GREEN, H. & B. GoLpBERG. 1964. Collagen synthesis by human fibroblast strains. Proc. Soc. Exp. Biol. Med. 117: 258-261. GREEN, H. & B. GoLpBEeRG. 1964. Collagen and cell protein synthesis by an established mammalian fibroblast line. Nature (London) 204: 347-349. GREEN, H. & G. J. Toparo. 1967. The mammalian cell as differentiated microorganism. Ann. Rev. Microbiol. 21: 573-600. CHATTERJEE, I. B., N. C. Kar, N. C. GHosH & B. C. Guna. 1961. Aspects of

ascorbic acid biosynthesis in animals.

Ann. N.Y. Acad. Sci. 92: 36-56.

LEVENE, C. I. & C. J. Bates. 1970. Growth and macromolecular synthesis in the 3T6 mouse fibroblast. I. General description and the role of ascorbic

acid. J. Cell Sci. 7: 671-682. LEVENE, C. I., S. SHOSHAN & C. J. BaTEs. 1972. The effect of ascorbic acid on the cross-linking of collagen during its synthesis by cultured 3T6 fibroblasts. Biochim. Biophys. Acta 257: 384-388. Bates, C. J., C. J. PRYNNE & C. I. LEVENE. 1972. The synthesis of underhy-

droxylated collagen by 3T6 mouse Acta 263: 397-405.

10. 1:

fibroblasts

in culture.

Biochim.

Biophys.

LEVENE, C. I., C. J. BATES & A. J. BAILEY. 1972. Biosynthesis of collagen crosslinks in cultured 3T6 fibroblasts : effect of lathyrogens and ascorbic acid. Biochim. Biophys. Acta 263: 574-584. Bates, C. J., A. J. BArLeEy, C. J. PRYNNE & C. I. LEVENE. 1972. The effect of ascorbic acid on the sythesis of collagen precursor secreted by 316 mouse fibroblasts in culture. Biochim. Biophys. Acta 278: 372-390. BaTEs, C. J., C. J. PRYNNE & C. I. LEVENE. 1972. Asorbate-dependent differences in the hydroxylation of proline and lysine in collagen synthesized by 3T6 fibroblasts in culture. Biochim. Biophys. Acta 278: 610-616. LEVENE, C. I., J. J. ALEo, C. J. PRYNNE & C. J. BATES. 1974. The activation of protocollagen proline hydroxylase by ascorbic acid in cultured 316 fibroblasts.

Biochim. Biophys. Acta 338: 29-36. BATES, C. J. & C. I. LEVENE. 1971. The synthesis of sulphated glycosaminoglycans by the mouse fibroblast line, 3T6. Biochim. Biophys. Acta 237: 214-226. Priest, R. E. & C. BuBLitz.

1967.

The influence

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acid and tetrahy-

dropteridine on the synthesis of hydroxyproline by cultured cells. Lab. Invest. 17: 371-379. Suimizu, Y., D. S. MCCANN & M. K. KeEcu. 1965. The effect of ascorbic acid on human dermal fibroblasts in monolayer tissue culture. J. Lab. Clin. Med.

65: 286-306. SCHAFER, I. A., L. SILVERMAN, J. C. SULLIVAN & W. VAN

B. ROBERTSON.

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Ascorbic acid deficiency in cultured human fibroblasts J. Cell Biol. 34: 83-95. Switzer, B. R. & G. K. SUMMER. 1972. Collagen synthesis in human skin fibro-

blasts: xylation.

20.

oN,

effects of ascorbate, «-ketoglutarate and ferrous ion on proline hydroJ. Nutr. 102: 721-728.

PETERKOFSKY, B. 1972. The effect of ascorbic acid on collagen polypeptide synthesis and proline hydroxylation during the growth of cultured fibroblasts. Arch. Biochem, Biophys. 152: 318-328. PETERKOFSKY, B. 1972. Regulation of collagen secretion by ascorbic acid in 3T3 and chick embryo fibroblasts. Biochem, Biophys. Res. Commun, 49: 1343-1350. DeELL’OrcO, R. T. & J. H. NAsH. 1973. Effects of ascorbic acid on collagen syn-

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GRIBBLE, T. J., J. P.Comstock & S. UDENFRIEND. 1969. Collagen chain formation and peptidyl proline hydroxylation in monolayer tissue cultures of 1-929

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Priest, R. E. & L. M. Davies. 1969. collagen. Lab. Invest. 21: 138-142.

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MANNER, G. 1971. Cell division and collagen synthesis in cultured fibroblasts. Exp. Cell Res. 65: 49-60. GRANT, M. E. & D. J. Procxop. 1972. The biosynthesis of collagen. New Eng. J. Med. 286: 194-199, 242-249, 291-300. BORNSTEIN, P. 1974. The biosynthesis of collagen. Ann. Rev. Biochem. 43: 567-604. Berc, R. A. & D. J. PRockop. 1973. The thermal transition of a non-hydroxylated form of collagen. Evidence for a role of hydroxyproline in stabilising the triple helix of collagen. Biochem. Biophys. Res. Commun. 52: 115-120.

1969. The effect of scurvy on hexoswounds in guinea pigs. Biochem. J.

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fibroblasts.

29%

30. aul

32)

33%

34.

35:

36.

Sie

38.

308

40.

BERG,

Arch. Biochem, Biophys. 129: 308-316.

R. A., Y. KisHIDA,

Y. KOBAYASHI,

Cellular

proliferation

K. INouE,

and

synthesis

A. E. TONELLI,

of

S. SAKAKI-

BARA & D. J. PRockop. 1973. A model for the triple-helical structure of (prohyp-gly )io involving a cis-peptide bond and interchain hydrogen-bonding to the hydroxyl group of hydroxyproline. Biochim. Biophys. Acta 328: 553-559. JIMINEZ, S., M. Harscu & J. ROSENBLOOM. 1973. Hydroxyproline stabilises the triple helix of chick tendon collagen. Biochem. Biophys. Res. Commun. 52: 106-114. ROSENBLOOM, J. & M. HarscuH. 1973. Hydroxyproline content determines the denaturation temperature of chick tendon collagen. Arch. Biochem. Biophys. 158: 478-484. DaRNELL, J. & J. ROSENBLOOM. 1974. Ultracentrifugation as a probe of conformation of unhydroxylated procollagen and collagen. Biochem. Biophys. Res. Commun. 57: 910-917. Juva, K., D. J. Prockop, G. W. Cooper & J. W. LAsu. 1966. Hydroxylation of proline and the intracellular accumulation of a polypeptide precursor of collagen. Science 152: 92-94. RAMALEY, P. B. & J. ROSENBLOOM. 1971. Inhibition of proline and lysine hydroxylation prevents normal extrusion of collagen by 3T6 fibroblasts in culture. Fed. Eur. Biochem. Soc. Letters 15: 59-64. Deum, P. & D. J. Procxop. 1971. Synthesis and extrusion of collagen by freshly isolated cells from chick embryo tendon. Biochim. Biophys. Acta 240: 358369. JIMINEZ, S. A., P. DEHM, B. R. OLSEN & D. J. Prockop. 1973. Intracellular collagen and procollagen from embryonic tendon cells. J. Biol. Chem. 248: 720-729. Marco .is, R. L. & L. N. LUKENS.

cretion of collagen by mouse 147: 612-618.

1971.

The role of hydroxylation

fibroblasts in culture.

Arch. Biochem.

on the se-

Biophys.

Annals

304

New

York

Academy

of Sciences

42.

1973. Inhibition of collagen synthesis by Switzer, B. R. & G. K. SUMMER. a,a'-dipyridyl in human skin fibroblasts in culture. In Vitro 9: 160-166. 1974. Biological significance of the BarLey, A. J., S. P. Rospins & G. BALIAN.

43.

intermolecular cross-links of collagen. Nature 251: 105-109. STEINBERG, J. 1973. The turnover of collagen in fibroblast cultures.

41.

44. 45.

46.

J. Cell Sci.

12: 217-234. Wers, Z., M. C. BuRLEIGH, A. J. BARRETT & P. M. STARKEY. 1974. The interaction of a-macroglobulin with proteases. Binding and inhibition of mammalian collagenases and other metal proteinases. Biochem. J. 139: 359-368. 1967. Evidence for a faster Hurycu, J., M. Cuvapit, M. Ticny & F. Bentac. degradation of an atypical hydroxyproline- and hydroxylysine-deficient collagen formed under the effect of 2,2’-dipyridyl. Europ. J. Biochem. 3: 242-247. Barnes, M. J. & E. Kopicek. 1972. Biological hydroxylations and ascorbic acid with special regard to collagen metabolism. Vitamins and Hormones 30:

47.

1—43. Hutton, J. J., A. L. TappeL & S. UDENFRIEND. requirements of collagen proline hydroxylase.

48.

Ruoaps,

1967. Cofactor and substrate Arch. Biochem. Biophys. 118:

231-240. R. E. & S. UDENFRIEND.

coupled to collagen 1473-1478.

proline

1968.

hydroxylase.

Decarboxylation

Proc.

Nat.

of a-ketoglutarate

Acad.

Sci.

U.S.A.

60:

49,

PETERKOFSKY, B. & S. UDENFRIEND. 1965. Enzymatic hydroxylation of proline in microsomal polypeptide leading to formation of collagen. Proc. Nat. Acad.

50.

Ruoaps, R. E. & S. UDENFRIEND. 1970. Purification and properties of collagen proline hydroxylase from new-born rat skin. Arch. Biochim. Biophys. 139:

al.

MussInI, E., J. J. HUTTON lase in wound healing,

Sci. U.S.A. 53: 335-342.

329-339.

Sy,

53.

& S. UDENFRIEND. 1967. Collagen granuloma formation, scurvy and

proline hydroxygrowth. Science

157: 927-929. LEVENE, C. I., C. J. BATES & J. J. ALEO. 1973. Effect of ascorbic acid on protocollagen proline hydroxylase in 3T6 mouse fibroblast cultures. (Abstract) Fed. Proc. 32: 829. STASSEN,

F. L. H., G.

J. CarpDINALE

& S. UDENFRIEND.

1973.

Activation

prolyl hydroxylase precursor by ascorbic acid in L-929 fibroblasts.

54.

eis)

56.

Wf

32: 660. Abstract. STASSEN, F. L. H., G. J. CARDINALE & S. UDENFRIEND. 1973. Activation of prolyl hydroxylase in L-929 fibroblasts by ascorbic acid. Proc. Nat. Acad. Sci. U.S.A. 70:.1090-1093. Comstock, J. P., T. J. Grippte & S. UDENFRIEND. 1970. Further study of the activation of collagen proline hydroxylase in cultures of L-929 fibroblasts. Arch. Biochem. Biophys. 137: 115-121. Comstock, J. P. & S. UDENFRIEND. 1970. Effect of lactate on collagen proline hydroxylase activity in cultured L-929 fibroblasts. Proc. Nat. Acad. Sci. U.S.A. 66: 552-557. McGee, J. O’D., U. LANGNess & S. UpENFRIEND. 1971. Immunological evidence

58.

oe)

60.

of

Fed. Proc.

for an

inactive

precursor

of collagen

proline

hydroxylase

in cultured

fibroblasts. Proc. Nat. Acad. Sci. U.S.A. 68: 1585-1589. McGez, J. O’D. & S. UpENFRIEND. 1972. Partial purification and characterisation of peptidyl proline hydroxylase precursor from mouse fibroblasts. Arch. Biochem. Biophys. 152: 216-221. STASSEN, F. L. H., G. J. CarpINALE, J. O'D. MCGEE & S. UDENFRIEND. 1974. Prolyl hydroxylase and an immunologically related protein in mammalian tissues. Arch. Biochem. Biophys. 160: 340-345. Berc, R. A. & D. J. PRockop. 1973. Affinity column purification of protocollagen proline hydroxylase from chick embryos and further characterisation of the enzyme. J. Biol. Chem. 248; 1175-1182.

Levene 61.

& Bates:

Collagen in Cultured Fibroblasts

305

PoPENog, E. A., R. B. ARONSON & D. D. VAN SLYKE. 1969. The sulphydryl nature of collagen proline hydroxylase. Arch. Biochem. Biophys. 133: 286-292.

DISCUSSION

Dr. J. Gross: Since molecular oxygen is required for the hydroxylation mechanism, how do you get hydroxylation in the absence of oxygen? Dr. LEVENE: | think there is enough oxygen. First of all, we must be very careful about saying that we have anoxic situations because there is a series of papers showing some oxygen present even when cells are grown in an apparently anerobic atmosphere. Dr. J. E. HALVER: Those who are studying underhydroxylated collagen might well consider these fish that can be raised at different temperatures. Although the melding of the triple helix and its destruction is around 42—48°, that of the underhydroxylated triple helix is around 27°. Maybe this explains what we see when we raise these salmonids at 15° C and have these animals in apparently good health but with distorted and hyperplastic collagen areas. Do you think this is possible? Dr. Gross: We have actually done the experiment with tadpoles, because they are poikilothermic and we thought that we could regulate the degree of hydroxylation by regulating the temperature of the animal. There was in fact some evidence indicating that the rate of triple helix formation as governed by the hydroxylation of proline would determine the rate of hydroxylation of lysine and there would be beautiful interdigitation of two different enzyme systems working on a macromolecule. We lowered the temperature of one group of tadpoles to 7 degrees and raised another group to 37 degrees. You can manage that if you are careful with them, and have determined the proline hydroxy:proline ratio. To our surprise there was relatively little change. We were tempted to interpret a small change as being significant, but I do not think you can. Dr. HALVER: Tadpoles are sort of an intermediate stage of amphibian that can synthesize some ascorbic acid. You might use a more ascorbic-aciddependent animal. Dr. Gross: This did not really explore the role of ascorbic acid, but an experiment could be designed to see what that role might be. QUESTION: I have several related questions: First, what is the concentration of ascorbic acid in the tissue medium

and why? Second, does the concentration of ascorbic acid cause a change in pH of your medium? Third, due to the very high instability of the ascorbic acid in the medium, how often do you change the medium? Dr. LEVENE: We used the traditional concentration of 50 mcg per ml. In fact all we probably needed was 10 mcg per ml. In the experiments we did there was a 24 hour period and it has been said that the half-life of ascorbate is very short. I think this is probably true. The pH was mostly 7.2 and became more acidic when the cells were more active. I think this would keep the ascorbate in its own form. But we found we could always measure some ascorbate left after 24 hours. But even had we been unable to detect any

306

Annals

New

York Academy

of Sciences

chemical ascorbate we could have inferred its presence by the effect on the pro:hypro ratios. QUESTION: Did you have to adjust the pH due to the concentration of the ascorbic acid? Dr. LEVENE: No, never. At 50 mcg per ml the bicarbonate buffer is sufficient. The medium changes anyway as you get lactate production. I am sure you can probably go up to 10 times the dose and not notice much pH difference. Dr. V. ZANNONI: Is it possible that it is not really an activation but rather protection against inactivation? Has anyone bubbled oxygen in an enzyme prep, with or without ascorbic acid? Dr. LEVENE: We have not done this.

ASCORBIC-2-SULFATE METABOLISM BY HUMAN FIBROBLASTS * A. D.

Bond

Division

of Science and Mathematics Columbus College Columbus, Georgia 31907

Introduction

Ascorbic-2-sulfate was first synthesized and studied in connection with its sulfate transferring properties.!~* Its discovery in brine shrimp cysts ® and later in a variety of animal tissues“. * suggested such a role in vivo. While the function of ascorbate is still incompletely understood, it has long been known to play a role in the sulfation of connective tissues. On the assumption that ascorbic-2-sulfate participated directly in the biosynthesis of mucopolysaccharides, ascorbic-2-[*°S]sulfate was synthesized and its metabolism studied in ascorbutic guinea pigs. Results were inconclusive because of its rapid excretion from these animals.+ Fibroblasts in culture are known to synthesize both collagen and mucopolysaccharides.

In addition,

ascorbic

sulfate

in the culture

medium

can

not be

excreted. Consequently, we set out to study metabolism of ascorbic-2-sulfate in human fibroblasts in vitro and to relate this to the function of ascorbate in human metabolism. Methods

and

Procedure

Human skin fibroblasts were subcultured on Eagle’s minimum essential medium with 15% fetal calf serum at 37°C and, usually, with 250 pg/ml ascorbate. Medium was changed daily after the cells had reached approximately ¥% of confluency. Because the effect of ascorbic sulfate on cells and its mode of metabolism were unknown, several experimental regimes were used. Cells were first cultured in a medium containing a supplement of 1 mM diammonium ascorbic-2-sulfate and their rates of growth were compared to cells receiving ascorbate. Representative cultures were treated with 0.25% trypsin and shaken loose, and the number of cells per flask were counted.

The medium was supplemented at various stages of growth and at various periods after confluency by ascorbic-2-[**S]sulfate, prepared as previously described.8 Permeability of cells to ascorbic sulfate was determined by separating cells from the medium, washing briefly, then counting aliquots of medium and of homogenized cells in BBOT-Toluene in a Packard Tricarb liquid scintillation counter. * This work was jointly sponsored by the Biology Division of Oak Ridge National Laboratories and USPHS,

NIH, Grants

1F03

AM51218

and 2F03 AM

51218-02.

travel contract from Oak Ridge Associated Universities also assisted these studies. + Unpublished observations.

307

A

308

Annals Sodium

[#°S]sulfate

New (New

York Academy England

Nuclear)

of Sciences was

added to the medium

at

various stages of cell growth and after confluency, in the presence and absence of ascorbate. The total incorporation and types of molecules into which sulfate was incorporated were both examined. Some cultures were supplemented with 0.05 mCi 1-[!'C]ascorbate (New England Nuclear) with a total ascorbate content of 175 ug/ml. These cultures were then examined in the same way as those receiving ascorbic sulfate or inorganic sulfate supplements. The medium was removed and an aliquot centrifuged at 100,000 X g for

5 hours in a 5~20% sucrose buffer containing 0.05 M Tris buffer, pH 8.0. The gradient was dripped onto paper disks and counted in BBOT-Toluene. Combined medium and homogenized cells were filtered through a UM-10 ultrafilter (Diaflow®), followed by washing until filtrate counted background. The retentate was concentrated, counted for total activity and electrophoresed on Whatman No. 1 paper at 200 V for 90 minutes. Migration was determined by cutting thin strips and counting them in BBOT-Toluene, using heparin and chondroitin sulfate B as standards. Duplicates were stained for mucopolysaccharides or protein. Attempts were made to precipitate mucopolysaccharides from the retentate with tetraalkyl ammonium ions and to coprecipitate them with chondroitin sulfate B. Aliquots were additionally fractionated on G-200 resin (Sephadex®) and the fractions assayed for radioactivity and protein. Low-molecular-weight compounds were fractionated either from the UM-10 ultrafiltrate or from the medium following deproteinization with HCIO,. The fractionation procedure was the same used to purify ascorbic-2-sulfate from synthetic preparations, as previously described.* Particular interest was directed to detection of labeled ascorbic-2-sulfate from preparations containing [*°S] or [1#C]. Because specific activities were ‘high and anticipated yields low, a marker of unlabeled ascorbic sulfate was added just prior to fractionation. Fractions containing unknown labeled compounds were separated and purified and characterization was begun. In particular, a fraction labled ““Y” was chromatographed, hydrolysed, and subjected to qualitative analyses, and some derivatives were prepared. A trimethylsilylated derivative was prepared and fractionated by GLC, and the volatile fractions were submitted to analysis by mass spectroscopy.

Results

and

Discussion

FIGURE | represents the growth curves for cells receiving respectively ascorbate and ascorbic sulfate. The time necessary to reach confluence was always a little longer in the presence of ascorbic sulfate but did not appear to be significantly so. Ascorbic-2-[**S]sulfate seems passively permeable; the ratio of counts in the washed cell layer to those in the medium was not significantly different from the ratio of cell volume to media volume. Metabolism of ascorbic sulfate in young, rapidly growing cultures is limited. With cells approximately %4 grown, only about 5% of ascorbic-2-[*°S]sulfate was changed even after 36 hours. At this stage of growth, little [°°S]sulfate was taken up either and no detectable synthesis of ascorbic-2-[°°S]sulfate occurred, Earlier observations of Upton” indicated that in live animals little sulfate uptake occurred during wound healing until several days after wound closure. Therefore, we maintained fibroblasts for 5 days postconfluence before supplementing with the isotopic tracer. Twenty to twenty-five percent of a

Bond:

FIGURE 1. Growth of fibroblasts on MEM (Ea-

gle’s) with

calf serum.

in medium

15%

fetal

Cells

grown

Ascorbic-2-Sulfate

Metabolism

309

_ !0O

supplemented

with 250 ug/ml sodium ascorbate represented by (—) and those in 1 mM diammonium ascorbic-2sulfate, by (---).

10° CELLS x

10

TIME AFTER 1 mM

10 PLANTING (days)

ascorbic sulfate solution was then altered within 24 hours.

Also, 1-[1*C]-

ascorbate disappeared rapidly from these fibroblasts, but, as anticipated, it was destroyed in sterile medium as well. However, a small amount of [*#C]ascorbic2-sulfate was detected in these cells. Similarly, when these aged fibroblasts were given [*°S]sulfate, a small amount of [°°S]ascorbic-2-sulfate was detected. Only

very small concentrations of ascorbic sulfate were formed. It was interesting that 1-['*C]lascorbate of fairly low specific activity gave higher counts in a sulfated derivative than high-specific-activity [*°S]sulfate (TABLE 1). A logical explanation would be that the sulfate is predominantly derived from a nonsulfate source. McCulley '° has reported a similar observation in an ascorbate-requiring process of cells having an inborn error in sulfur-amino-acid metabolism. TABLE

INCORPORATION

|

OF [“C] FROM 1-["“C]ASCORBATE AND [*SJSULFATE INTO COMPOUND “Y”

OF

[*S]

FROM

Source

Approximate Specific Activity

Counts per Minute in Compound “Y”

1-[“C]Ascorbate [*S]Sodium sulfate

0.3 mCi/mmole 3 mCi/mmole

300 34

310

Annals

New

York

Academy

TABLE

of Sciences

2

INCORPORATION OF [*S] FROM SULFATE AND FROM ASCORBIC-2-SULFATE INTO MUCOPOLYSACCHARIDES (MPS) BY FIBROBLASTS

Specific activity Total counts MPS in medium MPS in cell layer Percent total counts incorporated

[*"S]Sulfate

Ascorbic-2-[*S] Sulfate

3.3 mCi/mmole 26.6 * 10° cpm 31,700 cpm 13,300 cpm

0.34 mCi/mmole 13.2 « 10° cpm 3,500 cpm 1,080 cpm

0.2%

0.03 %

The number of sulfated molecules retained by ultrafiltration did increase rapidly as the cells matured in the presence of ascorbate. Similar increases were seen using ascorbic-2-[**S]sulfate. The incorporation pattern suggested that ascorbic sulfate was used at least as well as inorganic sulfate (TABLE 2). That is, sulfate of 10 times greater activity was incorporated at less than 10 times the rate of ascorbic sulfate and, of course, only about 20% of the latter was hydrolysed. It was observed that in the absence of ascorbate the amount of inorganic sulfate incorporated in polymer did not increase as rapidly. Furthermore, the labeled material did not migrate with the mucopolysaccharides, heparin, or chondroitin sulfate B, nor were they coprecipitated with it. Fractionation on G-200 Sephadex showed that the principle material formed in the absence of ascorbate was proteinaceous. In the presence of ascorbate, radioactivity was

not associated with the protein fraction (FIGURE 2). Fractionation of low-molecular-weight components

by the procedure used to purify ascorbic sulfate allowed us to assay for this important compound while isolating other metabolites. From ascorbic-2-[*°S]sulfate, 2 significantly labeled species were detected. One, fraction B, was absorbed in acidic charcoal but washed free in water, whereas ascorbic sulfate required an NH,OH-ethanol eluting solvent. This molecule had no uv absorption but behaved chromatographically like ascorbic sulfate. A second species behaved, in the isolation procedure, like inorganic sulfate but was not coprecipitated with BaCl, and H,SO,. Identification of these was postponed with attention diverted to an unknown compound, “Y,” which appeared together with labeled ascorbic sulfate in media supplemented with [*°S]sulfate or 1-[**C]ascorbate. In addition, ‘“Y” was found to be formed in cultures treated with labeled ascorbic sulfate. From labeled ascorbate or sulfate, “Y" bore more counts than the ascorbic sulfate detected. This molecule is difficult to distinguish from ascorbic sulfate in many ways, except that it does not absorb uv light significantly. Unfortunately, the quantity of this substance is very small, making its identification difficult. However, its ubiquitous distribution with greater label than ascorbic sulfate suggests that it could provide an important clue to ascorbate metabolism. Compound Y is readily hydrolysed in dilute HCl. When it is derived from

[*°S]sulfate, all radioactivity appears in the precipitate formed by treating the hydrolysate with BaCl, and H,SO,. We could detect the hydrolysed compound by I, on chromatographic sheets. Qualitative tests suggested a carbohydrate-like molecule.

Bond:

Ascorbic-2-Sulfate

Metabolism

311

Conversion of the hydrolysed Y to volatile trimethylsilylated derivatives allowed separation of 4 major components. Mass spectroscopy of these indicated they represented the parent molecule increasingly trimethylsilylated. Comparison of the tetra-substituted derivative’s disintegration patterns with those of ascorbic sulfate and ascorbic acid derivatives was made. Both ascorbic acid and ascorbic sulfate produced the same fragmentation pattern. Sulfate is apparently rapidly cleaved in the trimethylsilylating medium. The pattern from the TMS derivative of compound Y (FicurRE 3) has a mass exceeding that of the corresponding ascorbic acid derivative by 2 units. This is just the mass increase

2000

f

1000

] \

pe

|

&

|

\n

aN

i

:

|

\n

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|

Ay

=

|

i Vi

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ale

as=: a.

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:

7

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LF

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FRACTION

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Ficure 2. [*S]Sulfate containing fractions from G-200 (Sephadex). Counts per minute, CPM, represented by (—) and concentration of protein (----) from cells receiving no ascorbate supplement. Cells receiving ascorbate had CPM represented

by (—-—)

and concentration of protein (-------

312

Annals

New

York

Academy

of Sciences

expected if ascorbic sulfate had had its double bond reduced. The use of deuterated TMS derivatives produced evidence that there were 4 -OH groups silylated as in ascorbate. However, the heavier-mass fragments represented primarily the loss of CH,— or TMS-groups typical of these types of derivatives, and we were unable to decipher an identifiable pattern. Ascorbate derivatives have previously been observed to undergo various internal cyclizations or rearrangements,'!12. which could explain a loss of uv absorption, and we

FIGURE 3. Mass spectrum of volatile TMS derivatives of ascorbic acid, ascorbic-2-sulfate and compound Y. The portion of the spectrum reproduced is, in each case, for the tetra-substi-

466

tuted

A

c

derivative.

308

Compound Y-4T MS(‘H)

cannot rule out ascorbic-3-sulfate as one of the other metabolites. We have searched diligently in both natural and synthetic reaction mixtures for it without success, We conclude that fibroblasts utilize and synthesize ascorbic-2-sulfate at distinct periods in their development, at times utilizing ascorbic sulfate at least as readily as inorganic sulfate. The prominent occurrence of other sulfated ascorbate metabolites and evidence of reduction suggest that sulfate transfer is only one role of this compound.

Bond:

Ascorbic-2-Sulfate

Metabolism

313

Acknowledgments

The cooperation and guidance of F. J. Finamore and J. Regan, in whose laboratories the work was done, and the assistance of W. Rainey with mass spectroscopy is gratefully acknowledged. References 1. 2.

Forp, E. A. & P. M. Ruorr. 1965. Chem. Commun. : 630. Mumma, R. O. 1968. Biochem. Biophys. Acta 165: 571.

3.

Bonp, A. D., B. W. MCCLELLAND, J. R. EINSTEIN Arch. Biochem. Biophys. 153: 207.

4. 5. 6. 7.

Cuu, T. M. & W. R. SLAUNWHITE, JR. 1968. Steroids 12: 309. Meap, C. G. & F. J. FINAMoRE. 1969. Biochemistry 8: 2652. MuMMaA, R. O. & A. J. VERLANGIERI. 1971. Fed. Proc. 30: 370. TOLBERT, B. M., D. J. ISHERWOOD, R. W. ATCHELY & E. M. BAKER. 1971. Fed. Proc. 30: 529. BAKER, E. M., III, D. C. HAMMerR, S. C. Marcu, B. M. ToLBEeRT & J. E. CANHAM. 1971. Science 173: 826.

8.

9. 10. 11. 12.

Upton, A. C. & T. T. ODELL, McCuLty, K.S. 1972. Amer. HAwortTh, W. N., E. L. Hirst JacKson, K. G. A. & J. K. N.

& F. J. FINAMOoRE.

1972.

Jr. 1956. Arch. Path. 62: 194. J. Path. 66: 83. & J. K. N. JoNes. 1937. J. Chem. Soc. : 549. Jones. 1965. Can. J. Chem. 43: 450.

DISCUSSION

Dr. S. SHAPIRO (Hoffmann-La Roche, Nutley, N.J.): Did you subject the sulfate of the macromolecule to proteolytic digestion? Dr. Bonp: Yes we did. We then attempted to precipitate chondroitin sulfate, but were unable to. Dr. SHAPIRO: Well, you mentioned it resembled a sulfate with protein. Dr. Bonpb: In the absence of ascorbate you have a material that looks like a sulfated protein. In the presence of ascorbate or ascorbic sulfate, you have a material that appears to be a mucopolysaccharide. Dr. SHAPIRO: What was the basis for this conclusion? Dr. Bonpb: They stain histologically as protein or mucopolysaccharide. Dr. J. Gross: Are you sure your compound Y is not a high molecular weight compound? Dr. Bonp: It should not be because it passes through the filter readily and acts very much like ascorbate sulfate. The mass spectrum and so forth supports this. Dr. B. M. ToLBErRT: I was rather curious, regarding your last slide, whether there was any possibility your trimethylsilyl derivative of the ascorbate sulfate might have desulfated and actually been a trimethylsilyl derivative of ascorbic acid. The similarity between patterns were certainly remarkable. Dr. Bonp: They are identical.

INSTABILITY AND FUNCTION: ASCORBIC ACID AND GLUTAMINYL AND ASPARAGINYL RESIDUES Arthur

B. Robinson

and

Steven

L. Richheimer

Linus Pauling Institute of Science and Medicine Menlo Park, California 94025

The search for functions of biologically important molecules entails, for the most part, the accumulation of facts that have positive implications for biological processes. Facts with apparently neutral or negative implications for biological processes are usually given lesser importance. We think that this is proper, since such a small fraction of biological processes are currently understood on a molecular level. Apparently negative molecular characteristics may be compensated by some as yet unknown process or may not actually occur in vivo. Sometimes, however, an apparently “negative” fact is so prominent and pervasive that it may be useful to reclassify the fact as “positive” and then to develop a new hypothesis on that basis. An example of this kind of reasoning is the development of the hypothesis that, through deamidation, glutaminyl and asparaginyl residues may serve as molecular clocks for biological processes.1-* In this paper we have applied similar reasoning to some of the facts about L-ascorbic acid. The fact that glutaminyl and asparaginyl residues deamidate under physiologically interesting solvent conditions has been known for 40 years. During most of that time, deamidation was considered to be a nuisance to protein chemists. The apparently negative implications of the introduction of extensive heterogeneity into protein molecules in vivo were, for the most part, ignored. Why should 2 of the 20 most common amino acid residues be unstable? Recently it was suggested |)? that this instability is actually the biological function. Glutaminyl and asparaginyl residues may serve as molecular clocks that time the development, turnover, and aging of proteins and organisms. Recent evidence in support of this hypothesis is reviewed in Reference 3. During some experiments on deamidation, L-ascorbic acid was added to the peptide and protein solutions under study. It was found that L-ascorbic acid markedly accelerates the degradation of the protein transferrin * and the peptides GlyArgAsnArgGly * and GlyAlaAsnAlaGly.° Dehydroascorbic acid showed the same effect.° Molecular oxygen was shown to be required for this degradation,® and it was shown that the active substance in this degradation is short-lived under the solvent conditions that were used, pH 7.4, I = 0.15, 37° C, phosphate

buffer.° The degradation was monitored by measurement of ammonia evolution from the solutions and changes in electrophoretic mobility of the peptides and proteins. This degradation was attributed to deamidation, and it was suggested that it might be related to the importance of L-ascorbic acid for good health.‘ The conclusion that this degradation results from deamidation involved the assumption that peptide bonds are stable in aerobic solutions of L-ascorbic acid. It has now been shown,’ however, that this assumption is incorrect for transferrin, and other work* subsequently confirmed this finding for the basic protein of central nervous system myelin. Richheimer? has carried out several experiments that suggest that the reaction proceeds through H.O, and free-

314

Robinson

& Richheimer:

Instability and Function

ses)

radical intermediates. It results in the breakage of peptide chains and release of ammonia from the fragmented peptides. C. W. M. Orr found a similar result for catalase.° It is known that L-ascorbic acid is much more stable in vivo than it is under similar solvent conditions in vitro. The in vitro instability results from oxidation of L-ascorbic acid by O,. Apparently L-ascorbic acid is stored in vivo in places with a low partial pressure of O., or else is chemically altered so as to increase its stability. Storage of L-ascorbic acid in the sulfated form would be an example of this latter possibility. However, during periods of stress, such as infection, the L-ascorbic acid is destabilized and used up very quickly. Some animals compensate for this by markedly increasing the rate at which they synthesize L-ascorbic acid, while others must buy the L-ascorbic acid and increase their ingestion of it. Beneficial effects of markedly increased dosages of L-ascorbic acid have been reported for many different kinds of stress.1°: 1! These effects are sometimes quite rapid. The symptoms of many diseases decrease very quickly after the administration of large doses of L-ascorbic acid.!? Why should so much of this inherently unstable substance be required for good health? This is especially puzzling, because the oxidation of L-ascorbic acid may result in increased destruction of many protein molecules that are essential to life, yet an animal

under

stress requires

an increased

amount

of

L-ascorbic acid. We think that an important biological function of L-ascorbic acid may be to increase protein degradation. This degradative substance may be stored in an inactive form in the white blood cells and released specifically onto the proteins of invading organisms. Alternatively, if the initial defense mechanisms are overwhelmed

or other stress is present, oxidizable L-ascorbic acid could be released generally in the tissues. This would serve to degrade foreign and host proteins alike. This process would raise the specific activity of the host proteins as they would be replaced by freshly synthesized proteins and would interfere with the multiplication of foreign organisms. This suggested role of L-ascorbic acid as a regulatable substance for increasing protein catabolism is supported by the suggestion that L-ascorbic acid oxidation products increase the rate of deamidation of proteins.*~® It is considerably strengthened by the discoveries that these oxidation products actually cause extensive breakage of peptide chains.

REFERENCES

1. 2. 3.

Rosinson, A. B., J. H. U.S.A. 66: 753. Rosinson, A. B. 1974. Rosinson, A. B. & C. J. B. L. Horecker and E. INEYe

McKEeRrow

& P. Cary.

4.

RoBINSON,

5.

FA PAP McKerrow, Calif.

6. 7.

IRVING, K. & A. B. RoBINsON. 1972. Unpublished. RICHHEIMER, S. L. 1974. Ph.D. Thesis. Stanford

1973.

Proc.

Nat. Acad.

Sci.

Proc. Nat. Acad. Sci. U.S.A. 71: 885. Rupp. 1974. Jn Current Topics in Cellular Regulation. L. Stadtman, Eds. 8: 247. Academic Press. New York,

A. B., K. Irnvinc & M. McCrea.

J. H.

1970.

Ph.D.

Thesis.

1973.

Proc. Nat. Acad. Sci. U.S.A.

University

of California.

University.

San Diego,

Palo Alto, Calif.

316

Annals

New

York Academy

8. WESTALL, F. C. & A. B. Ropinson. 9. 10. 11.

12.

1975.

of Sciences

Arch. Biochem.

Biophys.

Submitted.

Orr, C. W. M. 1967. Biochemistry 6: 3000. Srone, I. 1972. The Healing Factor. Grosset & Dunlap, Inc. New York, N.Y. Pautinc, L. 1971. Vitamin C and the Common Cold. W. H. Freeman & Co. San Francisco, Calif. KLENNER, F. R. 1971. J. Appl. Nutr. 23: 61.

DISCUSSION

Dr. J. Gross: I’m sure you are aware of Shimke’s work and Goldberg’s studies on intracellular protein turnover. Shimke’s made some interesting correlations between the molecular weight and the rate of turnover, finding a direct correlation, the higher the molecular weight, the faster the molecule turns over

and Goldberg has correlated turnover rate with abnormal amino acid analogs and was able to show direct correlations. I just wondered whether you’ve been able to at least make a correlation between the amide contents, their denaturation rate and their molecular size, all of which seem to correlated from the studies of these other people. Dr. RoBINSON: Probably the larger a protein is, the greater number of amide residues it might have. Dr. Gross: The concentration or the greater number? Dr. RoBINsoN: Either or both are possible. Dr. W. WEIs: I wonder if you have seen alpha oxo acids in your system and if you have systems free of 2+ metal ions, as you could have stimulated pyridoxal phosphate catalyzed reactions. Dr. RoBINSON: We haven’t looked for the molecules that you mentioned. First, we have tried to keep metals out of the system by purifying our solvents. If we add copper or iron to these solutions all these processes run faster, but there’s always a possibility that we have some metal contamination. Dr. WILLIS: I wondered if you’ve considered the problem described at the end of your talk, a proposal for explaining your results in extending this to plants where ascorbic acid is present in very large quantities. Dr. A. E. HARPER: Have you done any studies on turnover in animals receiving different levels of ascorbic acid in their diets? Dr. RoBINson: No. These studies should be carried out.

TISSUE CHANGES MARGINAL VITAMIN Norman The Bowman

M.

Sulkin

INDUCED BY C DEFICIENCY *

and Dorothy

F. Sulkin

Department of Anatomy Gray School of Medicine of Wake Forest University Winston-Salem, North Carolina 27103

INTRODUCTION

The serious consequences of acute ascorbic acid deficiency in man and in experimental animals have been the subject of numerous histologic studies.1~4 Tissue fragility, capillary hemorrhage, and defective wound healing are all important manifestations of the pathologic condition of scurvy. In recent years, a number of electron-microscopic studies have supplemented the information revealed with the light microscope.®® Peach,® J¢rgensen,® and Ross and Benditt *.* have been concerned with the fine structural changes seen in the fibroblasts of scorbutic animals and the relationship of these altered fibroblasts to collagen formation. Other studies have been conducted on changes in the endothelial cells related to capillary hemorrhage caused by vitamin C deGIeENCIESiaa== Recent studies 121° in this laboratory have shown the influence of ascorbic acid on additional cell types. A vitamin-C-deficient diet in guinea pigs resulted in cellular changes in autonomic ganglion cells and their satellite cells and in sensory ganglion cells and associated neuroglia, corneal epithelium, corneal basal lamina,

and corneal

stroma.

In nerve

cell studies, it was

interpreted

that an

increase in agranular vesicles, the dilation of rough endoplasmic reticulum with its ensuing loss of ribosomes and distention of the Golgi complex were all related to faulty protein synthesis. The degeneration noted in the mitochondria was also thought to indicate a disturbance in protein synthesis and energyyielding enzymatic systems. The alterations that occurred in these cells were reversible after the administration of ascorbic acid. An additional observation pointed out the marked degeneration of collagen in the extracellular spaces during scorbutus and its formation during recovery. Since the collagen fibers did not seem to be spatially associated with fibroblasts and were in close proximity to satellite cells, it has been hypothesized that the satellite cells played a role in the maintenance and deposition of these fibers. The cornea of scorbutic but otherwise normal guinea pigs showed variable degrees of changes, depending on the duration of the nutritional insult. The fine structural alterations observed included marked edema in the surface cells of the corneal epithelium with enlarged intercellular spaces but with intact desmosomal attachments, dilated endoplasmic reticulum, and swollen mitochondria. Degenerating cells were also observed in the middle layer. Extensive changes were noted in the basal cell profiles with dense cytoplasm, loss of * This investigation was supported in part by a grant from Hoffmann-LaRoche, Inc., and by General Research Support Grant RR5404 from the National Institutes of Health.

Sill

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hemidesmosomes, interrupted basement lamina, and protruding cytoplasm into the stroma with concurrent changes in the stroma. The collagen pattern was disrupted; some of the modified keratocytes showed great activity. Vascular invasion was sometimes present. Until recently, experimental studies dealing with vitamin C deficiency, particularly those in the realm of morphology, dealt with acute deficiency of this vitamin. Although acute deficiency in man is rarely reported in this country, subacute or subclinical deficiency is an important nutritional and health problem. Therefore, the present investigation was undertaken to determine the effects of such latent deficiency on selected cells and tissues.

MARGINAL

ASCORBIC

ACID

DEFICIENCY

STUDIES

In initial experiments, guinea pigs weighing 200-300 g were placed on a vitamin-C-free diet and were given a daily supplement of 0.2 to 0.4 mg of ascorbic acid/100 g body weight by tubal oral administration. It was found that the animals could not be maintained on this diet for long periods of time. When an animal showed overt signs of deficiency, the ascorbic acid supplement was increased to 0.8 mg/100 g for a period of several days before reverting to the more rigid diet. Thus, in the first series of animals, although the concentration of vitamin C fluctuated considerably during the course of the experiments, animals were kept alive without overt signs of deficiency for as long as 104 days. As a result of studies reported from Ginter’s laboratory'® and after some preliminary studies in this laboratory, groups of animals were started on two additional dietary regimes. In one, guinea pigs were placed on a vitamin-C-free diet supplemented each day by tubal administration of 1.0 mg of vitamin C in 1 cc of 20% sucrose solution. In the other regime guinea pigs were placed on a scorbutigenic diet for 14 days. After this period, the scorbutigenic diet was supplemented by 0.5 mg of ascorbic acid in 1 cc of 20% sucrose solution daily. Since these animals soon reached weights of 600 g or more they were receiving less than 0.1 mg/100 g per day. Yet, some of these animals survived on such a diet for more than 150 days without overt signs of deficiency. Observations with the electron microscope of autonomic ganglia from guinea pigs on a prolonged marginal vitamin C deficiency revealed degenerative changes that were not as severe as those in acutely scorbutic animals. However, certain other alterations were observed. Instead of a breakdown of collagen fibers as noted in the scorbutic animals, there was an increase in these fibers in extracellular spaces (FIGURES 1 and 2). Findings of basal bodies and an occasional fully developed cilium have been reported in neurons. There has been speculation about their appearance and function. In the present studies, these were observed with much greater frequency (FiGuRES 3 and 4). Milhaud and Pappas" found formation of numerous cilia in the central nervous system following pargyline (monoamine oxidase inhibitor) treatment. It was not clear whether ciliation in the central nervous system was due to the result of pargyline per se or as a result of inhibition of monoamine oxidase. Collagen proliferation and induced ciliation in nerve cells in chronic marginal ascorbic acid deficiency require further experimental investigation. Speculation in the literature on the effects of ascorbic acid on cholesterol metabolism ‘* led to an examination of the fine structure of liver cells from normal, acutely scorbutic, and marginally vitamin-C-deficient guinea pigs. In

Sulkin & Sulkin:

Tissue Changes

319

general, sections of liver taken from acutely scorbutic animals (animals on a vitamin-C-free diet for 21-30 days) showed a fairly normal characteristic profile. A few cells exhibited swollen mitochondria and the mitochondrial matrix appeared less dense (FIGURE 5). Observations in these initial studies

revealed that the hepatic cells from the marginally deficient animals

(animals

Figure 1. Electron micrograph of a section of an autonomic ganglion from a guinea pig on a marginal vitamin-C-deficient diet for a preiod of 104 days. The vitamin C supplement (0.2-0.4 mg/100 g body weight) was dissolved in water. Note large concentration of collagen in extracellular space.

on a marginally deficient diet for 90 days or more) differed from hepatic cells of the other two groups. These differences included a marked reduction and displacement of the granular endoplasmic reticulum. Wherever present, this organelle usually encircled the mitochondria, the latter having developed a dense matrix (FIGURE 6). The major alteration observed was a sharp proliferation

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Ficure 2. Electron micrograph of a section of an autonomic ganglion from a guinea pig on a marginal vitamin-C-deficient diet for a period of 118 days. The vitamin C supplement (1 mg per animal) was dissolved in 1 ml of 20% sucrose. Note

large concentration of collagen in extracellular space.

of smooth endoplasmic reticulum, sometimes occupying large areas of cytoplasm, often to the extent of displacing the other organelles to the periphery of the cell (FiGuRE 6). This was interpreted to be indicative of increased steroid metabolism. Jones and Fawcett !® suggested that the enzymes necessary for cholesterol biosynthesis are associated with smooth endoplasmic reticulum. Jones and Armstrong?° have shown an increased cholesterol biosynthesis following phenobarbital-induced hypertrophy of the smooth endoplasmic reticulum in the liver. The observations made in this laboratory of the morphologic changes in the hepatic cells of the marginally deficient animals led to a study of the levels of serum cholesterol and liver cholesterol. In the small number of samples studied, there appeared to be no significant changes in the serum cholesterol. Total cholesterol in the liver was measured following saponification and extraction of the tissue with Skellysolve B (Skelly Oil Co., Tulsa, Oklahoma) and using the

Autoanalyzer Il methodology.*! The results of these studies are shown in FicuRE 7. The difference between the mean for the control group and the mean for the groups on a marginally deficient diet was significant at the 5% level (p < 0.05). The experimental animals from which these data were obtained were on the marginally deficient diet from 68 to 104 days. Linear regression

Sulkin & Sulkin:

Tissue Changes

321

tests indicated no correlation between the number of days on the diet and the concentration of liver cholesterol. The results of the above preliminary study suggested that an examination be made on the aortas of guinea pigs that had been on a marginally deficient diet. Light-microscopic examinations of sections of aortas of animals that had been subjected to a chronically deficient diet for at least 100 days, and up to 150 days, showed numerous alterations in the aortic walls. The most striking were the presence of large intimal plaques (FIGURE 8), which appeared to be of the musculofibrotic type, as further demonstrated by the Van Gieson-Verhoff stain and the Mallory stain (FIGURE 9). This proliferation of collagen in the aortic wall was of particular interest when considered with respect to the concomitant proliferation of collagen in the extracellular spaces in autonomic ganglia following chronic ascorbic acid deficiency (FIGURES 1 and 2). Another observation of note was the presence of marked endothelial proliferation in many of the sections (FIGURE 10). This was sometimes accompanied by intimal thickening and sometimes without such thickening. Other changes included a consistent high degree of metachromasia in the ground substance in the walls of the aortas from the experimental animals and, in some instances, the presence of a fibrous, amorphous material of varying thickness underlying the endothelium (Figure 11). This material has not yet been identified. Unfortunately, since it takes from 3 to 5 months to obtain suitable material for

Ficure 3. guinea pig on cilium in the cut obliquely

Electron micrograph of a section of an autonomic ganglion cell from a a marginal vitamin-C-deficient diet for 102 days. Note the fully formed cytoplasm adjacent to the nucleus and the portion of a second cilium next to it.

FicuRE 4. Electron micrograph of a section of an autonomic ganglion from a guinea pig on a marignal vitamin-C-deficient diet for 104 days. The cytoplasm of the satellite (neuroglial) cell contains a fully formed cilium and the basal body from forming second cilium.

FIGURE 5. Electron micrograph

of a section

of a hepatic cell from

a

an acutely

scorbutic guinea pig. The cell appears essentially normal except that the mitochondria are slightly swollen.

Figure 6. Electron micrograph of a section of a hepatic cell from a guinea pig that had been on a marginally vitamin-C-deficient diet for a period of 104 days. Note the reduction of the granular endoplasmic reticulum and the encirclement of these organelles around the mitochondria when they are present. The sharp proliferation of smooth endoplasmic reticulum to the extent of displacing other organelles is of special significance.

Yh

FIGURE 7. The _ tissue levels of liver cholesterol in a group of 4 guinea pigs on a control diet and 11 experimental guinea pigs on a marginal ascorbic-acid-deficient diet for periods of 68 to 138 days.

a a

as

TISSUE LIVER CHOLESTERO, 2k CONTROL

Sm6et

SSO Wi lenses EXPERIMENTAL

Annals

324

*

7

~

New

York

Lit

se

Academy

st

of Sciences

hee

~~

Ficure 8. A section of the aorta from a guinea pig that had been on a marginal vitamin-C-deficient diet for 104 days. The large intimal plaque appears well formed and consists mainly of musculo-fibrotic tissue. (Stained with hematoxylin and eosin).

FicureE 9. A section of an aortic arteriosclerotic plaque from a guinea pig that had been ona marginal vitamin-C-deficient diet for a period of 109 days stained with the Van Gieson-Verhoff stain to demonstrate the fibrotic nature of the lesion.

Sulkin & Sulkin:

Tissue Changes

B25

this type of study, sufficient material was not available for lipid studies as well as other histochemical and ultrastructural studies of the aortic wall. Such studies are now in progress.

Ficure 10. A section from an aorta of a guinea pig that had been on a marginal vitamin-C-deficient diet for 110 days. This section is characterized by endothelial proliferation and intimal thickening.

SUMMARY

Chronic marginal vitamin C deficiency in guinea pigs results in alterations in certain tissues that are quite different from those observed following acute deficiency. In autonomic ganglia, although changes observed in the organelles of some cells are similar to those seen in acute deficiency, the specific changes are the presence of large numbers of cilia in the cytoplasm of both ganglion

326

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York

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cells and their associated neuroglia and the marked proliferation of collagen fibers in the extracellular spaces. The evidence presented points to a link between vitamin C and arteriosclerosis. One mode of interaction may be the effect of latent vitamin C deficiency on cholesterol metabolism. The data presented indicate important changes in the liver with marked increases of agranular endoplasmic reticulum. Together with the liver cholesterol studies, the indications suggest an increased cholesterol deposition in the liver. Light-microscopic sections of the aorta reveal alterations in many of these vessels ranging from endothelial proliferation to large, well-formed musculofibrotic arteriosclerotic plaques.

_Ficure 11. A section from the aorta of a guinea pig that had been on a marginal vitamin-C-deficient diet for 105 days. Note the thick band of amorphous substance underlying the endothelium.

ACKNOWLEDGMENT

The technical assistance of Mrs. Lynn King is gratefully acknowledged. REFERENCES

1. Wovrpacn, S. B. & O. A. Bessey. 2)

Physiol. Rev. 22: 233-289. WIGGERS, C. J. 1949. Physiology Febiger. Philadelphia, Pa.

1942.

Tissue changes in vitamin deficiencies.

in Health

and

Disease.

5th

edit.

Lea

&

Sulkin & Sulkin: 3.

4.

327

Ltoyp, B. B. & H. M. Sincrair. 1953. Vitamin C. In Biochemistry and Physiology of Nutrition. G. H. Bourne and G. W. Kidder, Eds. 1: 370-471. Academic Press. New York, N.Y. CHATTERJEE, G. C. 1967. Effects of ascorbic acid deficiency in animals. In The Vitamins. W. H. Sebrell, Jr. and Robert S. Harris, Eds. 1: 407-457. Academic

Press. New York, N.Y. 5. PEACH, R. 1962. An electron struct. Res. 6: 579-590. 6.

Tissue Changes

J@RGENSEN, O.

1964.

optical study of experimental

Electron-microscopical

scurvy.

J. Ultra-

studies of granulation tissue forma-

tion in open wounds of ascorbic acid deficient guinea pigs. Acta Path. Microbiol. Scand. 60: 365-375. 7. Ross, R. & E. P. BENpITT. 1962. Wound healing and collagen formation. II. Fine structure in experimental scurvy. J. Cell Biol. 12: 533-551. 8. Ross, R. & E. P. BENpiTT. 1964. Wound healing and collagen formation. IV. Distortion of ribosomal patterns of fibroblasts in scurvy. J. Cell Biol. 22: 365-389. 9. Gore, I., Y. TANAKA, T. FUJINAMI & M. L. GooDMAN. 1965. Aortic acid mucopolysaccharides and collagen in scorbutic guinea pigs. J. Nutr. 87: 311-316. 10. Gore, I., T. FUJINAMI & T. SHIRAHAMA. 1965, Endothelial changes produced by ascorbic acid deficiency in guinea pigs. Arch. Path. 80: 371-376. 11. Frreperici, H. H. R., H. Taytor, R. Rose & C. L. PrrRaAni. 1966. The fine structure of capillaries in experimental scurvy. Lab. Invest. 15: 1442-1458.

12.

SuLxin, D. F. & N. M. SULKIN. 1967. An electron microscopic study of autonomic ganglion cells of guinea pigs during ascorbic acid deficiency and partial inanition. Lab. Invest. 16: 142-152.

13.

SuLxin, D. F., N. M. SULKIN & M. L. RoTHROCK. 1968. Fine structure of autonomic ganglia in recovery following experimental scurvy. Lab. Invest. 19:

14.

55-66. SuLkin, D. F., N. M. SuLKIN & H. NusHAN. 1972. Corneal fine structure in experimental scorbutus. Invest. Ophthal. 11: 633-643.

15.

SuLkINn,

D. F., N.

M. SULKIN

& H. NusHan.

ganglia during experimental scurvy.

1973.

Fine

Acta Neuropath.

16.

GinTER, E., P. BopeK & M. OveckKa. 1968. Model guinea pigs. Int. Z. Vit. Forschung 38: 104-113.

17.

MiLHaup, M. & G. Pappas. 1968. Cilia formation pargyline treatment. J. Cell Biol. 37: 598-609.

18.

GiInTER,

19.

acids. Science 179: 702-704. Jones, A. L. & D. W. FAwcetTr.

20.

21.

E.

1973.

Cholesterol:

Vitamin

structure

of sensory

23: 141-151. of hypovitaminosis

C

in

in the adult cat brain after

C controls

its transformation

to bile

1966. Hypertrophy of the agranular endoplasmic reticulum in hamster liver induced by phenobarbital. J. Histochem. Cyto-

chem. 14: 215-232. Jones, A. L. & D. T. ARMSTRONG. 1965. Increased cholesterol biosynthesis following phenobarbital induced hypertrophy of agranular reticulum in liver. Proc. Soc. Exp. Biol. Med. 119: 1136-1139. Rusu, R. L., L. LEon & J. TURRELL.

1971.

Automated

and triglyceride determination on the AutoAnalyzer.

simultaneous

cholesterol

II. Jn Advance

mated Analysis-Technicon International Congress, 1970. Eds. : 503—507. Thurman Associates, Miami, Fla.

in Suto-

E. C. Barton, et al.,

DISCUSSION

Dr. A. E. HARPER: You implied that your deficiency is marginal, yet you indicated that survival of 160 days is unusual. It is important to know what you mean by marginal deficiency—have these animals stopped growing and

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are they losing weight and beginning to diet during the course of the experiment? If so, this would be far from a marginal

deficiency.

Second, it was not clear to

me what type of controls you had used, because obviously if the animals show a depression of food intake they are having lower intakes of a great many other nutrients, and consequently have less available energy. Reduced energy and protein intakes affect the endoplasmic reticulum and result in disaggregation of protein-synthesizing systems. I think these are two rather crucial points in relation to interpretation of the nature of these effects. Dr. SULKIN: We did not have time during this presentation to go into details of the diet. We did pair feeding studies and inanition had absolutely no effect on these alterations, in either chronic or acute deficiency studies. We used three different types of deficient diets. When the animals showed scorbutic signs we increased the supplement from 0.2—0.4 mg to 0.8 mg/day for two or three days. When the animals appeared to recover we went to a more rigid diet. I am only going to mention one diet, in which we followed the regimes suggested by Ginter: we gave the animals 0.5 mg of vitamin C dissolved in 1 cc of 20% sucrose per animal. The animals attained a weight of about 600 gm. Most of the animals increased in weight, but we lost about half of the animals during the course of the experiment. They incurred infections very easily, including those of the ear, or pneumonia. Dr. HARPER: I would call that more than a marginal deficiency. Dr. VOLUNGER (Rutgers University, New Brunswick, N.J.): Did you determine any blood cholesterol levels on termination of the experiment? Dr. SULKIN: We did and saw nothing consistent. Dr. E. M. NADEL (Medical University of South Carolina, Charleston, S.C.): What level of the aorta were you taking your sections from? Did you look at them under polarized light for cholesterol? Dr. SULKIN: We did not have the material to do that. We took the entire aorta and cut out pieces every millimeter from the ascending to the descending portion. We saw nothing that resembled foam cells—these seem to be wholly fibrotic. We are now repeating these experiments using a larger number of animals, and we plan to perform histochemical and ultrastructural studies. This might prove to be an exceilent model for the electron microscopic study of arteriosclerosis. Dr. NADEL: Do you really think this is atherosclerosis? Dr. SULKIN: I did not call it atherosclerosis. I am following our pathologist’s advice and calling it a musculofibrotic arteriosclerosis. Dr. E. DeGkwirz: Did you look into the pancreas of the deficient guinea pigs? Dr. SULKIN: We have not looked at the pancreas. As far as hemorrhage is concerned, they may look fairly normal externally, but when opened are full of hemorrhages. Dr. M. P. LAMDEN: I believe that Dr. Ginter would be the one who could answer the question of increased synthesis of cholesterol. Some of his work indicates that hydroxylation mechanisms for the conversion of cholesterol to the bile acids are decreased by an ascorbic acid deficiency, and this hence leads to increased accumulation of cholesterol. Dr. Deckwitz: If I remember correctly, Ginter, in a paper given in England this spring, indicated that it might be the lack of P-450 that lowered the metabolism of cholesterol.

ALTERATION IN HUMAN GRANULOCYTE FUNCTION AFTER IN VITRO INCUBATION WITH L-ASCORBIC ACID * W. Byron Smith, Stephen B. Shohet,+ Ellen Zagajeski, and Bertram H. Lubin Bruce Lyon Memorial Research Laboratory at the Children’s Hospital Medical Center of Northern California Oakland, California 94609 and Division of Hematology University of California Medical Center San Francisco, California 94122

INTRODUCTION

The exposure of human granulocytes to ascorbic acid in vitro results in stimulation of the hexose monophosphate shunt,' augmentation of random migration and chemotaxis,” and inhibition of aldehyde formation and iodination of protein; ® however, neither the bactericidal nor the phagocytic capacity of these cells is altered.* Pauling ® has suggested that the prophylactic ingestion of massive doses of L-ascorbic acid may prevent viral infections in man. The possible consequences of such doses on granulocytic function are of considerable interest. Accordingly, we have studied these potential effects by measuring the influence of varying concentrations of L-ascorbic acid on bactericidal and candidacidal activity in human granulocytes. MATERIALS Preparation

AND

METHODS

of Bacteria

and

Fungi

The Staphylococcus aureus ATCC 25923 used for all bactericidal assays was grown for 18 hours in trypticase soy broth, centrifuged at 4000 g for 10 minutes

at room

Hank’s

balanced

Sabouraud’s

2%

temperature,

washed

salt solution dextrose

broth

twice in 0.9%

(HBSS).

Candida

for 48 hours

saline, and suspended

albicans

to two

weeks,

was

cultured

washed

and

in

on re-

suspended in HBSS, and enumerated in a hemocytometer. Greater than 95% of the organisms were viable as determined by trypan blue dye exclusion.® Granulocyte

Isolation

Heparinized whole blood was obtained from healthy adult donors who were not ingesting supplemental ascorbic acid. The granulocytes were isolated after * This work was supported, in part, by Hoffmann-La Roche, Inc. + Recipient of Grant AM-16095 and Career Development Award AM-37237 the National Institutes of Health.

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centrifugation on a Hypaque-Ficoll cushion and sedimented with 5% dextran, using a modification of the technique of Steigbigel.’ The white cell button was washed twice with HBSS containing 20% fetal calf serum, resuspended in HBSS, and counted. Contaminating erythrocytes were removed by osmotic shock. These preparations contained 88-95% granulocytes. Oxygen

Consumption

A Clark’s membrane oxygen electrode and a Dohrman recorder were utilized to measure oxygen consumption. Each cuvette contained 2 ml of either Hank’s balanced salt solution (HBSS) or distilled water. Oxygen consumption was measured for 5 minutes at 37°C with constant stirring to obtain “resting” values. L-ascorbic acid (as the Na salt, Sigma Chemical Co., St. Louis, Mo.) at pH 7.4 in HBSS or distilled H,O was then added to the cuvette and O, consumption measured for an additional 5 minutes. Results were expressed as microliters of O, consumed/hour. Hexose

Monophosphate

Shunt

Production of “CO, from 1-'C-glucose of resting and stimulated granulocytes was determined as previously described by Root.* The cells were stimulated with either latex spherules, bread yeast, or Staphylococcus aureus. After a 60-minute incubation at 37°C, the amount of 4#CO, was measured. Lascorbic acid in HBSS, pH 7.4, was added to resting and stimulated cells. The results were reported in either nmoles of glucose oxidized/hr/S X 10° cells or as a percent increase over the control incubation. Bactericidal

Activity

Human granulocytes (5 10°) were placed into 12 X 75 mm polyethylene tubes containing 1 X 10% organisms and 10% AB serum in a final volume of 1 ml. Following the addition of L-ascorbic acid, the flasks were incubated at 37° C on a rotating drum. Similar mixtures with the exclusion of granulocytes were used in cell-free experiments. Aliquots were withdrawn at 30 and 120 minutes, plated on tryptic soy agar, and processed for colony counting by a modification of the technique of Michenberg.” Iodination

of Zymosan

The iodination of zymosan granules was determined by incubating 5 x 107 zymosan particles, 5 X 10° granulocytes, sodium iodide 10 mM, 17% AB serum, and 0.2 wC!°I at 37° C using a modification of the technique of Pincus.1° The reaction was terminated after 1 hour by the addition of 0.1 ml of 0.1 M sodium thiosulphate and placed on ice. Following the addition of 10% cold trichloroacetic acid, the radioactivity in the precipitate was determined. The results were expressed as nmoles of iodide converted to a TCA-precipitable form per 5 X 10° cells/hour. A blank containing all reagents except granulocytes was subtracted from the experimental results.

Smith ef al.: Human

Granulocyte

Function

331

RESULTS The influence of L-ascorbic acid on the growth of Staphylococcus aureus is indicated in FicguRE 1. At physiologic concentrations (5 mM), L-ascorbic acid had no effect on bacterial growth. When the concentration of L-ascorbic acid was increased to 50 mM, a significant bactericidal effect was noted, with killing of 30% and 45% of organisms after incubation for 30 and 120 minutes. The addition of L-ascorbic acid in physiologic concentrations to an incubation mixture containing human granulocytes resulted in a prompt burst of respiratory activity, which was maximal at 100-200 mM and was inhibited at

10 mM LAA

50 mM LAA

Staph. of Percent killed aureus

0

30

60

120

Minutes

Ficure 1. Effect of L-ascorbic acid on killing of Staphylococcus aureus. The incubation contained 110" organisms suspended in HBSS. Number of experiments are noted in parentheses. The results are expressed as the percent of organisms killed compared with a control without L-ascorbic acid at each time interval.

200-400 mM (FiGcuRE 2). This effect was small, however, when compared with the oxidative burst that occurred following the addition of phagocytic particles to human granulocytes (TABLE 1). The addition of 100 mM L-ascorbic acid to resting granulocytes increased the HMPS activity by 450% as against a 1280% increase following the addition of Staphylococcus aureus. In data not shown, the increased activity of the HMPS induced by Staphylococcus was not augmented when L-ascorbic acid was simultaneously added to the stimulated granulocytes. The in vitro effect of L-ascorbic acid on the bactericidal activity of granulocytes is demonstrated in Ficure 3. In contrast to controls, the addition of

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nanomoles oxidized, Glucose

0

50

100

200

mM

400

t-Ascorbic acid

FicureE 2. Effect of L-ascorbic acid ‘on the HMPS in resting human granulocytes. Leukocytes (510°) were incubated at 37° C in 10% AB serum-HBSS with 1-“Cglucose (7.6 nmol and 0.5 wCi glucose) and L-ascorbic acid. After 1 hour, the quantity of “CO. generated was determined. The open and closed geometric figures represent individual experiments, The results are expressed as nmol of glucose oxidized/hr/5 x 10° granulocytes.

TABLE

STIMULATION

OF HMPS

1

IN HUMAN

GRANULOCYTES

Increase in “O-1-Glucose Oxidation (% ) 10 mM

LAA

(9)

64.4

20 mM LAA (10) 50 mM LAA (10) 100 mM LAA (6) 200 mM LAA (6) Polystyrene beads (7)

2323, 296.7 453.0 305.6 599.6

Bread yeast (9) S. aureus (11)

974.3 1276.2

Note: Experimental conditions are those described in FiGuRE 2. The figures in parentheses indicate the number of experiments. The ratio of phagocytic particles to white blood cells was 400:1 for polystyrene beads, 1:1 for bread yeast, and 100:1

for Staphylococcus aureus, phagocytic particles.

L-ascorbic

acid was not added to flasks stimulated with

Smith et al.: Human

Granulocyte

TABLE

EFFECT

OF

LAA

on

Function

333

2

Puacocytosis

oF

Candida

albicans

Organisms Ingested per 100 Cells mM

AA

at 15 Minutes

Control Ce) 10 (4) 20 (7) 100 (3)

OI Nee 2ieoe 86.60 + 26.02 85.80=24.32 87.23 +31.73 5302223749

Note: The experimental conditions are those described in FigurE 4. The figures in parentheses indicate number of experiments.

L-ascorbic acid at a final concentration of 5 mM had no effect on the killing of Staphylococcus aureus. At 50 mM, however, there appeared to be a slight inhibition of killing at 30 minutes (p < 0.1) and somewhat more at 120 minutes (p < 0.01). The mean inhibition in killing at 30 minutes was 22%. The p values were determined by Student’s paired t-test. The effect of L-ascorbic: acid on the phagocytosis of Candida albicans by human granulocytes is demonstrated in TABLE 2. At 5, 10, and 20 mM, there appeared to be a slight increase in phagocytosis, while at 100 mM there appeared to be minimal inhibition of this process. These differences were not statistically significant. The effect of L-ascorbic acid on the intraleucocytic destruction of

of Percent killed organisms

[ot heath 30 minutes

|

120 minutes

FicureE 3. Effect of L-ascorbic acid on bactericidal activity of human granulocytes. Leukocytes (510°) were incubated with 1 x 10’ organisms (Staphylococcus aureus) in 10% AB serum-HBSS and L-ascorbic acid at 37° C for 2 hours. Open figures (O, (J) represent controls without L-ascorbic acid. The results are expressed as the mean +1 SD. The number of experiments is noted in parentheses.

334

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York

Academy

of Sciences

Candida albicans was measured at concentrations of 2.5 to 100 mM (FIGURE 4). The pH varied between 7.3 and 7.8 during these experiments. In the absence of ascorbate, human granulocytes killed 28-35% of ingested Candida albicans in 1 hour. With the addition of 5 mM t-ascorbic acid, candidacidal activity was inhibited 36% more than in controls. This effect was dosedependent with increased inhibition at higher concentrations. The iodination of zymosan granules by human granulocytes was also inhibited by L-ascorbic acid (FiGuRE 5). In controls stimulated with zymosan only, the mean '°°I converted to a precipitable form in 1 hour was 2.08 = 0.66 (1 SD) nmol. With the addition of 2.5 mM L-ascorbic acid to the incubation in 4 experiments, there was a significant decrease in iodination; the mean 1*°I converted to a precipitable form was 0.366 = 0.238

(1 SD)

nmol

(p < 0.01).

This effect was dose-dependent, with complete inhibition at a concentration of 20 mM.

a

ae ~

~,

~,

killed Percent yeast

0

20

50

mM Ficure cytes,

4,

Equal

Effect of L-ascorbic numbers

acid on

of granulocytes

100

t-Ascorbic acid candidacidal

and organisms

activity of human (Candida

albicans),

granulo2.5 10%

were incubated in 10% AB serum HBSS with L-ascorbic acid at 37°C for 1 hour. The results are expressed as the percentage of the original organisms killed.

Smith et al.: Human

Granulocyte Function

335

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349 et al.: Ascorbic Acid in Guinea Pigs Veen-Baigent

level of mg. All reached to be of

York

New

Annals

350

Academy

of Sciences

0.5 mg. Growth was not statistically enhanced by intakes above 0.5 clinical symptoms of deficiency did not disappear until levels of intake 0.5 mg. An intake of 0.15 mg/100 g BW per day would thus appear only borderline adequacy for the parameters measured, and over the

rr |

3

;?

TISSUE ASCORBIC

ig ?

ACID

i°

5

a

>

3

H

3 }

:

:

i: st

a ee ees

BLOOD

O18

ASCORBIC

as

w

208

we

sae

188

ACID

(ag/1@8 Plesme mi)

cells) pe/te’ (Lewcecytes

O05

as

os Ascorbic Acid

1.0 2.0 =(mg/100g BW)

me

Ficure 2. Pattern of response of tissue and blood ascorbic acid to graded levels of ascorbic acid intake for 6 weeks.

experimental period studied. It might well be suspect over an extended period of time. For example, Ginter et al.’ have induced a chronic latent hypovitaminosis C by feeding 0.5 mg ascorbic acid per day (approximately 0.15 mg/ 100 g BW per day) over three months to guinea pigs whose tissue ascorbic acid

Veen-Baigent

et al.: Ascorbic

Acid in Guinea

Pigs

Bol

had been initially reduced to low levels by feeding a scorbutogenic diet for 14 days. In the present study, all parameters responded well to 0.5 mg ascorbic acid/100 g BW per day|!; larger intakes had no specific benefit. Thus, within the present experimental setting the ascorbic acid requirement for the parameters measured appears to reside at virtually one level of intake rather than extend over a range of intakes as is suggested by earlier reports.1 An intake of 0.5 mg/100 g BW, which supported maximal response of parameters measured, resulted in 2

saturation of leukocytes with ascorbic acid and approximately % saturation of plasma. Other tissues were well below their eventual apparent saturation levels (liver, 20%; adrenal and spleen, 23%; heart, 32%; kidney, 34% of saturation levels). These observations give no support to the essentiality, or even benefit, of achieving tissue saturation in the guinea pig. It is doubtful that other functions of ascorbic acid, e.g., those that measure enzyme activity directly, have a requirement for tissue saturation to achieve normal rate of activity. Zannoni et al.!2 observed the activity of microsomal drug hydroxylation enzymes in guinea pigs with liver ascorbic acid levels of 6 mg/100 g to be unchanged from control animals (liver ascorbic acid, 20 mg/100 g), but reduced by 50% when liver levels fell to 2.5 mg ascorbic acid/ 100 g. Thus, normal enzyme activity was still evident when total liver vitamin A was less than 30% of control levels (which were likely close to saturation), although it declined when liver ascorbic acid fell to 12% of control levels. The pattern of response of plasma and tissue ascorbic acid levels to increasing ascorbic acid intake (FIGURE 2) suggested that ascorbic acid may not be necessarily freely exchangeable among tissues or between blood and tissue. The periods of plateau and rise of ascorbic acid in blood were out of phase with those observed in tissue. At very low levels of intake (0.05 mg), ascorbic acid in tissuse rose at the apparent expense of leukocyte ascorbic acid (plasma ascorbic acid was not determined). A second plateau in leukocyte and in plasma levels at intakes of 0.5 to 2.0 mg was again accompanied by an elevation in liver and adrenal ascorbic acid. This relationship is of interest on 2 counts. First, while leukocyte ascorbic acid was, in general, indicative of tissue ascorbic acid stores,!* it did not closely reflect the changes occurring in tissue ascorbic acid with increasing intakes. Second, the data are suggestive of a preferential transfer of ascorbic acid from blood (including leukocytes) to tissues supporting essential ascorbic-aciddependent functions, when ascorbic acid intakes are low (0.05 to 0.15 mg/100 g BW). At “adequate” intakes (0.5 to 2.0 mg/100 g BW), blood and some tissues (kidney, heart, and spleen) remained relatively constant as liver and adrenal ascorbic acid rose. Once an intake of 2.0 mg was exceeded, ascorbic acid in all tissues rose until eventual “saturation” levels were attained. The data do not permit conclusions as to whether saturation represented true saturation of binding sites in the tissue or of the rate of uptake by tissue cells,1! establishment of a new kidney threshold, or saturation of the active transport absorptive mechanism of ascorbic acid across the gut wall.’ Earlier reports have demonstrated that the ascorbic acid intake-tissue concentration curve follows the shape of dissociation curves.1* More recent studies

|| This intake is approximately equivalent to 2.0 to 2.5 mg ascorbic acid/day at the midpoint of the experiment, or 2.5 to 3.5 mg/day by the end of the experimental period (56 days).

352

Annals

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York

Academy

of Sciences

have indicated that liver ascorbic acid continues to rise while plasma levels have achieved saturation levels over intakes of 2 to 20 mg ascorbic acid per day (approximately equivalent to 1 to 10 mg/100 g BW per day).*’ Similarly, leukocyte ascorbic acid did not change as ascorbic acid intake increased from 1.6 to 10 mg/100 g BW, although adrenal ascorbic acid rose almost twofold.** These reports did not cover the entire dose range included in the present study and, thus, did not establish whether further increases could be induced at high dose levels. Higher levels of intake (10, 50, and 100 mg/100 g BW) conferred no benefit on the parameters measured in the present study. There was, however, some evidence for at least one detrimental effect of a very high intake in reduction in serum copper observed in the 100-mg-dose group (experiment 1). Recently Evans et al.!® have shown that ascorbic acid decreased binding of zinc and copper by metallothein from bovine duodenum and liver, and thus probably interferes with absorption of these metals from the gut. Growth was not inhibited by high intakes of ascorbic acid in this study. This is in agreement with the observations of Nandi et al.17 that 100 mg ascorbic acid/ 100 g BW had no influence on growth. However, an apparent depression of growth rate has been shown by Yew 2° with 50 mg ascorbic acid/100 g BW per day, and by Sorenson et al.,?1 who fed guinea pigs diets containing 86 g ascorbic acid/kg diet (approximately equivalent to 400 mg ascorbic acid/ 100 g BW per day). The variability in response of individual pigs within each dose group was notable, particularly with respect to growth and blood and tissue ascorbate levels. Final body weight within any one dose group varied in some instances as much as 25%. At the lower ascorbic acid intakes, tissue and blood ascorbic acid of individual animals varied several-fold within one dose level: this discrepancy diminished with higher intakes. There was, however, over all groups, a highly significant correlation (p < 0.01) between leukocyte ascorbic acid and ascorbate in specific tissues, and between plasma and tissue ascorbic acid. Variable responses within groups at low dose levels (0.05 to 0.5 mg/100 g BW) may reflect different minimal requirements in individual animals, as has been suggested by Williams and Deason.*? Variability at high intakes is more likely indicative of differences in absorption rate or capacity, tissue binding, or rate of metabolism of ascorbic acid.

CONCLUSION

The maximal response of a selection of ascorbic-acid-dependent parameters to a range of intakes of ascorbic acid has been shown to occur at virtually one level of intake in the present experiments. Further, this response occurred with intakes that produced only /% saturation or less of tissues with ascorbic acid. Excessive intakes did not enhance effects, and, as in the case of serum copper levels, may have a detrimental effect. In view of the known factors that may influence requirements ' and the intralaboratory variability in experimental conditions, the more reasonable approach to the interpretation of dose response data is likely to relate the response to the accompanying degree of tissue saturation, rather than to an absolute level of ascorbic acid intake.

Veen-Baigent

et al.: Ascorbic Acid in Guinea Pigs

S96)

ACKNOWLEDGMENTS

This was a cooperative project involving the Department of Nutrition, School of Hygiene, Faculty of Dentistry, and Faculty of Food Sciences. Appreciation is expressed to Hoffmann-La Roche for their support and to the investigators and technical staff who participated in the investigation. In particular, we are indebted to Mrs. D. Riley Brymer and to Miss N. Fritz, Department of Nutrition, for their excellent technical assistance in the laboratory and in the care and dosing of the animals.

REFERENCES

CHATTERJEE, G. C. 1967. Ascorbic acid requirements of animals. Jn The Vitamins. W. H. Sebrell, Jr. & R. H. Harris, Eds. 1: 495-500. Academic Press. New York, N.Y.

Rep,

M. E. & G. M. Briccs.

1953.

Development

of a semi-synthetic

diet for

young guinea pigs. J. Nutr. 51: 341-354. ALBRINK, M. J. 1959. The microtitration of total fatty acids of serum with notes on the estimation of triglyceride. J. Lipid Res. 1: 53—59. FrRincs, C. S., T. W. FENDLEY, R. T. DUNN & C. A. QUEEN. 1972. Improved determination of total serum lipids by the sulfophosphovanillin reaction. Clin.

Chem. 18: 673-674. SCHLIERF, G. & P. Woop. 1965. Quantitative determination of plasma free fatty acids and triglycerides by thin-layer chromatography. J. Lipid Res. 6: 317-319. KRITCHEVSKY, D. & S. MALHOTRA. 1970. Recovery of lipids from thin-layer chromatography for radioassay. J. Chromatogr. 52: 498-499. ZLATKIS, A., B. ZAK & A. J. BoYLeE. 1953. A new method for the direct determination of serum cholesterol. J. Lab. Clin. Med. 41: 486-492. Fiske, C. H. & Y. SuBpBARow. 1925. Colorimetric determination of phosphorus. J. Biol. Chem. 66: 375—400. Lou, H. S. & C. W. M. WiLson. 1971. An improved method for the measure-

ment of leucocyte ascorbic acid concentrations. 90-98. Bessey, O. A., O. H. Lowry

& M. J. Brock.

tion of ascorbic acid in small amounts

13.

14. .

Int. J. Vitam. Nutr. Res. 41:

1947.

The quantitative determina-

of whole white blood cells and plate-

lets. J. Bol. Chem. 168: 197-205. GINTER, E., P. BopEcK & M. OvecKa. 1968. Model of chronic hypovitaminosis C in guinea pigs. Int. J. Vitam. Nutr. Res. 38: 104-113. ZANNONI, V. G., E. J. FLYNN & M. Lyncu. 1972. Ascorbic acid and drug metabolism. Biochem. Pharmacol. 21: 1377-1392. CHEVILLARD, L. & F. HAMON. 1943. Le taux de l’acide ascorbique dans les leucocytes et les plaquettes comme test de la carence en vitamine C. C. R. Soc. Biol. 137: 307-309. SHARMA, S. K., R. M. JOHNSTONE & J. H. QuUASTEL. 1963. Active transport of ascorbic acid in adrenal cortex and brain cortex in vitro and the effects of ACTH and steroids. Can. J. Biochem. Physiol. 41: 597-604. STEVENSON,

N. R. & M. K. BrusH.

1969.

Existence

and characteristics

dependent active transport of ascorbic acid in the guinea pig.

Nutr. 22: 318-326. Grroup, A., C. P. LEBLOND, R. RATSIMAMANGA & E. GERO. 1938. mal en acid ascorbique. Bull. Soc. Chim. Biol. 20: 1079-1087. Nanpi,

B. K., A. K. MasumMpeER,

N. SUBRAMANIAN

Amer.

of Na*-

J. Clin.

Le taux nor-

& J. B. CHATTERJEE.

1973.

354

Annals

New

Effects of large doses 1688-1695. 18.

19. 20. 21.

22.

York

of vitamin

Kerry, M. O. & O. PELLETIER.

1974.

Academy

of Sciences

C in guinea Ascorbic

pigs and

rats.

acid concentrations

J. Nutr.

103:

in leukocytes

and selected organs of guinea pigs in response to increasing ascorbic acid intake. Amer. J. Clin. Nutr. 27: 368-372. Evans, G. W., P. F. Masors & W. E. CorNATzerR. 1970. Ascorbic acid interaction with metallothionein. Biochem. Biophys. Res. Commun. 41: 1244-1247. Yew, M.S. 1973. “Recommended daily allowances” for vitamin C. Proc, Nat. Acad. Sci. 70: 969-972. SorENSEN, D. I., M. M. Devine & J. M. Rivers. 1974. Catabolism and tissue levels of ascorbic acid following long-term massive doses in the guinea pig. J. Nutr. 104: 1041-1048. Wi LtiAMs, R. J. & G. DEASON. 1967. Individuality in vitamin C needs. Proc. Nat. Acad. Sci. 57: 1638-1641.

DISCUSSION

Dr. DeGKwitz: What do you think the saturation level is? Looking at your slides, it seems pretty clear to me that absorption capacity is limited just like in man. Of course if the absorption capacity is limited, you will not get enough ascorbic acid into the tissues. Dr. VEEN-BAIGENT: The saturation point is simply the top level which is obtained at high dose levels, whether it occurs because the absorption capacity is saturated or whether the tissues are saturated. Dr. M. Brin: I would like to commend both Drs. Sulkin and Baigent for undertaking investigation in the “twilight zone” of marginal deficiency. I think the traditional approach has been to vary one factor at a time in an acute deficiency situation. That results in a moribund animal. It is only in such marginal states that we have something more analogous to the human condition, where otherwise healthy individuals are “at risk” in a certain group of nutrients. The tissues and the metabolic systems must then adapt to the lack of adequate levels of micronutrient. I think the very provocative questions raised by these studies still remain to be answered.

ParRTIV.

PHARMACOLOGICAL

CLINICAL

ASPECTS

PHARMACOLOGICAL OF ASCORBIC ACID C. W.

M.

ASPECTS

Wilson

Department of Pharmacology University of Dublin and The Allergy Clinic Mercer's Hospital Dublin 2, Ireland

Ascorbic

acid is a vitamin.

In view of the current interest in the effects

produced by administration of supplements that are relatively large in comparison with the recommended daily dietary intake, the effects produced by this vitamin must be considered from a pharmacological point of view. This implies that its kinetics should be studied in the blood and tissues in relation to any clinical observations that appear to be associated with, or to succeed, its administration. As in the case of other biologically active substances, the physiological factors of age,! sex,” and circadian, monthly, and yearly rhythms 3 can all alter its pharmacokinetics and pharmacodynamics. Unlike any other exogenous medicament, however, ascorbic acid is present in the blood and tissues under normal circumstances. In consequence, the interaction of supplementary vitamin C with intrinsic ascorbic acid complicates analysis of the effects produced by its administration, and affects the pharmacokinetics of the

tissue ascorbic acid. Ascorbic acid plays an essential role in many metabolic reactions* during which there is a rapid tissue turnover of the vitamin, with consequent potentiation or inhibition of enzymatic reactions. As a result, the metabolism of extrinsic drugs may be affected by the availability of ascorbic acid in the liver and in other tissues.’ It is therefore essential to consider not only the pharmacokinetics and pharmacodynamics of ascorbic acid itself but also the effect of its interaction with drugs administered for specific purposes, in relation to tissue ascorbic acid. Some of these aspects of the clinical pharmacology of ascorbic acid are discussed below. ASCORBIC

ACID

METABOLISM

DURING

HEALTH

Vitamin C is absorbed passively through the human buccal mucous membrane. Its absorption is dependent upon time of contact with the mucosa and on the pH of the buccal contents.° Absorption is, however, dependent upon the concentration of ascorbic acid in the tissues lining the lumen. This is shown by the fact that percentage absorption of ascorbic acid from the solution of ascorbic acid in the mouth is greater in males, in whose buccal cells resting ascorbic acid concentrations are lower than in females. Active uptake of ascorbic acid takes place from the alimentary canal.‘ Administration of single loading doses of ascorbic acid in the range of 500 to 2000 mg in normal subjects shows that uptake into the plasma reaches its peak after 4 hours (FiGuRE 1). Absorption from the alimentary canal is reduced during peptic ulceration and disease of the gastrointestinal tract.s It is known that tissue destruction asso-

355

Annals

356 Leucocyte

Ascorbic

Acid

New

York

of Sciences

Academy Plasme

( vg [10% colts)

, Ascorbic

; ° Acid. (mg. [.)

Females Males

soo mg. AA

Females

Males wooo mg.

AA

Females

3

Males

2

2000 mg. AA

1

°

Hours

After Vit. C Loading

Dose

Hours

2

After Vit.

C Looding

a4

Dose

Figure 1. Male and female leukocyte and plasma ascorbic acid concentrations during a 4-hour period after administration of single loading doses of vitamin C of 500, 1000, or 2000 mg, at zero time (the ascorbic acid blood response curve, AABRC). Means and standard deviations for each point from at least six normal subjects. (From Loh et al. By permission of Clinical Pharmacology and Therapeutics.)

ciated with disturbed function occurs during scurvy. The reduced intestinal uptake of ascorbic acid, which occurs during the development of scurvy in guinea pigs after the fall in plasma and tissue ascorbic acid levels, may result from the associated disturbances in cell morphology and malfunction.® Impaired absorption of orally administered vitamin C resulting from defective active absorption of ascorbic acid from the alimentary canal in human scurvy may account for the delay in elevation of plasma levels during repletion ?° in comparison with the rapid uptake of vitamin C observed in healthy subjects. Administration of acute loading doses of vitamin C to healthy young adults results in dose-related rises in plasma ascorbic acid during the succeeding 4 hours. Doses of 500 and 1000 mg do not cause elevation of leukocyte ascorbic acid when the initial plasma and leukocyte concentrations are 0.9 mg/100 ml and 31 pg/10* cells (Figure 2); 2000 mg causes a significant rise in leukocyte concentrations in female, but not in male, subjects.!1! The difference between the sexes when they are on an intake of 30-60 mg/day becomes more pronounced when a loading dose of vitamin C is administered that is large enough to exceed the existing metabolic demand for ascorbic acid by the tissues. This suggests that a threshold value exists for the active uptake of ascorbic acid into the leukocytes under normal circumstances in healthy human beings. It occurs at a plasma level of about 1.9 mg/100 ml in young adult females but is not

Wilson:

Clinical Pharmacological

Aspects

Sail

achieved at a level of 1.8 mg/100 ml in young adult males after 1 hour. Absorption of exogenous vitamin C into the plasma determines its metabolic availability for the tissues. When the threshold value is exceeded, ascorbic acid passes from the plasma into the leukocytes by passive diffusion and through an active absorption mechanism.!” The ascorbic acid is retained in the leukocytes as a labile storage pool. When guinea pigs or human beings are deprived of exogenous vitamin C, depletion of leukocyte ascorbic acid occurs. The labile storage. pool ultimately becomes depleted to about 21% and 5% of the initial values, respectively; the female pool always remaining larger than the male pool.!* Ascorbic acid is retained in the stable pools of the tissues in order to preserve vital metabolic functions until the final stage of vitamin C deprivation. Such stable pools exist in the leukocytes, brain, and ovaries. Ascorbic acid is found in the liver of female guinea pigs but only to a very small extent in the liver of male guinea pigs after vitamin C deprivation." The mechanism controlling uptake in the leukocytes differs in activity between males and females at values below the saturated level, but under stable metabolic conditions, and at saturation, it appears to be similar in the sexes.1® 1° Incubation of leukocytes in ascorbic acid in vitro alters the uptake capacity because leukocyte concentrations can then increase to a maximal value of about 60/70 yg/10* cells. The uptake of ascorbic acid has been studied in vitro by incubating leukocytes in 3% ascorbic acid and comparing the absorption after 2 hours with the initial resting value 12 (FIGURE 3). Uptake is significantly increased in the presence of ferrous iron.‘* It appears that an unknown plasma factor plays a role in control of the uptake mechanism in vivo.1* The in vitro

Leucocyte A.A.

‘Lt Hrs. Level

ja

3 FiGuRE 2. Leukocyte and plasma ascorbic acid concentrations 4 hours after administration of loading doses of 500 to

2000 mg vitamin C. Each point is the mean value from at least ten normal male and female subjects.

Plasma A.A. of O Hrs. Level

EB ER sR SRR 500 1000 2000

Log Dose Vit.C.

(mg)

Annals

358

New

York

Academy

of Sciences

uptake of ascorbic acid by leukocytes can be used as a method for measuring their ability actively to absorb and retain ascorbic acid for storage. This procedure provides different information from that obtained from the ascorbic acid blood response curve (AABRC), which indicates the degree of tissue metabolic demand for, and availability for storage of, ascorbic acid in the plasma.

PHYSIOLOGICAL

DESATURATION

OF

TISSUE

ASCORBIC

ACID

There is a direct correlation between leukocyte ascorbic acid concentrations and lingual ascorbic acid concentration in healthy subjects.'**° It is stated that

240 +-Passive————4tACTIVE—_41

220

5

Z

> S & _ 5

u

200 FIGURE 3. Leukocyte ascorbic acid uptake after 2 hours incubation in 3 mg % ascorbic acid. Passive uptake is significantly affected by change in pH of the me-

180 “0

dium. Active uptake is significantly increased by addition of ATP and oxygen to

“Oo

rad

the medium and by the presence of ferrous ions. 120

100

Incubation in AA 3mg % pH a a 78 7-4 alone ATP O, ATP Fe alone °,

leukocyte ascorbic acid levels provide a measure of tissue ascorbic acid,?!22 but until now the evidence for this statement has been shown to exist only in healthy subjects. Plasma responses to loading doses of ascorbic acid vary greatly between individuals, and within one individual at different times. Tissue concentrations vary in the same way. Plasma and leukocyte ascorbic acid concentrations undergo a circadian rhythm, the maximum occurring at 1200 hours and the minimum at 2400 hours.?* The degree of circadian swing varies greatly between individuals. A monthly rhythm occurs in females in association with the menstrual cycle,2 and ascorbic acid tissue concentrations decrease at ovula-

tion *° and during pregnancy.*° Leukocyte and plasma levels are normally higher in females than males.” 1°: 27.28 The levels are also high in children

Wilson:

Clinical Pharmacological

Aspects

359

below the age of 11 years.2° They are more elevated in the tissues of the newborn human fetus and still higher in utero, where they considerably exceed those of the maternal tissues.*°: 1 Values become progressively reduced as age increases.1_ When plasma and leukocyte ascorbic acid concentrations are compared between individuals, it must be appreciated that apparent tissue desaturation, as demonstrated by a low value for the leukocyte ascorbic acid, may be attributable to an exaggerated physiological fluctuation in an otherwise normal individual. Such physiological desaturation can normally be detected by examining the correlation between leukocyte and plasma concentrations.®2_ Administration of supplementary vitamin C is not required for control of the physiological desaturation in such individuals under normal circumstances.

However,

admin-

istration of supplementary vitamin C is indicated during pregnancy.

DIETARY

DESATURATION

OF TISSUE

ASCORBIC

ACID

Deprivation of the recommended dietary intake of vitamin C from males leads to a fall in plasma and leukocyte ascorbic acid concentrations *? and reduction in the body pool of ascorbic acid to 300 mg in about 90 days,** with production of a variety of scorbutic signs and symptoms. Blood ascorbic acid values are also reduced in females and scorbutic features appear,*® although scurvy occurs less commonly in females.* Provided that the gastrointestinal mucous membrane is not damaged so that active uptake of vitamin C is diminished, oral administration of vitamin C repletes the body pool on a linear basis, an intake of 66.5 mg daily producing repletion of the body pool and acceptable plasma concentrations in about three weeks.*+ The tissue saturation can subsequently be maintained by the recommended dietary intake of vitamin C in normal individuals. Reduction in the recommended dietary intake of vitamin C in normal individuals leads to dietary desaturation of tissue ascorbic acid. This can easily be treated by daily oral administration of an adequate dose of vitamin C for an appropriate period.

TISSUE

SATURATION

WITH

DIETARY

VITAMIN

C

The extent to which variations in tissue ascorbic acid are within the normal range, or are pathophysiological and thus indicative of abnormal metabolism, is unknown. Administration of supplementary vitamin C can give rise to high plasma concentrations. Raised leukocyte ascorbic acid levels are produced by passage of ascorbic acid out of the plasma, but this is limited in vivo by a saturable mechanism in the leukocytes. The highest concentration for ascorbic acid reported in the leukocytes is in the region of 60 ug/10* cells though the doses administered have ranged from 200 to 1500 mg daily.*® ** More recently, Hume and Weyers *8 reported a leukocyte concentration of 30.2 + 12.3 pg/ 10° cells on administration of 10 g daily for one week, and Wilson and Greene 1 reported a maximal concentration of 32.1 + 2.1 in males and 40.9 + 3.0 yg/ 10° cells in females after administration of a single loading dose of 2 g in normal subjects. No reports have shown that males and females differ significantly in their maximal leukocyte ascorbic acid concentrations when leukocyte saturation is achieved. It is probable that the mechanism limiting excess saturation in the leukocytes, and possibly in other tissues, is a defense mechanism that prevents

360

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the occurrence of undesirably high concentrations of ascorbic acid in the tissues. This would not prevent production of high plasma concentrations of ascorbic acid. High plasma levels could lead to elevated urinary concentrations of ascorbic acid with associated production of oxaluria and oxalate stones. There is however no evidence that side effects of this nature occur in normal individuals.*® Tissue oversaturation with ascorbic acid therefore appears not to give rise to signs of disease in previously normal individuals.

PATHOPHYSIOLOGICAL

DESATURATION

OF

TISSUE

ASCORBIC

ACID

Acute or chronic tissue desaturation can occur in pathopsysiological states when tissue demand for ascorbic acid is increased. It gives rise to a variety of signs and symptoms.’ Among other states, increased tissue demand occurs when cold symptoms, whether of viral or bacterial origin, are present,‘°** during atopic states, during neoplastic tissue growth,” and during rheumatoid arthritis.4° Demonstration of reduced plasma and often of reduced leukocyte concentrations in these disease states has been considered adequate justification for administering empirically determined doses of vitamin C. These vary in the extent to which they exceed the recommended daily intake of the vitamin. In very few of these diseases has any attempt been made to discover whether blood or tissue levels can be raised by administration of supplementary vitamin C, whether the limiting mechanism for saturation has been affected by the disease, or whether symptom intensity is correlated with dose administered or subsequent tissue levels. Such studies were advocated for the common cold.*® It appears now that they are equally applicable in the other diseases.

The

Common

Cold

It has been demonstrated that plasma, leukocyte, and tissue concentrations of ascorbic acid are significantly reduced when cold symptoms are present **: #7: #8 and that plasma and leukocyte concentrations are raised by administration of prophylactic and acute loading doses of vitamin C.°*: 48-5! There is a significant inverse correlation between plasma and leukocyte concentrations and size of the blood response to loading doses of vitamin C, and symptom intensity.®: 43, 5° The reduction in blood and tissue ascorbic acid concentrations during colds and the beneficial prophylactic and therapeutic effects achieved by administration of supplementary vitamin C indicate that ascorbic acid must be maintained in adequate concentration in the tissues for metabolic utilization during the period when cold symptoms are present, and possibly during the period of postcold tissue repair. The mechanism by which supplementary vitamin C exerts its beneficial effect on cold symptoms still remains to be elucidated. Several factors may be operative. Ascorbic acid may reduce the severity of catarrhal symptoms through

its mucolytic action in the upper respiratory tract.°2 It has been shown that supplementary vitamin C increases the pulmonary volume in humans °? and that this can increase oxygen exchange even when the upper respiratory membranes are inflamed. Although it has been stated that ascorbic acid does not have viricidal effects on strains of viruses commonly responsible for the production of cold symp-

Wilson: toms,°*

it does

have

Clinical

nonspecific

Pharmacological antibacterial,

Aspects

antifungal,

361 and

antiviral

ef-

fects.®°. °° It has recently been shown that ascorbic acid can prevent replication of RNA and DNA bacteriophages.**: %* As a consequence of the alteration in ascorbic acid metabolism in the leukocytes that occurs during the antigen antibody reaction,®’ it is probable that deficiency of ascorbic acid reduces immunological activity of the leukocytes and production of antibody, during viral attack.®°. “! An adequate concentration of ascorbic acid is necessary for the leukocytes to maintain efficient and active phagocytosis,®*: °’ which maintains the defensive reaction of the body against infection."' The production of interferon is dependent at least to some extent on the action of ascorbic acid."* It is possible that ascorbic acid plays a role in prostaglandin synthesis and by this means influences the inflammatory reaction in the upper respiratory membranes."* Destruction of the respiratory membranes occurs during colds. Formation and growth of epithelial basement membrane is diminished when the tissue concentration of ascorbic acid is reduced.®* Administration of vitamin C, by increasing the integrity and thickness of the mucous membranes, may prevent penetration and spread of the rhinovirus. Through its anabolic action on formation of intercellular cement and growth of cell membranes, administration of supplementary vitamin C enables healing to occur more rapidly, and so reduces the severity and duration of cold symptoms.

Atopic States

Loh et al.°* have demonstrated that ascorbic acid is involved in the antigenantibody reaction. This observation has been used as the basis of the Leukocyte Ascorbic Acid Direct Antigen Challenge Test (LAADACT) for atopic allergy. It depends upon measurement of differences in ascorbic acid uptake produced when sensitized leukocytes are incubated in the presence or absence of the sensitizing antigen. The leukocyte ascorbic acid concentration after incubation in ascorbic acid increased to 208% of the normal value of 35.5 + 11.4 yg/108 cells (TABLE

1).

When

the cells were

incubated

with ascorbic

acid and

1%

antigen to which the patients had been shown to be sensitive by skin tests, their concentration

of ascorbic

acid increased

only to 177%

(ascorbic

acid versus

ascorbic acid + Ag: p < 0.05). There was no alteration in uptake of ascorbic acid by leukocytes from atopic subjects when incubated with nonsensitive antigen, and the uptake of ascorbic acid by leukocytes from normal subjects was unaffected by addition of antigen to the incubation medium. On repetition of the test after successful desensitization to the specific antigens, the uptake of ascorbic acid into the leukocytes was significantly increased. After desensitization, addition of the originally sensitive antigen to the medium did not significantly affect the uptake of ascorbic acid into the leukocytes.** In the other direct challenge tests for the antigen-antibody reaction, release of histamine or other pharmacological mediators takes place following damage produced by the reaction in the leukocyte membrane. The LAADACT differs from the other reactions in that uptake of a biologically active agent is inhibited, possibly by inactivation of active absorption by the leukocytes. However, it could also be that the reduced levels found in the leukocytes after introduction of the antigen originate from increased utilization of ascorbic acid by the cells during the reaction.®* 7° Ascorbate has been shown to play an important role

Annals

362

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York

TABLE Leukocyte

Ascorsic

Acip

(AA)

of Sciences

1

Direcr

Atopic Subjects (67)

Mean SD %

Resting

AA Alone

AA+ Sensitive Antigen

3555 11.4 100

Tisai 31.4 208

62.8 28.9 177

28.2 100

65.5 232

52.9 187

Mean %

Nonsensitive Antigen (8) 34.2 SB ies) S37 100 156 156

TEST*

Normal Subjects (30)

Resting

AA Alone

AA+ Sensitive Antigen

aa 11.0 100

59.5 18.8 168

58.7 19.4 168

Atopic Subjects Before Desensitization (8)

Mean %

CHALLENGE

ANTIGEN

Atopic Subjects After Desensitization (8)

Ses 118

82.0 291

79.7 282

Atopic Subjects With

* Resting leukocyte ascorbic acid concentrations on removal

from blood; concen-

trations after incubation for 2 hours in ascorbic acid 3 mg %; or after incubation with

ascorbic acid and antigen 1%

PNU

per 100 ml mixture.

Ascorbic acid ug/10° cells.

in activating the cyclic AMP system.*! This leads to alteration in membrane permeability and gives rise to histamine release from the leukocytes.*? Ascorbic acid has a role in structural and numerical control of lysosomes, in regulating the activity of their enzymes, and in preservation of cell membranes, all of which are altered in consequence of the antigen-antibody reaction.® It therefore seems that-ascorbic acid is involved in protection against the effects of the antigen-antibody reaction rather than in impaired absorption after it has taken place.

Cancer

Tumor tissue in experimental animals has a high level of ascorbic acid during the period of rapid growth.** Human skin also has a high concentration of ascorbic acid while rapid cell proliferation is taking place during healing after burning.’ Plasma and leukocyte ascorbic acid concentrations are reduced in children with acute lymphatic leukemia.*°-** Comparison of ascorbic acid concentrations in control and leukemic children at the time of relapse demonstrated neither that reduced dietary intake, nor impaired absorption of vitamin C, nor the effect of chemotherapy were responsible for the reduced ascorbic acid concentrations in the leukocytes or plasma of the leukemic children.2® Subsequent investigations, following acute administration of loading doses of vitamin C, have demonstrated that its rate of uptake is slower and the urinary excretion of ascorbic acid is significantly less in leukemic patients (TABLE 2). Patients suffering from carcinoma of the lung also have reduced plasma and leukocyte ascorbic acid concentrations. It was found by analysis of the tumor

Wilson:

Clinical Pharmacological

Aspects

363

tissue and biopsy of metastases that the ascorbic acid content of malignant tissue is significantly higher than in adjacent normal tissue and that the urinary excretion of ascorbic acid is abnormally low after administration of loading doses.*$ These results indicate that growing tumor tissue actively takes up ascorbic acid from the plasma. Enhanced utilization of this ascorbic acid would account for the depletion of the labile stores, the stable metabolic pools in the leukocytes of the leukemic patients, and the labile stores in patients with other types of cancer. The immunocompetence of scorbutic guinea pigs is diminished, as shown by their tolerance to allografts. This change appears to be due to depletion of ascorbic acid in the lymphocytes,’® suggesting that ascorbic acid could be involved in immunological defense mechanisms in cancer. Physiological hyaluronidase inhibitor, which requires ascorbic acid for its synthesis, may also play a role in the prevention of cancer. In the absence of ascorbic acid, hyaluronidase, present in the tissues in an uninhibited state, can promote cell growth in tumor and other cell-proliferative disorders.8° It was suggested by Goth and Littinon *! that 88% of all cancers originate from organs containing less than 4.5 mg per 100 g of ascorbic acid, whereas only 12% of cancers originate from organs containing higher concentrations of ascorbic acid.

THE

INTERACTION

OF ASCORBIC

ACID

WITH

DRUGS

The interaction of ascorbic acid with the pharmacological effects of drugs can occur through the following mechanisms: 1. Through the interaction of ascorbic acid with other biologically active agents that are normally present in the tissues but may be administered as therapeutic agents. The interaction of synthetic ACTH or corticosteroids with ascorbic acid provides an example of this,*?:** and it has been shown that administration of oral contraceptives produces a significant reduction in plasma

TABLE

2

PLASMA, LEUKOCYTE, AND SKIN ASCORBIC ACID VALUES FROM NORMAL CHILDREN AND GERIATRIC SUBJECTS, AND FROM PATIENTS SUFFERING FROM LEUKEMIA AND CARCINOMA OF THE LUNG *

Ascorbic Acid Concentrations

Blood

Children Normal Leukemic Geriatric subjects Normal Lung cancer

Urine

Number

Leukocytes

Plasma

10 10

$6:42221-9= 35.9+15.9

£0:40250.20 0.95+0.25

U 1

26.02217.1" 125

0:47 =20:20 0.13

0

4 hr

8:2 52: Omen 30 2=Et 2 3.42.4 9.67.9 — 235

— 4.6

* Values for urinary excretion of ascorbic acid from normal and leukemic children at zero time and 4 hours after administration of a loading dose of 500 mg vitamin C. Leukocytes: yug/10* cells. Plasma: mg/100 ml. Urine mg/100 ml. Skin: mg/100 g. Means and SD.

364

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and leukocyte ascorbic acid concentrations that is attributable to the action of the estrogen fraction.*! The interaction of ascorbic acid with iron has been described in more detail elsewhere.*° 2. Through the effect of ascorbic acid in drug metabolism.° The change in folate metabolism, which has been described as a side effect of phenytoin administration, is primarily dependent on the initial alterations in the metabolism of ascorbic acid that determine the changes in hepatic microsomal activity.8° 3. Through alteration in tissue ascorbic acid concentrations as a result of which the pharmacological action of the drug takes place. This is exemplified by the association of leptazol-induced convulsions in guinea pigs and the concurrent fall in ascorbic acid concentrations in the midbrain. The severity of the convulsions can be reduced by prior administration of vitamin C.*? The interaction of fenfluramine with tissue ascorbic acid, as a result of which fenfluramine exerts its antiobesity effect, is discussed below. 4. Through indirect interaction between ascorbic acid and the administered drug, the extent of which is determined by differences in tissue activity during health and disease. This interaction is exemplified by the relationship between the actions of aspirin and ascorbic acid metabolism in healthy subjects and the same subjects in pathophysiological states. The factors that play a role in these interactions have already been reviewed from pharmacological® aspects. The interactions between ascorbic acid and fenfluramine, and ascorbic acid and aspirin, provide examples of drug-induced desaturation of tissue ascorbic acid.

DRUG-INDUCED

DESATURATION

Ascorbic

Acid

OF

TISSUE

ASCORBIC

ACID

and Fenfluramine

When discussing the action of ascorbic acid on the growth of guinea pigs, Evans and Hughes ** suggested that primarily it exerts a metabolic effect on tissue formation. Administration of a scorbutogenic diet to guinea pigs results in an initial gain in weight followed by a rapid loss of weight associated with a reduction in food intake.*® Females can make metabolic adjustments so that they maintain higher tissue ascorbic acid concentrations and survive the scorbutogenic diet longer than males °°! (FIGURE 4). In view of the important role played by ascorbic acid in maintenance and gain of body weight in guinea pigs, Odumosu and Wilson * studied the role of fenfluramine on weight changes and on ascorbic acid metabolism in male and female guinea pigs receiving a scorbutogenic diet. It was found that fenfluramine, 15 mg daily, produced immediate weight loss and diminished food intake in both sexes. These effects were more pronounced in the males. When guinea pigs were given the scorbutogenic diet together with vitamin C supplements of 30 mg/kg daily, the effect of fenfluramine was reversed, and slight but nonsignificant weight gain took place. The administration of fenfluramine is associated with reduced hepatic ascorbic acid levels in supplemented and scorbutic animals in comparison with those that did not receive fenfluramine. This effect was more evident in the males. Fenfluramine affected cholesterol and triglyceride metabolism. In supplemented animals hepatic cholesterol was significantly raised in comparison with nontreated controls; in the scorbutic groups, hepatic cholesterol was signifi-

Wilson:

Clinical Pharmacological

Aspects

cantly reduced when fenfluramine was administered. Fenfluramine triglyceride levels in scorbutic but had little effect in supplemented

365 reduced animals.

During fenfluramine administration, ascorbic acid catabolism is enhanced.

This gives rise to associated changes in cholesterol metabolism %* (FIGURE 5). Significant loss of weight has been reported during the development of experimental scurvy in male subjects **.°* and during the early repletion

120

no

qe ReGs >.

3

fe @ 8"ap -

PERCENT WEIGHT INITIAL

Le)

20

Roe) DAYS

hewsadershemenbersemborcosheemad 40 3 60 7 8 930 Wo

OF EXPERIMENTAL

DIET

Ficure 4. The effect of vitamin C supplementation and deficiency on the growth and survival of guinea pigs. An ascorbic-acid-free diet was administered from the first day. Male and female supplemented guinea pigs received 100 mg/kg vitamin C daily by stomach tube. Control females received 30 mg/kg daily by intraperitoneal injection. Male scorbutic guinea pigs that received the diet alone had all died by day 28. On day 24 the female guinea pigs receiving the diet alone were divided into those that showed rapid weight loss (F Diers) and those that were maintaining their weight (F Surv). F Diers had all died by day 36. F Surv continued to survive for over 100 days. The numbers of survivors at each point in time are indicated. (From Odumosu and Wilson.” By permission of Nature.)

phases.?° These findings suggest that ascorbic acid could be operative in maintenance of and changes in body weight in humans in the same way as in guinea pigs. Comparison of blood and tissue ascorbic acid levels in a group of obese subjects matched by age and sex with a group of normal subjects demonstrated that leukocyte ascorbic acid concentrations were significantly higher in the obese subjects and that their tongue test times were significantly shorter, indicating

Annals

366

New

York Academy

of Sciences

40

FEMALES 30 LIVER

20 AA

10

me

t)

30 Per

20 100g

10

= fe] DAYS

OF

DIET

Ficure 5. Hepatic ascorbic acid concentrations in female and male guinea pigs on the day preceding administration of a scorbutogenic diet FE=4 and after 24 days on the diet. On day 24, concentrations are shown in the groups receiving the diet with a supplement of 30 mg/kg vitamin C daily E==4; those receiving no supplement (___] ; those receiving supplement with 15 mg/kg fenfluramine daily [777] ; and those receiving fenfluramine with no supplement daily QM. Six guinea pigs per group. Means and standard deviations.

that the lingual ascorbic acid concentration in the obese subjects was abnormally high. In a pilot investigation to test the hypothesis that ascorbic acid could have a role in weight control, Wilson et al.°° placed two groups of obese subjects on 1000 calorie diets. Group I was put on a vitamin-C deficient diet from which all food containing vitamin C was excluded and Group 2 received a daily dietary intake of 40 mg vitamin C. Fenfluramine was administered to both groups from the second week of the dietary restriction, the dosage being increased in individual patients as required in order to maintain maximal loss (FIGURE 6). Weight fell progressively in both groups during the ten weeks of the investigation. The patients in Group 1, on the vitamin-C-deficient diet, lost more weight, and their mean

intake of fenfluramine was less, than the patients

in Group 2. Comparison of the leukocyte and plasma ascorbic acid concentra-

Wilson:

Clinical Pharmacological

Aspects

367

tions in the two groups showed that plasma and leukocyte levels diminished rapidly and remained lower in the group on the vitamin-C-deficient diet. Ascorbic

Acid

and Aspirin

It has been shown that the uptake of ascorbic acid into leukocytes obtained from heaithy young adults is inhibited when the incubation mixture contains 3.5 mg per 100 ml aspirin in addition to ascorbic acid 3 mg per 100 ml. When aspirin alone was added to the medium, ascorbic acid did not leak out of the leukocytes, indicating that aspirin does not damage the cell membrane.®® Administration of an acute loading dose of 600 mg aspirin together with 500 mg vitamin C caused a greater increase in plasma ascorbic acid than was obtained with the vitamin C alone but completely arrested its uptake into the leukocytes (FIGURE 7). This inhibitory effect on leukocyte uptake occurred even when the loading dose of vitamin C was raised to 2000 mg.°? Simultaneous administration of aspirin with the vitamin C resulted in increased urinary excretion of ascorbic acid. When 600 mg of aspirin was administered by itself every six hours daily for seven days, a significant reduction in plasma concentrations

( )= FENFLURAMINE mgfday (24) 20 GROUP

18

Ficure 6. Mean weight loss in obese patients on a 1000-calorie diet from day 0. At the end of the

second week fenfluramine

was administered in doses progressively increased to attain maximal weight loss in individual subjects. Group two received 40 mg vitamin C daily in their diet. Group one was on vitamin -C - deficient diet. Mean daily dose of fenfluramine shown.

WEIGHT LOSS (Ibs.)

ONE

(24)

14

= 12

Gacukinwe

(From Wilson et al.” By

6

permission of the /nternational Symposium on Obesity.)

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Wilson:

Clinical

Pharmacological

Aspects

369

occurred after three days. In contrast, the leukocyte ascorbic acid dropped precipitously during the first four days and then diminished gradually until the seventh day, when it had reached a value of 10 ug/10° cells. These results confirmed the earlier finding that aspirin increases the urinary excretion of ascorbic acid.°* When aspirin is administered with vitamin C, the ascorbic acid levels obtained in the plasma are elevated as a result of unimpeded and possibly enhanced intestinal absorption, by reduced uptake and storage in the leukocytes, and by renal clearance of ascorbic acid, for which there is competition by simultaneous presence of the aspirin. It is commonly recommended that aspirin be prescribed when cold symptoms are present in order to relieve the toxic symptoms. The rationale for this therapy is unknown, apart from the generalized proposal that aspirin inhibits prostaglandin synthesis and release and the consequent inflammatory reaction.2? When 600 mg aspirin is administered together with 2000 mg vitamin C three days after cold symptoms have commenced, the ascorbic acid blood response curves differ in the same subjects between the time when they are suffering from the cold symptoms and after recovery from the last of the symptoms. Three weeks after recovery the leukocyte uptake of ascorbic acid is similar to that in healthy individuals, being lower in males than in females. Administration of aspirin with the vitamin C reduces the leukocyte uptake in postcold individuals in the same way as it does in healthy individuals who have no recent history of a cold. When cold symptoms are present, this inhibitory effect of aspirin on leukocyte uptake of ascorbic acid is completely reversed. Active leukocyte uptake of ascorbic acid takes place. The concentrations of ascorbic acid in the leukocytes 2 and 4 hours after administration of the vitamin C with the aspirin are significantly higher than the resting level at zero time (FIGURE 8). The plasma ascorbic acid levels tend to be higher in healthy individuals than in subjects who have had a cold or are recovering from it when aspirin is administered with vitamin C. The higher plasma levels can be attributed to the diminished uptake into the leukocytes and the lower metabolic utilization of ascorbic acid in the healthy subjects.113 During colds, absorption of ascorbic acid achieves the normal saturated level when the leukocytes are incubated in a medium containing ascorbic acid. When aspirin is added to the medium with the ascorbic acid, uptake of ascorbic acid is increased in leukocytes taken from the subjects while suffering from their colds. The increased uptake had disappeared three weeks after recovery from the last cold symptom (TABLE 3). The reduced uptake in the presence of aspirin, as occurs in normal subjects,”® had not then become apparent. Absorption of ascorbic acid by incubated leukocytes taken from atopic subjects is reduced in the presence of the sensitizing antigen. However, when aspirin is added to the medium, uptake of ascorbic acid is restored to normal 1°° (TABLE 3). These in vitro studies confirm the in vivo findings that ascorbic acid metabolism can be profoundly altered by the pharmacological action of aspirin. The quality of this effect of aspirin is affected by the state of health of the individual. During health, aspirin inhibits tissue uptake of ascorbic acid. It also fails to exert any antipyretic action. When the tissues are in a state of immunological reaction

or defensive

response

against

viral

attack,

both

of which

result

in

inflammatory reaction, aspirin exerts the reverse effect. It promotes the uptake of ascorbic acid by the tissues, and it also exerts an antipyretic action when fever is present. It is possible that this change in the action of aspirin may be associated with the tissue availability of ascorbic acid, which could control prostaglandin release.®°

Academy

York

New

Annals

370

of Sciences

150 INITIAL 100. LEUC. AA 50 M

F

couob ™ 400.”

M -

a

POST

F COLD ee

mM

F

HEALTHY we ee ee

INITIAL 300 PLASMA 200 AA

100. |

M cotuob

F

M POST

F COLD

M

F

HEALTHY

FicurE 8. Values for leukocyte and plasma ascorbic acid as percentage of the concentrations at zero time, 4 hours after administration of a loading dose of 2000 mg vitamin C, or 2000 mg vitamin C with 600 mg asprin. The loading doses were administered to male and female subjects during and 3 weeks after recovery from cold symptoms. These values are compared with the values in healthy subjects without a recent history of respiratory symptoms. 2000 mg vitamin C alone [_}; 2000 mg vitamin C with 600 mg aspirin Ml. Significance of differences from resting values are indicated.

CONCLUSIONS

Several factors affect ascorbic acid storage and metabolism in health and disease. The national recommended daily dietary intakes do not maintain a state of tissue saturation under normal circumstances.!°! The present dietary intake for Americans of 45 mg daily was recommended on the basis of the intake required to restore and maintain the ascorbic acid body pool of the 12 volunteers described in two studies by Hodges et al.‘®: 1°? The subjects in these studies consisted of two male groups having age ranges of 33 to 44, and 26 to 52 years, respectively. They were stated to be in good health, although two of the subjects in the first study had minor electrocardiographic abnormalities. They were all heavy smokers.'!* It has been established that leukocyte and plasma ascorbic acid concentrations are significantly diminished in smokers in comparison with nonsmokers.!°*-!°° They are also significantly and greatly reduced in patients with gross electrocardiographic abnormalities.1°’ The claim that the ascorbic acid metabolism and hence the ascorbic acid pools of the volunteers described by Hodges et al.** are representative of normal male subjects may therefore be open to doubt.

Wilson:

Clinical Pharmacological

Aspects

MAE

It has been demonstrated that ascorbic acid metabolism differs in many ways between males and females.2 Tissue ascorbic acid concentrations in plasma and leukocyte progressively diminish from the time of intrauterine life until old age. The studies of Hodges et al.*° indicate that the size of the body pool of ascorbic acid is related to plasma ascorbate values, and other studies indicate that leukocyte ascorbic acid concentrations are representative of tissue ascorbic acid saturation.*!: 2* To claim therefore that a dietary intake of vitamin C, determined from studies of the ascorbic acid pool, in 12 male subjects who were heavy smokers and two of whom had minor electrocardiographic abnormalities is applicable as a recommended dietary intake of vitamin C to the whole population does not appear to be justifiable. On the basis of the experimental results it is justifiable to apply it only to men within this age range. In such subjects it has been shown that ascorbic acid metabolism in relation to hemoglobin formation is at an optimal level.1°8 Administration of supplementary vitamin C can lead to a state of tissue saturation, but it is very difficult, if not impossible, to produce a state of hypersaturation in healthy subjects owing to an inbuilt mechanism that limits leukocyte saturation. Dietary desaturation of tissue ascorbic acid is easily produced by acute or chronic limitation of dietary intake of vitamin C. Tissue resaturation is easily achieved in normal males by oral administration of vitamin C for 3-4 weeks at the recommended dietary level. Larger intakes may be required in males who have pathophysiological features. It has been suggested that

TABLE

3

LEUKOCYTE AscorBic Acip (AA) UPTAKE IN THE PRESENCE OF ASPIRIN IN MALE AND FEMALE SUBJECTS DURING AND AFTER RECOVERY FROM COLD SYMPTOMS, AND UPTAKE INTO LEUKOCYTES FROM ATOPIC SUBJECTS *

Males

Females

Incubation in:

Resting Value

AA Alone

Incubation in:

AA+ Aspirin

Resting Value

AA Alone

AA+ Aspirin

62.7 280

84.0 2/5)

63.9 195

66.1 202

Leukocytes removed during colds

ug/ 10° cells Percent

223 100

ug/ 10° cells Percent

31.8 100

60.6 271

Gfalep? 319

22.4 100

Leukocytes removed after recovery from colds

64.1 201

65.2 205

Saul 100 Atopic subjects Incubation in:

ug/10% cells Percent

Resting Value

AA Alone

AA-+ Sensitive Antigen

AA+ Antigen-+ Aspirin

3555 100

TH 208

62.8 ay)

72.0 203

* Acetylsalicylic acid 3.5 mg % was added to the incubation medium containing 3 mg % ascorbic acid in a separate experiment in each group of subjects.

372

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York Academy

of Sciences

women who develop scurvy may have an abnormal metabolic pathway for ascorbic acid.1°° In pathophysiological conditions, acute or chronic desaturation occurs because of increased tissue utilization of ascorbic acid in response to increased demand for the vitamin. Desaturation is treated, as in other replacement therapy, by administration of adequate doses of supplementary vitamin C. The size of dose and duration of administration is determined by the type and nature of the desaturation. In these circumstances, replacement therapy not only must be adequate to produce resaturation of the tissues; it also has to compensate for the enhanced metabolic demand for ascorbic acid by the tissues for as long as it exists. Drug-induced desaturation of tissue ascorbic acid may be incidental to another pharmacological effect of the drug. As such, it is classified as an undesirable side effect of the drug. This is exemplified by the ascorbic acid desaturation produced by smoking and by the effect of aspirin on ascorbic acid metabolism in normal people. It is noteworthy that tetracycline administration is also associated with desaturation of leukocyte ascorbic acid and increased urinary excretion of ascorbic acid in normal men.1!° Drug-induced desaturation of tissue ascorbic acid may be necessary in order that the pharmacological action of the drug can take place. This is exemplified by the action of fenfluramine in obese subjects whose tissue ascorbic acid concentrations are elevated. Fenfluramine-induced

ascorbic acid desaturation

also occurs

in normal

subjects, in

whom a small loss of weight may take place during short periods of fenfluramine administration.®° The requirements of vitamin C vary according to the physiological or pathophysiological requirements of the tissues. The effect of various drugs on tissue saturation with ascorbic acid is also affected by the state of the tissues. In pathophysiological conditions such as the common cold, tissue requirements for ascorbic acid are enhanced. Administration of aspirin while cold symptoms are present promotes the uptake of ascorbic acid by the leukocytes and tissues. This action of aspirin is completely contrary to the effect of the drug on ascorbic acid metabolism in healthy individuals. The effect of aspirin on ascorbic acid metabolism in disease may be mediated through the action of ascorbic acid on prostaglandin metabolism,*° just as the antipyretic action of aspirin is mediated through the prostaglandin mechanism. When antibiotics are given together with supplementary vitamin C in the treatment of Pseudomonas infection, the vitamin potentiates the action of the antibiotics so that smaller doses are required for effective therapy.‘ In this pathophysiological condition also, it appears that the action of the drug may be influenced in diseased human beings by the state of their ascorbic acid metabolism. Ascorbic acid is a very labile compound that plays an important role in tissue metabolism and defense mechanisms in physiological and pathophysiological conditions. A defense mechanism comes into operation in the leukocytes and probably in other tissues that prevents tissue oversaturation when supplementary vitamin C is administered to normal individuals. The increased requirements during pathophysiological states produce alterations in the metabolism, and possibly the functions, of ascorbic acid. Supplementary vitamin C is required in appropriate dosage when tissue desaturation occurs. Administration of drugs can influence the tissue requirements of ascorbic acid in health and disease. Tissue oversaturation does not occur in pathophysiological or during druginduced desaturation. Experiments designed to determine the normal dietary

Wilson:

Clinical

Pharmacological

Aspects

373

requirements of the vitamin in one small sample of the human population cannot be applied indiscriminately to the remainder of the population, young or old, smokers or nonsmokers, male or female, pregnant or nonpregnant, whether taking contraceptives or not, healthy or pathophysiological. It must also be taken into account that most of the population are taking drugs of one kind or another.

ACKNOWLEDGMENTS

The author would like to express his thanks to his collaborators in the University of Dublin, in the pharmaceutical industry, and in the hospitals who have given their assistance and advice during the last ten years. Especially he would like to thank Dr. H. S. Loh, Dr. Abi Odumosu,

Dr. K. Watters, Dr. Ann Mullen, Mr. Maurice Greene, and Dr. S. Kakar, and the undergraduate students who have assisted in and volunteered to take part in the investigations. He would like to thank the pharmaceutical industry for supplying vitamin C and drugs, and for its financial assistance. He would also like to express grateful thanks to Mr. Foran, Mr. Molloy, and Mr. Dempsey for their technical assistance and Miss Collender for her secretarial assistance.

REFERENCES

1.

3.

4. 5. 6. 7.

8. 9. 0.

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20. 21. 22. 23.

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WILSON, C. W. M. 1974. Proceedings of the International Chronobiology Society, Little Rock, Arkansas. Reprinted from: Chronobiology. L. E. Scheving & F. Halberg, Eds. : 249-255. Igaku Shoin, Ltd. Tokyo, Japan. Enpoar, J. A. 1970. Nature 227: 24. WiLson, C. W. M. 1974. Proc. Nutr. Soc. 33: 231. Opumosu, A. & C. W. M. WILson. 1971. Nutr. Soc. 30: 81A. MAyYerRSOMN, M. 1972. Eur. J. Pharmacol. 19: 140. WixLson, C. W. M. 1974. Practitioner 212: 481. Opumosu, A. & C. W. M. Witson. 1970. Brit. J. Pharmacol. 40: 171P. Hopces, R. E., J. Hoop, J. E. CANHAM, H. E. SAUBERLICH & E. M. BAKER. 1971. Amer. J. Clin. Nutr. 24: 432. Wrtson, C. W. M. & M. Greene. 1974. Brit. J. Nutr. 33: 109A. Lou, H. S. & C. W. M. Witson. 1970. Brit. J. Pharmacol. 40: 169P. Witson, C. W. M. 1973. Vitamins. Editiones Roche 3: 75. Hoffmann-La Roche, Basle, Switzerland. Opumosu, A. & C. W. M. WiLson. 1973. Nature (London), 242: 519. Lou, H. S. & C. W. M. Witson. 1971. Brit. Med. J. 3: 733. Lou, H2S) 1972 Int_I. Vit. Nutr. Res. 42: 86. Lou, H. S. & C. W. M. Witson. 1970. Brit. J. Pharmacol. 40: 566P. MOHANRAM, M. & S. G. SRIKANTIA. 1967. Clin. Sci. 32: 215. CHERASKIN, E., W. M. Rincsporr & G. Exr-Asniry. 1964. Int. J. Vit. Res. 34: Bile Witson, C. W. M. & J. P. Kevany. 1972. Brit. J. Prev. Soc. Med. 26: 53. GrirFitus, L. L., J. C. BROCKLEHURST, D. L. Scott & J. BLACKLEY. 1967. 9: ile ANpDREWwS, J. & M. Brook. 1968. Geront. Clin. 10: 128. Lou, H. S. & C. W. M. WILson. 1973. Int. J. Vit. Nutr. Res. 43: 355.

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30. Sill B2h 33) 34. 353 36. Bie 38. 32). 40. 41.

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Lon, H. S. & C. W. M. WiLson. 1971. Lancet 1: 110. WILSON, C. W. M. & H. S. Lou. 1973. Lancet 2: 859. Mason, M. & J. M. Rivers. 1971. Amer. J. Obstet. Gynec. 109: 960. Dopps, M. L. 1959. In Yearbook of Agriculture. U.S. Department of Agriculture. Washington, D.C. Dopps, M. L. 1969. J. Amer. Diet. Ass. 54; 32. Kakar, S. C., C. W. M. WILSON & J. M. Bett. 1974. Irish J. Med. Sci. In

press. ADLARD, B. P. F., S. W. DE Sauza & SASAN Moon. Yew, M.S. This monograph.

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WILson, C. W. M. & H. S. Lou. 1974. Eur. J. Clin. Pharmacol. 7: 421. BarRTLEY, W. H., A. Kress & J. R. P. O'BRIEN. 1953. Medical Research Council Special Report Series No. 280. Her Majesty’s Stationery Office. London, England. BAKER, E. M., R. E. Hopces, J. Hoop, H. E. SAUBERLICH, S. C. MARCH & J. E. CANHAM. 1971. Amer. J. Clin. Nutrit. 24: 444.

WALKER, A.

1968.

Brit. J. Derm. 80: 625.

BROCKLEHURST, J. C., L. L. GRIFFITHS, G. F. TAYLor, J. Marks, D. L. Scott & JACQUELINE BLACKLEY. 1968. Geront. Clin. 10: 309. MaseEK, Von J. & F. Hrupa. 1964. Int. Z. Vitaminforsch. 34: 39. Hume_, R. & E. WEYERS. 1973. Scot. Med. J. 18: 3. WILSON, C. W. M. 1974. In Vitamin C. G. G. Birch & K. Parker, Eds. : 203-

220. Applied Science Publishers, Ltd. London, England. WILSon, C. W. M. & H. S. Lon. 1973. Brit. Med. J. 4: 166. WILSson, C. W. M., H. S. Lon & F. G. Foster. 1973. 6: 26. WILSON, C. W. M., H. S. Low & F. G. Foster. 1973. 6: 196. WILSON, C. W. M., H. S. LoH & M. GREENE. 1973. bridge, Comm. 49. Brit. J. Nutr. In press. WILSson, C. W. M. 1974. Clinical Allergy 4: 221. SaHup, M. A. & R. J. COHEN. 1971. Lancet 1: 937.

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on

MaAsEK, J., F. HRuBA, M. NeRADILOVA & S, Hema. 1974. Acta Vit. Enzymol. (Milano) 28: 85. Lon, H. S. & C. W. M. WiLson. 1971. Int. J. Vit. Nutr. Res. 41: 445. CouLeHAN, J. L., K. S. REISINGER, K. D. RoGers & D. W. BrapLey. 1974. New Eng. J. Med. 290: 6. ANDERSON, T. W., G. SuRANY!I & G. H. BEATON. 1974. C.M.A. Journal 111:

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52, ois) 54. sy 56. eWfp 58. Sey

60. 61.

Briccs, M. H.

1973.

Lancet 2: 677.

ZuskIN, E., A. J. Lewis & A. Bounuys. 1973, J. Allergy Clin. Immunol. 51: 218. WALKER, GEORGINA H., M. L. BYNoE & D. A. J. TyrRELL. 1967. Brit. Med. J. 1: 603. Ericsson, Y. & H. LuNpBeck. 1955. Acta Path. Microbiol. Scand. 37: 493.

KLENNER, F. R. 1949. Southern Medicine and Surgery 111: 209. Morata, A., K, KitaGAwa & R. SARUNO. 1971. Agr. Biol. Chem. 36: 1065. Murata,

A., K. KiraGAwa,

H. INMARU

& R. SARUNO.

1972.

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36: 2597. Lou, H. S., K. Watters & C. W. M. WiLson. 1973a. Irish J. Med. Sci. 142: alll: Cate, T. R., R. B. Couch & K. M. JoHNSON. 1964. J. Clin. Invest. 43: 56. PEREIRA, M. S., P. CHAKRAVERTY, G. C. ScHILD, SOWELLE. 1972. Brit. Med. J. 4: 701.

M.

T. CoLEMAN

& W.

R.

Wilson:

Clinical Pharmacological

Aspects

ais)

62. 63. 64.

COLLINGHAM, E. & C. A. Mitts. 1943. J. Immunol. 47: 443. NUNGESTER, W. J. & A. M. Ames. 1948. J. Infect. Dis. 83: 50. STEWART, F. S. 1968. Bacteriology and Immunology for Students of Medicine.

65.

: 146. Bailliere, Tindal & Castle Ltd. London, England. Lewin, S. 1974. In Vitamin C. G. G. Birch & K. Parker, Eds.

66. 67. 68.

Applied Science Publishers, Ltd. London, England. SHARMA, S. C., D. M. PucH & C. W. M. Witson. 1974. 53: 469. Priest, R. E.

1970.

: 221-251.

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Nature 225: 744.

69.

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70. Ake

ther. 27: 249. Quiz, P. G. & J. G. Hirscu. 1964. J. Exper. Med. 120: 149. Lewin, S. 1973. U.S. Army Research and Development

Technical Report ERO-3-73

1D2 73% 74.

Tey, 76. ge 78.

i): 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. oi 7, Wah, 94. 99:

96. Me 98. 99: 100. 101.

: 15-19.

Group (Europe) European Research Office, London.

ASSEM, E. S. K. 1973. Proc. Roy. Soc. Med., 66(12): 1191. MusuLIN, R. R., ETHEL SILVERBLATT, C. G. KING & G. E. Woopwarp. 1936. Amer. J. Cancer 27: 707. BaRTON, G. M. G., J. F. Laine & D. Barisonr. 1972. Int. J. Vit. Nutr. Res. 42: 524. WALpo, A. L. & R. E. ZrprF. 1955. Cancer 8: 187. Lioyp, J. V., P. S. Davis, H. EMERY & H. LANDER. 1972. J. Clin. Path. 25: 478. Kakar, S. C. & C. W. M. WiLson. 197. Brit. J. Nutr. Proc. 33: 110A. Kaxkar, S. C., C. W. M. WiLson, M. GREENE & J. M. BELL. 1974. Unpublished observations. KALDEN, J. A. & E. A. GuTHy. 1972. Eur. Surg. Res. 4: 114. CAMERON, E. & L. PAULING. 1973. Oncology 27: 181. Gotu, A. & I. LITrMAN. 1948. Cancer Res. 8: 349. Opumosu, A. & C. W. M. WILSON. 1970. Brit. J. Pharmacol. 40: 548P. Lon, H. S. & C. W. M. WiLson. 1972. 5th International Congress on Pharmacology, volunteer paper 847: 142. Briccs, M. & M. Briccs. 1973. Lancet 1: 998. Lou, H. S. & C. W. M. WiLson. 1971. Lancet 2: 768. Lou, H. S. 1973. European Nutr. Conf. Cambridge. Comm. 61. Brit. Nutr. In press. Opumosu, A. & C. W. M. WILSON. 1974. Brit. J. Pharmacol. 50: 471P. Evans, J. R. & R. E. HuGcHes. 1963. Brit. J. Nutr. 17: 251. Opumosu, A. & C. W. M. WiLson. 1970. Brit. J. Pharmacol. 40: 171P. Opumosu, A. & C. W. M. Witson. 1973. Nature 242: 519. Opumosu,

A. & C. W.

M. Witson.

1971.

Proc.

J.

Nutr. Soc. 32(2).

Opumosu, A. & C. W. M. Witson. 1973. European Nutr. Conf. Cambridge, Comm. 62. Brit. J. Nutr. In press. Opumos, A. & C. W. M. Witson. 1973. Brit. J. Pharmacol. 48: 326P. CRANDON, J. H., C. C. LuNp & D. B. Diti. 1940. 223: 353. WILSON, C. W. M., M. GREENE, A. MULLEN & A. OpuMosu. 1974. International Symposium on Obesity, London. In press. Lon, H. S., K. J. Watters & C. W. M. Witson. 1974. J. Clin. Pharmacol. 13: 480.

Lou, H. S. & C. W. M. Witson. 1974. J. Clin. Pharmacol. In press. DaniEts, A. L. & G. J. EVERSON. 1936. Proc. Soc. Exp. Biol. 35: 20. Vang, J. R. 1971. Nature New Biol. 231: 232. GREENE, M. & C. W. M. WiLson.

1975.

Unpublished observation.

Reports on Public Health and Medical Subjects No. Stationery Office.

London, England.

120.

1969.

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376 102. 103. 104. 105. 106.

107. 108. 109. 110. 111. 112. 113.

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Hopces, R. E., E. M. Baker, J. Hoop, H. E. SAuBERLICH & S. M. MARCH. 1969. Amer. J. Clin. Nutr. 22: 535. CaLper, J. H., R. C. Curtis & H. Forest. 1963. Lancet 1: 556. Brook, M. & J. J. Grimsuaw. 1968. Amer. J. Clin. Nutr. 21: 1254. PELLETIER, O. 1968. Amer. J. Clin. Nutr. 21: 1259. Burr, M. L., P. C. ELwoop, D. J. Hore, R. J. Hurtey & R. E. HUGHEs. 1974. Amer. J. Clin. Nutr. 27: 144.

Hume, R., E. Weyers, T. RAWAN, D. S. Rew & W. S. Hirts. 1972. Heart J. 34: 238. Lou, H. S. & C. W. M. Witson. 1971. Int. J. Vit. Nutr. Res. 41: 258. Mirra, M. L. 1969. Acta Geront. Geriat. Belg. 7: 65.

Brit.

Wrnpsor, A. C. M., C. B. Hopss, D. A. TrEBy & R. A. Cowper. 1972. 1: 214. Rawat, B. D., G. McKay & M. I. BLACKHALL. 1974. Med. J. Aust. 1: 169. Hopces, R. E. 1974. Personal communication.

GREENE, M. & C. W. M. Witson.

1975.

Brit. J. Pharmacol.

In press.

DISCUSSION

Dr. R. E. Hopces: I do not see the value of very high concentrations of ascorbic acid in tissues unless they can be shown to be necessary for a certain purpose. In the case of the leukocyte concentrations we have seen that there seems to be no advantage and perhaps a slight disadvantage. Dr. WILSON: I am sure you are correct; however, when you have a cold the concentration falls significantly. Dr. M. WINICK: I am concerned about the interpretation of differences in tissue levels between leukocytes as differences in uptake. All one can say is that the tissue level is changed, but certainly one cannot say it is due to a difference in uptake. It may very well be due to an increased rate in turnover, so all that has been measured, as I understand it, is the amount of vitamin C in the plasma and the amount of vitamin C in the leukocytes. Would you agree with that? Dr. WILSON: Not completely. If you put an animal or a human being on a diet deficient in vitamin C, you get a significant reduction of leukocyte levels. The only way this can be explained is that there is a leukocyte store of ascorbic acid which spills out as the tissues are depleted. Dr. W. B. SMITH: I was intrigued by a comment you made about the incidence of cancer in certain organs and I just wanted to respond. I think the organs that have a high affinity for ascorbic acid include the choroid plexus, cerebral cortex, the lens, leukocytes, and kidney, and the adrenals. The most common form of cancer in childhood is that of the brain, followed by leukemia, loci that have a very high affinity for ascorbic acid. In young children, Wilms’ tumor of the kidneys and neuroblastoma predominantly arising from the adrenals are very common tumors.

SOME ASPECTS OF CURRENT VITAMIN C USAGE: DIMINISHED HIGH-ALTITUDE RESISTANCE FOLLOWING OVERDOSAGE * G. N. Schrauzer,

D. Ishmael,

and G. W. Kiefer

Department of Chemistry University of California at San Diego, Revelle College La Jolla, California 92037

INTRODUCTION

During the past four years L-ascorbic acid (vitamin C) has become the most heavily consumed vitamin, following revivals of the belief in its prophylactic action on various diseases. Although vitamin C overdosage is considered to be essentially harmless, it is nevertheless necessary to investigate all physiological and pharmacological aspects of this abuse. A previous study from our laboratory ' suggested that the regular ingestion of gram amounts of ascorbic acid is without merit. Once the body stores for the vitamin are saturated, the rate of renal excretion and of other disposal mechanisms is accelerated in response to the continued oversupply. In accord with the findings of Masek and Hruba,” * ascorbic acid blood or serum levels begin to decline, and this tendency cannot be offset by a further increase of the dosage. However, not even a short-term ingestion of unphysiological quantities of ascorbic acid can be recommended for healthy individuals, if only because of the established absorption and secretion behavior. Moreover, the saturation of the body stores may actually be undesirable, since vitamin C, a powerful reducing agent in itself and a cofactor of many oxidases, may unduly raise the oxygen requirements of cells and tissues. Prior to World War I, vitamin C in moderate doses was recommended as a means to temporarily increase the “high-altitude resistance” of pilots, airplane passengers

and

mountaineers.'

However,

Pfannenstiel®:°

and

Doerholt*

also

reported that larger doses of vitamin C actually diminish the high-altitude resistance. Thus, rabbits that had been given large amounts of sodium ascorbate intravenously (100 mg each day for four days, an additional 200 mg 30 minutes before the experiment) developed convulsions typical of hypoxia with unprecedented violence in low-pressure chambers under conditions that were well tolerated by untreated animals. The same effect was in turn demonstrated with human volunteers. Pfannenstiel and Doerholt considered the diminished highaltitude resistance as a symptom of a genuine hypervitaminosis C. Since their observations have apparently not received much attention, however, we have repeated some of their experiments in a brief study and report our findings in the following.

* This work was supported by Grant GP 28458X of the National Science Foundation.

=e!

378

Annals

New

York

Academy

EXPERIMENTAL High-Altitude

of Sciences

METHODS

Physiology

Tests

The high-altitude physiology tests were conducted by use of a standard low (LPC) of type 9A9 (located at Miramar Naval Air Station, San Diego, California) under expert medical and technical supervision. The LPC is essentially a large vacuum chamber with airtight doors and seals. An external vacuum system allows the removal or pressurizing of the air inside the chamber under controlled conditions and by observers in an outside compartment. The simulated ascents and descents were made at rates corresponding to 5000 feet/minute. All experiments were conducted at a pressure corresponding to an altitude of 25,000 feet above sea level. Two subjects and one aide, all of whom were equipped with oxygen masks, entered the LPC. Once the desired pressure was reached, both subjects were asked to remove their oxygen masks and to write down numbers in descending order, beginning with 1,000. The time was recorded until the subjects became unable to write and were again given their oxygen masks. pressure chamber

Testing Procedure

and

Vitamin

C Administration

Our experimental subjects consisted of 10 volunteers (9 males and 1 female) of average age of 29 + 6 years. All subjects were given one LPC test prior to receiving vitamin C. Three subjects with the lowest initial high-altitude resistance were selected as controls and received a placebo (tablets of ‘“Health-rite Chewable Honeyed Proteins”). A study group of 4 individuals were asked to ingest 2 g of ascorbic acid per day (4 single doses of 500 mg) for 6 days. The remaining 3 subjects, those who showed the greatest high-altitude resistance during the control LPC test, were given similarly 3 g of ascorbic acid per day for six days. The ascorbic acid was administered in form of 250- and 500-mg tablets

(manufactured

by Stayner),

some

of which

were

orange-flavored

to

conceal their identity. A second LPC test was run after 6 days of vitamin C administration, at which time the regimen was stopped. A third LPC test was given two weeks after test 2. The results of all tests are summarized in TABLE 1.

RESULTS

AND

DISCUSSION

The results of the LPC experiments given in TABLE 1 are represented graphically in FiGure |. It may be seen that the oral administration of 2 g of ascorbic acid per day for 6 days does not significantly alter the high-altitude resistance. However, all 3 subjects receiving 3 g of ascorbic acid per day for 6 days exhibited a significant loss of high-altitude resistance (p < 0.05), which returned to normal within 2 weeks after termination of the regimen. We have thus confirmed that high oral doses of vitamin C indeed diminish the highaltitude resistance of normal subjects. The ingestion of large amounts of ascorbic acid may thus increase the risk of persons working under conditions in which the oxygen supply may suddenly or temporarily become limiting. Vitamin C overdosage would also seem to be contraindicated in patients suffering from diseases accompanied by actual or potential hypoxia. Further work is

Schrauzer

et al.: High-Altitude

TABLE

OBSERVED

LPC

RESIDENCE

OF

Subject No.

25,000

TIMES

FEET

Resistance

1

AT A SIMULATED

ABOVE

ALTITUDE

SEA-LEVEL

Observed Times (min) During Experiments 2D 3

1

25

2.05)

2.07

2

B21

2.52

2.02

4

2.34

3.45

2.60

5) 6

4.09 4.21

4.76 4.12

4.45 4.52

il

3.83

4.10

4.06

8

(63925)

4.31

9.10

3

3.30

9 10

3.50

6.34 432

5

Subject No’s

l

2

4.24 4.15

379

Ascorbic Acid Dosage

None

3.80

tice

2 grams/day for 6 days

6.03 5.23

epstonis (day) for 6 days

3

4

3 2 |

-——— Controls ——w4

Figure

1. Apparent

“high-altitude

resistance” of experimental subjects in LPC tests. Subjects 1-3 received a placebo, subjects 4-7, 2 grams/day, and subjects 8-10, 3 grams/day, of ascorbic acid, for 6 days prior LPCtest 2. Shaded areas indicate the experiments after the ascorbic-acidloading test.

(minutes) TIME

-+-———3g /day ———+I

380

Annals

New

York Academy

of Sciences

required to determine whether large doses of vitamin C interfere with the oxygen economy under normal conditions of life, although this may probably be considered unlikely. However, the observed diminished resistance of vitaminC-saturated subjects to hypoxic stress in itself provides one additional reason to regard current * concepts concerning the alleged beneficial effect of massive amounts of ascorbic acid with caution. On the other hand, the apparent positive effect of ascorbic acid in moderate amounts on the high-altitude resistance is not questioned by our experiments, although the mechanism of this action of the vitamin is obscure. Exposed to diminished oxygen supply at high altitudes, the body first compensates for the lack of oxygen by an increase of the CO, elimination.? Ascorbic acid could change the acid-base balance temporarily and thus assist in this adaptation process. ACKNOWLEDGMENTS

We wish to thank CDR Martin Passaglia, Jr., Aerospace Physiology Training Center, Medical Dept., Miramar NAS, San Diego, California, and his technical staff for their assistance and participation in this study. REFERENCES

1. ScHRAUZER,

2.

3.

4. 5. 6.

7.

8. 9.

G. N. & W. J. RHEAD.

1973.

Ascorbic

acid abuse:

effects of long-

term ingestion of excessive amounts on blood levels and urinary excretion. Intern. J. Vitamin Nutrition Res. 43: 201. MASsEK, J. & F. Hruspa. 1958. Zur Frage der Vitamin C-Bedarfsnormen. Ernahrungsforschung 3: 425.

MAseK, J. & F. Hrupa. 1964. Uber die Beziehungen zwischen der Saturation Serums und der Leukozyten mit Vitamin C. Intern. J. Vitamin Nutr. Res. 39. PEDERSEN, J. M. 1941. Ascorbic acid and resistance to low oxygen tension. ture 148: 84. PFANNENSTIEL, W.. 1938. Tierversuche iiber die Vitaminbeeinflussbarkeit

des 34: Nader

Hohenfestigkeit. Luftfahrtmediaz. Abhandl. 2(3/4): 234. LANG, K. 1965. Wirkungen sehr hoher Ascorbinsaure-Dosen Wissenschaft. Ver6ffentlich. Deutsch. Gesellschaft Ernaihrung 14: 149 (see footnote by W. Pfan-

nenstiel on p. 155). DOERHOLT, G. 1938. Tierexperimentelle Untersuchungen iiber den Einfluss des Vitamin C auf die HGhenfestigkeit. Luftfahrtmediz. Abhandl. 2(3/4): 240. Yew, Man-Li S. 1973. “Recommended daily allowances” for vitamin C. Proc. Nat. Acad. Sci. U.S. 70: 969. SHEPHARD, R. J. 1973. The athlete at high altitude. Can. Med. Assoc. J. 109: 207.

DISCUSSION

Dr. R. G. BROWN (University of Guelph, Guelph, Ontario, Canada): What were the blood gas concentrations in your high-altitude-exposed subjects? Did you do any blood chemistry on vitamin C levels in either leucocytes and plasma?

Schrauzer

et al.: High-Altitude

Resistance

381

Dr. SCHRAUZER: We took samples before the test which showed a normal level of ascorbic acid. Our 2-gram subject and our 3-gram subject were tested after 6 days and we found normal saturation levels in the 3-gram subject and about 60% saturation in the 2-gram subject. Dr. W. H. CHASEN (Veterans Administration Hospital, Boston, Mass.): Did you find any increases in uric acid with the high doses of vitamin C or any other abnormal chemical changes, such as altered cholesterol levels? Dr. SCHRAUZER: It was not possible to test this with our volunteers. Dr. M. Winick: I would like to pose a question about the human fetus and the newborn baby, both of which grow under reduced oxygen tension. Can you speculate on what high doses might do in that situation? Dr. SCHRAUZER: I would prefer not to. Because of the absorption-excretion characteristics, I do not think that we should recommend people take 3 grams of ascorbic acid. It is well known that they do not absorb it. I think 1 gram is enough. Ms. E. BARRETT

(Interesting Medicine, New

York, N.Y.):

If you compare

those patients who were on ascorbic acid to the controls, the controls go from slightly below 3 minutes to slightly above 4, and the controls for the people on 2 grams go higher, almost above 5 minutes. Yet, the people with the 3 to 3.5 gram dose, all stay at 442 minutes. So, in actuality the people on 3.5 grams (a) were stable and (b) did maintain a perfectly normal level as compared to the controls. Dr. SCHRAUZER: We selected for the 3-gram administration those who showed the most altitude resistance and they dropped to the value that you mentioned. We did this in order to see the effect at its largest. It is not known why there is such a vast individual variability in high altitude resistance. There are many other factors that need to be studied in this field. Ms. BARRETT: Then you really selected out a special group of patients. Dr. SCHRAUZER: Not really. My purpose in this study was to test Pfannenstiel’s results. In 1938, Pfannenstiel used several hundred volunteers and we confirmed his observations.

RELATIONSHIPS OF PROTEIN AND MINERAL INTAKE TO Lt-ASCORBIC ACID METABOLISM, INCLUDING CONSIDERATIONS OF SOME DIRECTLY RELATED HORMONES * G. C. Chatterjee, P. K. Majumder, S. K. Banerjee, R. K. Roy, B. Ray, and D. Rudrapal Department University

of Biochemistry College

Calcutta

700019,

of Science India

After the elucidation of the pathway of L-ascorbic acid metabolism in animals 1° investigations on the regulation of enzymes of the uronic acid pathway were started, mostly in relation to feedback control, hormonal regulations, and regulation by administration of drugs,1”-'* such as phenobarbital and cincophen. However, no systematic study in relation to the regulation of the enzymes of L-ascorbic acid metabolism, which is a part of the uronic acid pathway, has been carried out. It is known that the enzymes of this pathway are widely distributed. Some of the enzymes, such as UDP-glucuronyl transferase, glucuronidase, UDPGA-pyrophosphatase, D-glucuronic acid-1-phosphatephosphatase, uronolactonase, glucuronolactone-reductase, and gulonolactone oxidase,

are

lysosomes.

microsomal

in location,

while

Most of the other enzymes

glucuronidase

of this pathway

is also

present

in

are present in soluble

supernatant.

In livers of certain species of animals capable of synthesizing L-ascorbic acid, glucuronic acid phosphate phosphatase is absent. Glucuronic acid is instead made available for subsequent conversion to L-ascorbic acid through the formation and hydrolysis of glucuronides. It is known! that the enzyme glucuronidase can be effectively controlled by several drugs, and by sex hormones such as human chorionic gonadotrophin and testosterone. This is the first step in the in vivo regulation of biosynthesis of L-ascorbic acid through uronic acid formation. Most of the enzymes of the uronic acid pathway are widely distributed in various tissues, with the exception of L-gulonolactone oxidase, which is present in the microsomes of either liver or kidney depending on the species. It is known '®!* that almost all animals, with the exception of man, monkey, guinea pig, Indian fruit bat, and the red-vented bulbul (Pycnonotus cafer), can synthesize L-ascorbic acid. Those animals that cannot syn-

thesize L-ascorbic acid genetically lack the enzyme L-gulonolactone oxidase, which is also a key enzyme in the regulation of the synthesis of L-ascorbic acid in the animal species. Although studies on the metabolism of L-ascorbic acid have been carried out extensively, the functional role of this vitamin in many biochemical processes is still obscure. L-Ascorbic acid has been found 2°* to be involved in a variety

* This work was supported by the Council of Scientific and Industrial Research, India, the University Grants Commission, and the United States Department of Agriculture through PL-480 funds (FG-IN-467, A7-HN-26).

382

Chatterjee et al.: L-Ascorbic

Acid

Metabolism

383

of metabolic processes such as collagen biosynthesis, corticosteroid metabolism, electron transport processes, and cholesterol metabolism. Its role in the metabolism of proteins is reflected by the imbalance of serum proteins and enhanced catabolism and reduced protein synthesis under scorbutic conditions.2°: 4 The enzymes that are operative in the biosynthesis and metabolism of L-ascorbic acid have been found 7°-4° to be affected by dietary factors, varying nutritional conditions, the administration of several metal ions, and several hormones. These aspects of L-ascorbic acid metabolism regulation at the enzyme level and mostly under in vivo conditions are described in this communication in the sections dealing with dietary protein variation, dietary intake of metal ions, the relation to hormones, and sufficiency, or deficiency on the enzymes level of L-ascorbic acid metabolism. The effect of massive doses of L-ascorbic acid on scorbutic guinea pigs in relation to the phagocytic index and the level of lysosomal enzymes in liver and PMN leukocytes has also been investigated.

DIETARY

PROTEIN

VARIATION

AND

L-ASCORBIC

AcID

METABOLISM

The interrelationship of protein metabolism and L-ascorbic acid metabolism has been widely studied during recent years, and this has been extensively reviewed by Chatterjee.1* The results appear to be interesting and help in understanding the metabolism of this important vitamin under protein sufficiency and deficiency. The earlier findings of Cohen and Mendel *! and Hopkins and Slater +” initiated studies on the effects of variations in the quantity and quality of dietary protein on the tissue levels and urinary excretion of L-ascorbic acid in guinea pigs.**-48: However, these experiments do not provide clues regarding the role of protein nutrition in the metabolism of this vitamin at the enzyme level. Hence studies estimating the levels of enzymes involved in the metabolism of L-ascorbic acid in animals under these conditions have been carried out.?°-°* The importance of protein nutrition and the effects of starvation and subsequent repletion with protein in the biological control of the enzymes operative in the metabolism of L-ascorbic acid have been covered by the studies of Sasmal et al.,2° Stirpe and Comporti,*® and Stubbs and Griffin.®° °! Effect of protein quality and L-ascorbic acid deficiency on the stimulation of hepatic microsomal enzymes in guinea pigs has also been reported.°* These studies have been extended further in order to understand the metabolism of this important vitamin under conditions of protein sufficiency and protein deficiency. Details of the experimental conditions have been discussed in earlier publications.25 26.29 The content of such proteins as casein in the diet was varied in the percentages of 2, 9, 18, 25, 40, 60, and 88 with rats as experimental animals. For the maintenance of tissue L-ascorbic acid level in rats it has been found that increasing the dietary protein content increases the tissue level, with optimal level reaching at 25%; thereafter it starts to fall. The activities of liver p-glucuronolactone reductase and L-gulonolactone oxidase increased with increasing amounts of dietary protein until a 9% casein level was reached. Thereafter the activities remained stationary up to a 60% casein level and decreased beyond that level.2° Dehydroascorbatase was found to be increased markedly at 9% casein level, and a further increase in dietary casein did not increase the activity. Uronolactonase, which catalyses the hydrolysis of p-glucuronolactone to p-glucuronic acid, increased with increase in dietary protein up to 18% casein, and a further increase in the dietary protein had no effect. Xylulose

384

Annals

New

York

Academy

7

of Sciences

ie O~O

GLUCURORMOL ACTORE

O--O

RECUCTASE GULOROLACTOME

16 ‘-)

14

O—O

Rats

O—O

conmraor eunca

OxIOaseE

ries

O--O sconeuTosEenic DIET FED SUINEAPIOS

Protein) (~Amoles/mg Sp.activity 10

20

30

40

Casein

$0

60

70

content

860

90

100

Protein) Sp.activity(a.moles/ g 10 20 30

%—

40

Casein

$0

60

70

60

930

100

content %—

Ficure 1. Effect of variation of dietary casein on (/eft) the synthesis of L-xylulose in rats and guinea pigs and (right) on the synthesis of L-ascorbic acid in rats.

biosynthesis by rat kidney tissue was found to be not very markedly affected by dietary protein content *° (FIGURES | and 2). Studies 2° carried out using guinea pigs, a species unable to synthesize Lascorbic acid, showed that the tissue level of L-ascorbic acid is increased with a protein intake of up to 25% casein level. This indicates that the tissue retention of L-ascorbic acid is slightly improved by increased dietary protein. In the liver of scorbutic guinea pigs, a similar trend was observed as the values rose up to a 9% casein level and remained almost constant thereafter. The activity of liver dehydroascorbatase decreased slightly in guinea pigs fed higher amounts of casein. This was more marked in the case of scorbutic animals. This can be explained by assuming that the animals fed higher amounts of casein conserve more vitamin in the tissue. Xylulose biosynthesis, on the other hand, increased significantly in normal and scorbutic guinea pigs fed higher amounts of casein (FIGURES

| and 2).

The studies on the interrelationship of dietary protein content and L-ascorbic acid metabolism as presented here reflect the role of protein nutrition in the metabolism of this vitamin in rats and guinea pigs. It was shown ®*: ?* earlier

mare

E25 COMTROL

a

COMTROL

GUINEA

Pies

GUINEA FICS Rats

(L-AA Content

_@ ry 10

20

— — — ———@ SconeuTosenic ovinaa Pies DIET FED 30

40

$0

Casemcontent

60

70

%

80

90

100

~@ SconsuToecnic euinca Diet FED coe /mg moles activity Sp. (4 Protein)

4‘ °

10

20

30

40 80 60 70 80 Coseincontent % ——

90

100

Ficure 2. Effect of variation of dietary casein on (left) the L-ascorbic acid content in liver of rats and guinea pigs and (right) on the catabolism of L-ascorbic acid in rats and guinea pigs.

Chatterjee et al.: L-Ascorbic

Acid

Metabolism

385

that this vitamin has a definite role in protein metabolism, and in L-ascorbicacid-deficient guinea pigs protein metabolism is altered.**: 47 Studies have also been made in this laboratory on the metabolism of Lascorbic acid in protein-depleted and subsequently repleted rat. In these experiments,°°: °° the quality of the protein in the diet was varied. It is known 53-55 that fasting affects the concentrations of different enzymes in animal tissues and that these concentrations are subsequently changed by repletion. Stirpe and Comporti ** studied only the effect of fasting on the biosynthesis of L-ascorbic acid and xylulose in rats. Since casein and groundnut proteins are qualitatively different, it was thought worthwhile to study the alterations in the activities of the different enzymes of L-ascorbic acid metabolism in fasting rats subsequently repleted with diets containing either casein or groundnut protein as the source of protein. It was noted’ that during starvation all the enzymes operative in L-ascorbic acid metabolism in rats manifest lowered activities, the most affected being L-gulonolactone oxidase. In rats fed repletion diets containing groundnut protein as source of protein, not only are the activities of the enzymes studied restored to the original levels, but their levels are even slightly higher than those observed in rats fed repletion diet containing casein. These results (FIGURES 1 and 2) suggest that variations of quality and quantity of protein have a significant role in the regulation of the activities of enzymes of L-ascorbic acid metabolism, particularly of L-gulonolactone oxidase in rats. Recent studies by Stubbs and Griffin *°: *! on the regulation of aldonolactonase in protein-depleted fasting rats also confirm our previous findings.*° Studies on protein variation in the diet in relation to L-ascorbic acid metabolism as presented here have an additional implication in understanding the requirement of L-ascorbic acid in the population maintained on high- or on low-protein diet. The diet composition in the underdeveloped and developing countries is low in protein as compared with that of developed countries, whose population ingest very high amounts of protein. The observations as mentioned here contradict the earlier belief that a high-protein-fed population requires higher amounts of vitamin C, since it has definitely been found 2°: 7° that highprotein feeding (of course, not to the extent leading to hyperproteinemia) in rats leads to increased ascorbate tissue levels and increased bioformation in vivo and such feeding in guinea pigs leads to increased tissue retention because of decreased catabolism of vitamin C.

DIETARY

INTAKE

OF METAL

IONS AND

L-ASCORBIC

ACID

METABOLISM

The role of metal ions on L-ascorbic acid metabolism was studied mostly in relation to alterations in tissue levels and urinary L-ascorbic acid excretion under administration of various metal ions.'*:°°°* Recently it was reported °° that administration of certain metal ions, such as Cd**, Pb*+, and Hg?* can alter certain biochemical parameters in the living systems. Investigations dealing with the role of various metal ions, such as Cr?+, Mo*+, W*+, Pb?+, Cd*+, Hg*+, Mn?*, or Co?+ on the levels of different enzymes of L-ascorbic acid metabolism have been extensively carried out in our laboratory.**: 3°" The experimental conditions have been described in detail in earlier publications.2*-°2. The effect of different metal ions such as Cr*+, W°+, Mo%+. Pb*+, Cd2+, and Hg?+ on the regulation of different enzymes of L-ascorbic acid biosynthesis and metabolism and also on the status of L-ascorbic acid in different

386

Annals L- Gulonolactone

New

York

Academy

of Sciences

oxidase

240 200 160 120

FiGurE 3. Effect of various metal ions on the activity of liver Lgulonolactone oxidase and_ liver uronolactonase in rats. Results are expressed as percentage value.

Uronolactonase

rats and guinea pig tissues have been studied.*°-** The results are presented in FIGURES 3 and 4. Cr?+ and W*+ at dose levels of 5 ppm increases slightly but significantly the concentration of L-ascorbic acid in the liver tissues of rats, and such an increase is also reflected in the adrenal gland.** Administration of varying doses of Mo**, Cd?*+, or Hg?+ decreases the contents of L-ascorbic acid in the liver tissues L-AA

FicgurE 4. Effect of various metal ions on L-ascorbic acid content in liver and L-xylulose biosynthesis in rats. Each bar represents change over 100% of the

control

value,

Content

in Liver

L-Gulonate dehydrogenase & 200 160 120

decarboxylase

Chatterjee

et al.: L-Ascorbic

Acid

Metabolism

387

of rats in comparison to control group of rats. Administration of higher levels of Zn**+ (1500 ppm) or Cu?+ (250 ppm) resulted in a decrease in the L-ascorbic acid content in liver and kidney tissues. The synthesis of L-ascorbic acid from L-gulonolactone by rat liver microsomes was found to be significantly reduced with dietary excesses of Zn?+, Cu?*, and Cd?+, while with Cr*+ and W"* there is a very significant stimulation. Other metal ions, such as Mn?*

and Co?+, were

also found

to stimulate

the synthesis

of L-ascorbic acid from the substrate L-gulonolactone in the rat liver. Dehydroascorbatase was found to be stimulated by various concentrations of all the metal ions mentioned above, excepting Co2*, Mo*t, Zn2+, and Pb?+, which cases inhibited its formation.*°** Except with W*+, uronolactonase activity was

found to be decreased with all the metal ions studied. Xylulose biosynthesis was, however, increased only with W"* at a dose level of 5 ppm. The toxic effects of Cd*+, Pb?+, and Hg?+ in different biochemical processes have lately become topics of interest. In recent years, the absorption, distribution, metabolism, toxicity, and excretion of cadmium in particular has been studied in detail in many species.*”*! Study of the effects of these metal ions revealed that they reduce the levels of L-ascorbic acid in liver and kidney tissues of rats, but that supplementation of L-ascorbic acid to these groups of toxicated rats brings about an increased level of tissue L-ascorbic acid.°2 Although Cd?+ and Hg** toxicity decreased the synthesis of L-ascorbic acid in rats,**.°? Pb?+ toxicity caused an elevation of L-ascorbic acid synthesis.®* L-ascorbic acid supplementation, however, brought about synthesis almost to the control level. Activity of dehydroascorbatase was elevated by Cd?+ and Hg?* toxicity in rats, and supplementation of L-ascorbic acid to these groups did not change this elevated level. Uronolactonase activity was not altered by toxicity of these metal ions in rats, while xylulose biosynthesis was decreased appreciably in rats under the influence of Pb?*+ and Cd?+ toxicity (FIGURE 5). During study of the effects of various metal ions on the enzymes of metabolism of L-ascorbic acid in scorbutogenic-diet-fed guinea pigs, it was observed °° that administration of Mn+, Co?+, and Mo®%+ decreased markedly the concentration of L-ascorbic acid in the liver and kidney tissues, and supplementation of L-ascorbic acid to the scorbutogenic-diet-fed guinea pigs had no effect on such decreased levels. The three metal ions could all decrease the activity of dehydroascorbatase to the control level, which was significantly increased in the scorbutic group. The activity of liver uronolactonase was significantly stimulated by the administration of all three metal ions in the scorbutogenic-diet-fed group of guinea pigs, but supplementation of L-ascorbic acid to this group along with the administration of these metal ions could reverse this effect. On the basis of the results obtained °°: ** it is concluded that rats and guinea pigs behave differently in respect to dehydroascorbatase activity under the influence of cobalt and manganese but not molybdenum. In rats, cobalt and manganese administration led to increased dehydroascorbatase activity, while in guinea pigs there is a decrease in the activity, but this is observed only under scorbutogenic-diet condition. In the case of rats, neither of these metal ions was found to have any effect on the biosynthesis of L-xylulose. This is quite different in guinea pigs, particularly when manganese is used and a significant inhibition is noted in both the scorbutic and _ L-ascorbic-acid-supplemented animals. The explanation offered for these findings *! states that under scorbutic conditions, metal ion administration brings about a severe stress condition, leading to a more drastic fall of L-ascorbic acid status in the tissues as well as

Annals

388

New

York

Academy

of Sciences

an augmented decrease in the body weights as compared with the animals treated with metal ions and subsequently supplemented with 1-ascorbic acid. It is therefore suggested that L-ascorbic acid has some protective effect against a ' metal ion toxicity. In conclusion, our studies on the interrelationship of mineral nutrition and

160

L- G@ULOMOLACTONE

L-AYVLULOSE

OXIDASE

SYRMTHESIS

L-ASCORBIC ACID CONTENT IMLIVER

Ficure 5. Effect of supplementation of L-ascorbic acid on the enzymes involved in the metabolism of L-ascorbic acid under metal ion toxicity; activity of liver Lgulonolactone oxidase, L-xylulose synthesis, and L-ascorbic acid content in the liver of rats. Cadmium was administered at a dose of 6 mg/100 g body weight for 4 weeks. Lead was administered as lead acetate at a dose of 10 mg/100 g body wt daily for 4 weeks and mercury was administered as mercuric chloride at a dose of 0.1 mg/100 g body weight for first 2 weeks and at a dose of 0.2 mg/100 g body weight for last 2 weeks. L-ascorbic acid was supplemented at a dose of 10 mg/100 g body weight daily.

L-ascorbic acid metabolism seem to indicate a marked shift from the previous postulations °°-°* linking effects of metal ion administration on the changes in the tissue levels and urinary excretion of L-ascorbic acid. The studies reported here clearly indicate that under conditions where the tissue levels of L-ascorbic acid are slightly altered, as in the cases of Cr+,

there is a very gross change,

particularly

W*+, or Pb2+ administration,

in the specific activity of either L-

Chatterjee et al.: L-Ascorbic

Acid Metabolism

389

gulonolactone oxidase or certain other metabolizing enzymes. This could easily explain why, although these metal ions may stimulate the bioformation of the vitamin through stimulation of the activity of t-gulonolactone oxidase, the utilization of this vitamin is subsequently enhanced, keeping the tissue levels more or less unaltered. However, in order to examine the mechanism of the regulation of synthesis, utilization, and maintenance of tissue levels of L-ascorbic acid in the animal systems under conditions of metal ion administration, supplementation studies with L-ascorbic acid at certain metal ion toxicities were carried out. The supplementation of this vitamin brings about an increase in the tissue levels in the control group, while under metal-ion-treated and subsequently L-ascorbic-acid-supplemented animals the tissue L-ascorbic acid level is lower than that of the L-ascorbic-acid-supplemented control group. On the basis of this result (FIGURE 5) it is concluded that metal ion administration causes a . stress situation, with an increase in the utilization of L-ascorbic acid. Further studies on administration of L-ascorbic acid to Cd?+-toxicated rats in relation to growth rate, hemoglobin levels, and enzyme levels have shown 3 that L-ascorbic acid supplementation can cause a sort of protection against metal ion toxicity.

EFFECT

OF

LEVEL

HORMONES, OF

ENZYMES

SUFFICIENCY OF

OR

L-ASCORBIC

DEFICIENCY, ACID

ON

THE

METABOLISM

Series of investigations have been carried out earlier to study the vitamin C status of the animals under varying physiological and nutritional conditions as reflected by alterations in tissue levels and urinary excretion.1* Studies on the regulation of the enzymes involved in the metabolism of L-ascorbic acid under hormonal sufficiency and deficiency have recently been carried out in our laboratory. It was noted that enzyme levels in animal tissue could be altered by a wide variety of physiological, nutritional, and hormonal manipulations. There are basically two views on how the hormones regulate the biochemical processes at the molecular level: (1) involvement of RNA synthesis during enzyme induction; and (2) other effects not involving the synthesis of new RNA. It is also known that hormones regulate the synthesis of certain enzymes by influencing either gene, enzyme or enzyme-forming machineries.** °° Using rats as experimental animals, particularly under adrenalectomized, alloxandiabetic, castrated, thyroidectomized, hypophysectomized, and partially hepatectomized conditions (FIGURE 6), attempts have been made to understand the effect of various hormones on the activities of L-gulonolactone oxidase and certain other enzymes operative in the metabolism of L-ascorbic acid. It is known that the adrenal contains a high concentration of L-ascorbic acid and that this concentration is decreased under conditions of stress, including scurvy and hypophysectomy, while urinary vitamin C excretion is increased.'* L-ascorbic acid plays an important role in the process of corticosteroid metabolism in the adrenals.?? It has been noted *° that adrenalectomy brings about a drastic reduction in the level of L-gulonolactone oxidase, with a simultaneous increase in the activity of dehydroascorbatase. The administration of hydrocortisone to the adrenalectomized animals can stimulate the activity of Lgulonolactone oxidase appreciably, and this stimulation is sensitive to actinomycin D (FicurE 7). This suggests the involvement of transcriptional events during the induction of L-gulonolactone oxidase. This also holds good when

Annals

390

York

New

of Sciences

Academy

() 4

“Yl4

|

100]

Aw

man

CT 4

a4 g

3

—

50

° s° 2R

4

=

z

2 ° z

«


5

In analyzing serum ascorbate findings, it should be mentioned that serum and leukocyte vitamin C concentrations of women have been observed to be higher than those of men and may be related to ovarian hormone activity 2» 4:5, 41-48 (FIGURE 3). In contrast, the ingestion of oral contraceptives by women has been reported to reduce the concentration of serum and leukocyte ascorbic acid.‘+; 4° Vitamin C levels have been observed to be lower in the serum of cigarette smokers.*!: +. 47 The nutritional significance of these effects is unclear but needs to be considered when low serum ascorbate levels are encountered. Leukocyte ascorbic acid concentrations have been considered to be more closely related to tissue stores of the vitamin than have serum levels.‘ * *5: 4% 64,65 Others have suggested that leukocyte ascorbic acid levels do not reliably reflect tissue status and that both serum and leukocyte ascorbate levels should be used to do this.9: °4: 66 In general, serum vitamin C levels tend to respond more readily than leukocytes to recent dietary intakes of ascorbic acid. However, low serum ascorbate levels do indicate low or inadequate intakes of vitamin C with probably only partial reserves present. Nevertheless, under controlled intakes of ascorbic acid, there is a relationship between ascorbate intakes and ascorbate

442

Annals

New

York

Academy

of Sciences

— E ° o

>

E— 2 = z
> 8

| SIGNS OF

z

SCURVY

I+ROA INTAKE OF ASCORBIC ACID

OccUR 25

50

75

VITAMIN C INTAKE (mg/day)

100

levels in both the leukocytes and serum *:° (FiGcuREs 2, 4, 5). With an intake of 45 mg of vitamin C per day (RDA), leukocytes will contain approximately 20 mg/100 ml of cells (FIGURE 4). Subjects with leukocyte ascorbate levels of less than 8 mg/100 ml are considered to be at “high risk” (TABLE 2).*:° Levels may fall to zero in clinical cases of scurvy.

Although numerous procedures and modifications have been reported for the determination of vitamin C in white blood cells, the methods must still be considered technically difficult and require relatively large blood samples.** Consequently, present procedures are not practical for routine use in nutrition surveys. With adequate laboratory facilities, measurement of leukocyte ascorbate levels can be a useful diagnostic technique for clinical cases of vitamin C deficiency. To some extent measurements of serum ascorbate levels have been favored over measurements of white blood cells levels because of the greater percentage increase in serum ascorbate concentrations in response to graded intakes of vitamin C (FIGURE 5). Such factors, along with the relative ease of measurement, have led to the common use of serum ascorbate levels for determining vitamin C nutritional status. This approach was used in national nutrition surveys of Canada and the United States.18 In the Ten State Nutrition Survey, vitamin C nutriture was not observed to be a major problem among any of the groups studied. Nevertheless, males had a higher prevalence of lower vitamin C levels than did females. The mean serum vitamin C values were also observed to be lower for males than for females (FicuRE 3). The general existence of subjects with poor vitamin C

N a gy

Ficure 5. The influence of vitamin C intake on the percentage increase in the serum and white blood cell vitamin C level.* °

a

w°

nN

LEVEL VITAMINC IN INCREASE %

°9

10

20.30

40 50 60 70 60

VITAMIN C INTAKE (mgMay)

Sauberlich:

Vitamin

C Status

443

status increased with age. The Spanish-American population appeared to have the best vitamin C status of the groups studied, reflecting the general adequate intakes of ascorbic acid in their diets. Overall, serum vitamin C values were related to vitamin C intakes. This observation would support the usefulness of obtaining dietary intake information when it is not feasible to perform biochemical determinations. During the nutrition survey of Canada, little clinical evidence suggestive of a vitamin C deficiency was observed in the general population.! However, suggestive signs of a deficiency were noted in the Eskimo and Indian populations. Moreover, dietary and biochemical findings indicated serious vitamin C deficits among certain population groups. Serum vitamin C levels classified as high- and moderate-risk were observed in one-fourth of the infants and toddlers (0-4 years) in the Indian and general populations and in approximately one-half of the Eskimo children of the same age. School-aged children had only a slightly lower proportion at risk. Over one-half of the Eskimo adolescents (70% of the boys and 55% of the girls) were at risk. Even among the Indian and general populations, about one-third of the adolescents were considered at risk. =i=

-_—

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>

between age and the mean serum, blood, and urinary vitamin : 3 C values in children ages 7 to

z !.20 = f< 1.00 =

15

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years

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in

Pretoria,

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from

South

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et OIG som SERUM dalle VITAMIN Mee aeg S3 %**

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Dietary

intakes

Indian and Eskimo

of vitamin children.

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30.40

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AGE (YEARS)

considered

For the Eskimos,

wey

25%

highly

walls ets

inadequate

of the infants and




among 50%

of the older children and adolescents were observed to have inadequate intakes of vitamin C. It was concluded that the findings were indicative of a serious vitamin C deficit among Eskimo children and adolescents, while a moderate deficit existed among the Indian and general populations.* Erythrocyte and whole-blood ascorbic acid levels appear to be less sensitive indicators of vitamin C nutriture than are serum levels of the vitamin. In controlled vitamin-C-deficiency studies, the ascorbic acid levels in the erythrocytes never fell to the low levels observed in the plasma or serum.®:* Similar observations were made in South African children studied by Du Plessis *? (Ficure 6). Although no well-established guidelines are available relating blood ascorbate values to the nutritional status of vitamin C in a population, a tentative guide has been indicated in TABLE 2. Nevertheless, Hodges et al.® * noted in studies with adult men that when the whole-blood ascorbic acid level had fallen to 0.3 mg/100 ml or less, the body pool of the vitamin had been severely depleted and clinical signs of scurvy were observed. Under these same

444

Annals

New

conditions, plasma ascorbic

York Academy

of Sciences

acid levels had usually fallen to levels below

0.20

mg/100 ml. Y Rat Urinary excretion of ascorbic acid declines to undetectable levels in vitamin C depletion.® 7 **,%* Consequently, attempts have been made to relate the ascorbic acid:creatinine ratio of random fasting urine samples to ascorbic acid intake.°°-52_ In some instances, vitamin C excretion has been related to 24-hour urine collections or to 100 ml of urine. Recently, Nobile ** observed a relationship between plasma and urine vitamin C levels in 26 subjects studied (FIGURE 7).

The results indicated that vitamin C is excreted

in the urine before tissue

saturation is reached. Urinary vitamin C excretion data have received only limited use in determining the nutritional status of this vitamin.

As a result, guides for the inter-

pretation of urinary vitamin C levels in terms of nutritional status have not been established. Analytical procedures for measuring ascorbic acid in urine are more difficult and less reliable than those used for measuring the vitamin in serum. Nevertheless, in the scorbutic patient, the urinary excretion of vitamin C would be expected to be essentially zero and, hence, could provide supportive diagnostic information. In this respect, Baker et al.%*:** observed that the urinary level of ascorbic acid fell to an undetectable level in vitamin-C-depleted subjects and the presence of urinary ascorbic acid did not occur until the ascorbate body pool had been repleted to nearly normal levels. In a nutrition survey conducted in South Africa, Du Plessis *! measured the serum, blood, and urinary vitamin C levels of each subject investigated (FIGURE 6). Children were observed to have a urinary excretion of vitamin C that ranged

from 35 to 54 mg/g of creatinine.’ Sex appeared to have no influence on the urinary excretion of vitamin C, while age did influence the urinary excretion when expressed on a per-gram-of-creatinine basis. The results would indicate that, for children of various ages, an age-adjusted sliding-scale interpretive guide would be required to analyze urinary vitamin C excretion findings. However, Du Plessis concluded that “the blood and serum vitamin C levels are considered to give a satisfactory indication of vitamin C status in population groups, while the determination of urinary vitamin C excretion in such surveys may in fact be superfluous.” *4 Even though urinary ascorbic acid excretion rapidly declines to undetectable levels upon inadequate intake of the vitamin, ascorbate metabolites continue to

15 ae Q De

- ABORIGINAL CHILDREN «ABORIGINAL WOMEN oEUROPEAN WOMEN

E 10

Lys

FicurE

re z 2

:

:

Relationship

GA(N=26567= (Adapted from

FE 5 > > a a

z a >o

7.

between

plasma and urine levels of vitamin

(o)

05 1.0 1.5 PLASMA VITAMINC (mg/100 ml)

0S OsepeeesO0anF Nobile.**)

Sauberlich:

Vitamin TABLE

ANALYTICAL

C Status

3

PROCEDURES USED TO MEASURE IN BIOLOGICAL SAMPLES

Sample Serum (plasma)

Leukocytes Whole blood

Urine

445

VITAMIN

C LEVELS

Method

References

Colorimetric 2,6-Dichloroindophenol Dinitrophenylhydrazine Fluorometric Automated Colorimetric Colorimetric Automated Colorimetric

GN Sy, its, AV SOX 4,5, 14, 19, 31 27 4,5, 10,

Automated Titrimetric

27 2

AN, Ville, zee Me, Ome 455s LOD 14s 24526 228) WS} sl PA 16, 22, 25, 64

14, 15, 19=21, 24, 26, 28, 35

appear in the urine even in the presence of clinical signs of scurvy.®: 7:33.34 number

of metabolites

have been demonstrated

A

to be present, but only a few

have thus far been identified.**: °* Of these metabolites, ascorbate-2-sulfate appears to be an interesting compound for further study as an index of vitamin C status.°* °° Baker et al.°® reported that the adult human male excretes 30 to 60 mg of ascorbate-2-sulfate daily. With the onset of scurvy, the level of excretion fell. Unfortunately, at present a simple and reliable quantitative method is not available to measure ascorbate-2-sulfate in urine.°? The measurement of this metabolite could perhaps provide an index to the body vitamin C reserves and thereby yield more reliable information concerning ascorbate nutritional status than can serum levels of the vitamin. On occasion, ascorbic acid saturation tests are used to obtain information

as to tissue ascorbate deficit in individual patients.*: °°

For nutrition surveys,

such tests are not practical and are probably of little use.

At best, the saturation

or loading test must be conducted with considerable care and the results interpreted with caution. However, for clinical cases, the test may be of conclusive value in excluding scurvy as a diagnosis and of considerable usefulness in sustaining the diagnosis of scurvy. The various procedures used to measure ascorbic acid are summarized in TaBLE 3. Ascorbic acid is commonly measured with procedures employing either 2,6-dichloroindophenol or dinitrophenylhydrazine reagent. Microautomated procedures have also been developed that enable the rapid analysis of large numbers of samples. In addition, fluorometric procedures are available. Regardless of the method employed, ascorbic acid determinations must be conducted with considerable care in order to obtain reliable results and to avoid serious losses of the vitamin prior to analysis. Few additional procedures have been proposed to determine vitamin C nutritional status (TABLE 1). Among these is the lingual vitamin C test that has been studied extensively by Cheraskin et al.*° The technique is based on the earlier reports of Giza et al.°® ®° that the body reserves of ascorbic acid could be estimated by observing the time of decolorization of an aqueous solution of 2,6-dicholoroindophenol by the tongue. King and Little ° concluded from their

446

Annals

New

York

Academy

of Sciences

studies that the test does not seem to be specific for ascorbic acid and “would therefore have little or no value when used in an attempt to answer the question of whether a given patient has a mild deficiency of tissue ascorbic acid.” Although Cheraskin et al.*° have utilized the procedure extensively, the technique has received little use or acceptance by other investigators.”® Earlier, an intradermal ascorbic acid test was proposed.®! The procedure was based on the premise that the rate of disappearance of an intradermally injected solution of 2,6-dichloroindophenol was related to the degree of vitamin

C tissue saturation. Although the procedure proved to be variable and not entirely specific, some degree of correlation with vitamin C status was observed. Nevertheless, the method has not been well standardized

and has not come into

use in nutrition surveys or in clinical practice. The tyrosine load test has likewise proved to be of uncertain

value in the assessment

of vitamin

C status.**, °» °°

Vitamin C deficiency apparently is not the basic cause of the defective tyrosine metabolism that results in the excretion of tyrosyl metabolites. SUMMARY

Although vitamin C nutritional status in man may be determined on the basis of dietary intake findings and on clinical signs of a dietary deprivation, biochemical measurements represent the most objective approach. Without the availability of a functional biochemical procedure that relates to vitamin C status, information concerning inadequacies in this nutrient has been derived mainly from measuring ascorbate levels in serum

(plasma),

leukocytes,

blood,

and urine. The measurement of serum levels of ascorbic acid is the most commonly used and practical procedure for determining vitamin C nutritional status in individuals or population groups. Although leukocyte ascorbate levels provide information concerning the body stores of the vitamin, the measurement is technically more difficult to perform, and, hence, its use is largely confined to clinical situations as an aid in the diagnosis of scurvy. The clinical diagnosis of scurvy can be aided also by information on the urinary levels of ascorbic acid and the use of vitamin C loading or saturation tests. With recognized limitations, ascorbic acid can be measured in biological samples with the use of automated or manual colorimetric and fluorometric procedures. Nutrition surveys conducted in Canada and the United States have indicated vitamin C deficits among certain population groups.

REFERENCES

1, NurritioNn CANADA NATIONAL SuRVEY. 1973. Information Canada. Ontario, Canada. 2. Dopps, M. L. 1969. Sex as a factor in blood levels of ascorbic acid. J. Amer. Diet Ass. 34: 32. 3. TEN STATE NUTRITION SuRVEY, 1968-1970. IV. 1972. U.S. Department of Health, Education,

and Welfare, Center for Disease Control, Atlanta, Georgia.

4.

Publication No. (HSM) 72-8132. SauBeRLICH, H. E., J. H. SkALA & R. P. Downy. 1973. Laboratory tests for the assessment of nutritional status. CRC Critical Reviews in Clinical Laboratory

5.

SAUBERLICH, H. E., J. H. SkALA & R. P. Downy.

Sciences.

4(3).

CRC

Press, Inc.

Assessment of Nutritional Status.

Cleveland, Ohio.

CRC

1974.

Press, Inc.

Laboratory Tests for the

Cleveland, Ohio.

Sauberlich: .

Hopces,

R. E., J. Hoop,

Vitamin

J. E. CaANHAM,

1971. Clinical manifestations Clin. Nutr. 24: 432.

C Status

447

H. E. SAUBERLICH

of ascorbic

acid deficiency

& E. M.

in man.

BAKER.

Amer.

J.

Baker, E. M., R. E. HopcEs, J. Hoop, H. E. SauBerticn, S. C. Marcu & J. E. CANHAM. 1971. Metabolism of “C- and *H-labeled L-ascorbic acid in human scurvy. Amer. J. Clin. Nutr. 24: 444. Dutra DE OLIveiraA, J. E., W. N. PEARSON & W. J. Darsy. 1959. Clinical usefulness of the oral ascorbic acid tolerance test in scurvy. Amer. J. Clin. Nutr.

.

736303 Lez, K.-T. & P. C. LEONG. 1964. A new method for the determination of ascorbic acid in urine. Clin. Chem. 10: 575. SCHAFFERT, R. R. & G. R. KINGSLEY. 1955. A rapid, simple method for the determnation of reduced, dehydro-, and total ascorbic acid in biological material. J. Biol. Chem. 212; 59. MANUAL FOR NUTRITION SURVEYS. 1963. 2nd edit. Interdepartmental Committee on Nutrition for National Defense, Superintendent of Documents, U.S. Government Printing Office. Washington, D.C.

We

PELLETIER,

ily,

min C. Advances in Automated Analysis, 1972, Technicon Int. Cong. 9: 73. Mediad, Inc. Tarrytown, N.Y. Goap, W. C., J. H. SkaLa, R. S. Harpine & H. E. SAUBERLICH. 1973. A semi-

O. & R. BRASSARD.

automated

technique

1973.

A new

for the determination

automated

of vitamin

method

C

for serum

(ascorbic

vita-

acid)

in

serum or plasma samples. Laboratory Report No. 337. U.S. Army Medical Research and Nutrition Laboratory, Letterman Army Institute of Research. San Francisco, Calif.

Rog, J. H.

1961.

Appraisal of methods for the determination of L-ascorbic acid.

Ann. N.Y. Acad. Sci. 92: 277. Sarl, J. C., E. M. BAKER & H. E. SAUBERLICH. 1966. A simplified method for the isolation of urinary ascorbic acid as the 2,4-dinitrophenylosazone. Anal. Biochem. 15: 537. BEssEy, O. A., O. H. Lowry & M. J. Brock. 1947. The quantitative determination of ascorbic acid in small amounts of white blood cells and platelets. J.

Biol. Chem. 168: 197. Garry, P. J. & G. M. OWEN. 1967. Automated screening technique for vitamin C assay requiring small quantities of blood. Technicon Symposium: Automation in Analytical Chemistry 1: 507. Deutscu, M. J. & C. E. WEEKs. 1965. Microfluorometric assay for vitamin C. J. Ass. Off. Agri. Chem. 48: 1248. PELLETIER,

O.

1968.

Determination

of vitamin

C in serum,

urine

and

other

biological materials. J. Lab. Clin. Med. 72: 674. Howarp, A. N. & B. J. CONSTABLE. 1966. The use of homocysteine in the estimation of ascorbic acid in urine. Clin. Chim. Acta 13: 387. Hucues, R. E. 1964. Use of a cation-exchange resin in the determination of urinary ascorbic acid. Analyst 89: 618. Lon, H. S. & C. W. Witson. 1971. An improved method for the measurement of leucocyte ascorbic acid concentrations. Int. J. Vit. Nutr. Res. 41: 90. NoBILE, S. 1973. Fluorometric determination of vitamin C in plasma. The Vitamin Laboratories, Roche Products Proprietary. Dee Why, New South Wales, Australia. OLLIveR, M.

BY. 26.

1967.

Ascorbic acid.

IV.

Estimation.

In The Vitamins.

2nd edit.

W. H. Sebrell, Jr. & R. S. Harris, Eds. 1: 338. Academic Press. New York, INDY? DENSON, K. W. & E. F. Bowers. 1961. The determination of ascorbic acid in white blood cells. Clin. Sci. 21: 157. Baker, E. M., D. C. HAMMeR, J. E. KENNEDY & B. M. ToLBErRT. 1973. Interference by ascorbate-2-sulfate in the dinitrophenylhydrazine assay of ascorbic acid. Anal. Biochem. 55: 641.

448 ile 28. Zo. 30.

Annals

New

York Academy

of Sciences

Garry, P. J., G. M. Owen, D. W. LasuLey & P. C. Foro. Automated plasma m2 and whole blood ascorbic acid. In press. VUILLEUMIER, J. 1967. Analytische Probleme bei der Bestimmung von Vitamin Int. Z. Vitaminforsch. 37: C in Zusammenhang mit Ermahrungserhebungen. 504. Kine, D. R. & J. W. Lirrie. 1970. Lingual ascorbic acid test. J. Oral Med. 25: 107. CHERASKIN,

E. & W.

M.

Rincsporr,

Jr.

A fortunate

1971.

J. Oral

erratum.

Med. 26: 75. ail a2. 33%

34.

Du Puessis, J. P. 1967. An evaluation of biochemical criteria for use in nutrition status surveys. Council for Scientific and Industrial Research Report No. 261: 109. National Nutrition Research Institute. Pretoria, South Africa. NosiLe, S. 1974. Blood vitamin levels in aboriginal children and their mothers in Western New South Wales. Med. J. Aust. 1: 601. Honces, R. E., E. M. Baker, J. Hoop, H. E. SAUBERLICH & S. C. Marcu. 1969. Experimental scurvy in man. Amer. J. Clin. Nutr. 22: 535. Baker, E. M., R. E. Hopces,

J. Hoop,

Metabolism of ascorbic-1-"'C Clin. Nutr. 22: 549.

355

Baker, E. M., J. C. SAARI & B. M. ToLBert.

man.

36. 37. 38.

SO 40. 41.

42. 43. 44. 45. 46.

47.

H. E. SAUBERLICH

acid in experimental

TOLBERT,

1966.

& S. C. MARCH.

human

Ascorbic

scurvy.

1969.

Amer.

J.

acid metabolism

in

Amer. J. Clin. Nutr. 19: 371. B. M., A. W. CHEN,

E. M. BELL & E. M.

Baker.

1967.

Metabolism

of L-ascorbic-4-"H acid in man. Amer. J. Clin. Nutr. 20: 250. RECOMMENDED DIETARY ALLOWANCES. 1974. 8th edit. National Academy of Sciences. Washington, D.C. FRIEDMAN, G. J., S. SHERRY & E. P. RALLI. 1940. The mechanism of the excretion of vitamin C by the human kidney at low and normal plasma levels of ascorbic acid. J. Clin. Invest. 19: 685. Lewin, S. 1973. Evaluation of potential effects of high intakes of ascorbic acid. Comp. Biochem. Physiol. 46B: 427. SCHRAUZER, G. N. & W. J. RHEAD. 1973. Ascorbic acid abuse: Effects of long term ingestion of excessive amounts on blood levels and urinary excretion. Int. J. Vit. Res. 43: 201. Brook, M. & J. J. GRIMSHAW. 1968. Vitamin C concentration of plasma and leukocytes as related to smoking habit, age, and sex of humans. Amer. J. Clin. Nutr. 21: 1254. WoopuiLL, J. M. 1970. Australian dietary surveys with special reference to vitamins. Int. J. Vit. Res. 40: 520. Lon, H. S. & C. W. M. Witson. 1971. Relationship of human ascorbic acid metabolism to ovulation. Lancet 1: 110. McLeroy, V. J. & H. E. SCHENDEL. 1973. Influence of oral contraceptives on ascorbic acid concentrations in healthy, sexually mature women. Amer. J. Clin. Nutr. 26: 191. Rivers, J. M. & M. M. Devine.

1972.

Plasma

ascorbic acid concentrations

and

oral contraceptives. Amer. J. Clin. Nutr. 25: 684. BaILEy, D. A., A. V. Carron, R. G. TEECE & H. J. WEHNER. 1970. Vitamin C supplementation related to physiological response to exercise in smoking and nonsmoking subjects. Amer. J. Clin. Nutr. 13: 905. PELLETIER, O. 1970. Vitamin C status of cigarette smoking and nonsmokers.

Amer, J. Clin. Nutr. 23: 520.

48.

Lon, H. S. 1972. The differences in the metabolism the sexes at different ages. Int. J. Vit. Res. 42: 86.

49.

Lon, H. S. 1972. The relationship between dietary ascorbic acid intake and buffy coat and plasma ascorbic acid concentrations at different ages. Int. J. Vit. Res. 41: 80. Burcu, H. B. 1961. Methods for detecting and evaluating ascorbic acid deficiency in man and animals. Ann, N.Y. Acad. Sci. 91: 268.

50.

of ascorbic

acid between

Sauberlich: 51.

Lowry,

O. H.

1952.

Vitamin

Biochemical

evidence

C Status

449

of nutritional status.

Physiol. Rev.

32: 431. 52.

Davey,

B. L., M.

L. Wu

& C. A. Srorvick.

1952.

Daily

determination

of

plasma, serum and white cell-platelet ascorbic acid in relationship to the excretion of ascorbic acid and homogentisic acid by adults maintained on a con53.

trolled diet. J. Nutr. 47: 341. Kinc, C. G. 1973. The biological synthesis of ascorbic acid.

World

Rev. Nutr.

54.

Diet. 18: 47. Anonymous. 1973. Ascorbic acid sulfate (AAS), a metabolite of ascorbic acid with antiscorbutic activity. Nutr. Rev. 31: 251.

55.

Baker, E. M., III, D. C. Hammer,

56.

57. 58. 59.

60.

61.

CHERASKIN,

acid test. 62.

CRANDON,

HONEY. 63.

64.

65.

S. C. Marcu, B. M. ToLBert & J. E. CANHAM.

1971. Ascorbate sulfate: A urinary metabolite of ascorbic acid in man. Science 173: 826. BAKER, E. M., J. E. KENNEDY, B. M. ToLBERT & J. E. CANHAM. 1972, Excretion and pool size of ascorbate sulfate and other ascorbate derivatives in man. (Abstr.) Fed. Proc. 31: 705. Marcu, S. C. 1972. A quantitative procedure for the assay of ascorbate-3-sulfate in biological samples. (Abstr.) Fed. Proc. 31: 705. YeEw, M.S. & Y. Lo. 1973. Levels of optimal vitamin C intake in individuals as estimated by the lingual test. Proc. Soc. Exp. Biol. Med. 144: 626. Giza, T. & J. WECLAWoWIEZ. 1960. Perlingual method for evaluating the vitamin C content of the body: a rapid diagnostic test for vitamin C undernuttrition. J. Vit. Res. 30: 327. Grza, T., J. WECLAWoWIEZ & J. ZAIONC. 1962. The perlingual method for evaluating vitamin C: II. The relation between decolorization time and the vitamin C content of the organs. Int. J. Vit. Res. 32: 121. E., J. B. DUNBAR

& F. H. FLYNN.

1958.

III. A study of forty-two dental students. J. H., B. LANDAU,

1958.

Ascorbic

S. MIKOL, J. BALMANNO,

acid economy

The intradermal

ascorbic

J. Dent. Med. 13: 135. M. JEFFERSON

in surgical patients

& N. Ma-

as indicated

by

blood ascorbic acid levels. New Eng. J. Med. 258: 105. STEELE, B. F., C. H. Hsu, Z. H. Prerce & H. H. WILLIAMS; 1952. Ascorbic acid nutriture in human. I. Tyrosine metabolism and blood levels of ascorbic acid

during ascorbic acid depletion and repletion. J. Nutr. 48: 49. KeiTH, M. O. & O. PELLETIER. 1974. Ascorbic acid concentrations in leukoctyes and selected organs of guinea pigs in response to increasing ascorbic acid intake. Amer. J. Clin. Nutr. 27: 368. Burr, M. L., P. C. ELwoop, D. J. Hoe, R. J. HURLEY & R. E. HuGuHeEs. 1974.

Plasma and leukocyte ascorbic acid levels in the elderly. 27: 144.

Amer. J. Clin. Nutr.

DISCUSSION

Dr. D. Stusss: You indicate a difference in serum levels and the white blood cell level between adult males and females, but no sexual difference in children. We have found the same thing in rats of about 30 days of age. At

that age there is no sexual difference in serum ascorbate, but as they approach puberty at about 65 days a sexual difference appears. However, the males have a higher level than females, which is just the opposite of what you show in humans and guinea pigs. Can you account for this sexual difference in man, who cannot synthesize vitamin C?

450

Annals

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York Academy

of Sciences

Dr. SAUBERLICH: This same finding has been reported by a number of investigators, representing 25,000 people or more. Researchers in South Africa worked with about 1,000 people and found that when subjects reached 15 years of age a sex difference appeared in serum ascorbate. Dr. G. C. CHATTERJEE: There are differences between rats and human beings. The female rats synthesize about half as much ascorbate as the males. Dr. Stusss: I agree with Dr. Chatterjee. We measure synthesis in male and female rats and we found that the synthesis was lower in the female, but that does not answer the question of why there is this difference. Perhaps the rate of utilization in the male is greater than in the female. Unfortunately, all our human studies using labeled techniques in relating body pool size have been done with males. Dr. G. H. BEATON: I would like to give emphasis to a point Dr. Sauberlich made. You quoted the results of the American and Canadian surveys and commented somewhat on the Canadian one, which seemingly gives a very good parallel to vitamin C deficiency. What is extremely important is the point you made earlier: that the standards are quite different in those two surveys. The Canadian standard was pushed extremely high. I do not think the Canadian population and the American population are different. Perhaps the Eskimo is. Dr. SAUBERLICH: J do not think the emphasis would be on the Eskimo and the Indian population. We have worked with both and have to agree with some of their findings.

BIOLOGICAL

VARIATION

IN ASCORBIC

Man-Li

ACID

NEEDS

S. Yew

Clayton Foundation Biochemical Institute The University of Texas at Austin Austin, Texas 78712

It is now generally conceded that human beings need about 10 mg of ascorbic acid a day to prevent severe symptoms of scurvy. Studies have also indicated, however, that higher levels of intake, up to 100 mg of ascorbic acid daily are necessary to halt milder symptoms of scurvy as well as symptoms not recognized as typical of ascorbic acid deficiency.!-*:** Still higher levels of intake of a few grams of ascorbic acid daily have also been advocated for prophylactic or therapeutic use.!:° In opposition to such recommendations, however, are also reports suggesting undesirable effects of excess ascorbic acid intake.® Certainly, the range of needs for ascorbic acid from minimal levels to prevent scurvy to optimal levels to promote growth and health may be extremely wide. That various tissues do not retain ascorbic acid or become depleted to the same degree makes saturation studies an unsatisfactory criterion for estimating ascorbic acid needs.’ Biological variation may also contribute significantly to the difficulty of determining the ascorbic acid needs of individuals. Although all animals use ascorbic acid, only a few species need an exogenous supply of the vitamin. In the animals that can synthesize ascorbic acid, only liver or kidney cells have been shown to possess the synthetic enzymes.* Cells of other tissues require extracellular ascorbic acid, as do cells and tissues of species of animals that require exogenous ascorbic acid. Alterations in synthetic capacity of liver or kidney cells may increase or decrease ascorbic acid supply to other body cells and tissues. Studies have demonstrated lowered rates of ascorbic acid absorption and retention in tissues of rats during aging.°-!* Furthermore, during and after sexual maturation, rates of ascorbic acid synthesis in male rats were reported to be significantly higher than in female rats.1* The sex difference persisted in older rats despite the decreasing enzymatic activities in both sexes. Hormonal regulation of ascorbic acid metabolism was also shown by Salomon and Stubbs,!! who administered growth hormone to hypophysectomized rats and found an abrupt increase in ascorbic acid excretion. Furthermore, their results also indicated individuality in the rate of ascorbic acid turnover in rats of the same sex, age, and strain. Among 7 normal male Sprague-Dawley rats of the same age, the biological half-life of ascorbic acid varied from 38 to 89 hours. For 7 hypophysectomized rats, the variation was from 70 to 123 hours. Findings in animals that require exogenous ascorbic acid have shown similar phenomena regarding tissue ascorbic acid metabolism.'® In studying ascorbic acid needs of young guinea pigs, Williams and Deason '° have noted a 20-fold difference in needs within a group of 102 male guinea pigs. Their study pointed out that some guinea pigs may need relatively low levels of ascorbic acid while, presumably due to biochemical individuality, other guinea pigs may need much higher levels than previously reported. Such variation in ascorbic acid needs was also noticed by Yew "7 when she studied ascorbic acid needs in male guinea

451

452

LEVELS

New

Annals

OF AscorBIC

York Academy

IN TISSUES

ACID

TABLE

1

OF

CHICK

of Sciences

EMBRYOS

AND

7-DAy-OLD

CHICKS

Ascorbic Acid (mg/100 g tissue) Tissue

7-day embryo

14-day embryo

20-day embryo

7-day chick

Brain Liver Heart

25.0) 19.0 18.0

17.8 11.4 Sa

15.2 i 5.4

18.0 13.0 353

te

ee

2.8

2.4

Eye

pigs for growth and for overcoming surgical stresses. The biological half-life of ascorbic acid in guinea pigs was studied by Salomon.'* In 9 guinea pigs, a range of 70 to 144 hours was reported. Odumosa and Wilson? also showed that, when deprived of ascorbic acid in their diet, some female guinea pigs were able to retain sufficient ascorbic acid in the liver and survived while others died of deficiency. De Fabro 2° demonstrated the activity of an ascorbic-acid-synthesizing enzyme, gulonolactone oxidase, in livers of guinea pigs during embryonic stages that was absent in the livers of adult guinea pigs. In our laboratory, we have studied ascorbic acid concentrations in various embryonic tissues from both chicks and guinea pigs by using Pelletier’s method.*t Our findings (TABLES 1-3) indicate that ascorbic acid concentrations in some embryonic tissues were distinctively different from those of postembryonic tissues. It is noteworthy that embryonic chick and guinea pig brains contained higher ascorbic acid concentrations than many other tissues studied. These findings strongly hint that ascorbic acid needs of animals during various stages of embryonic development may differ from needs during postembryonic life. In addition, these findings suggest that ascorbic acid may be synthesized by the rapidly differentiating embryonic brain cells. Studies in humans have indicated influences of age and sex on ascorbic acid metabolism.?*: In addition, individual variations in ascorbic acid needs have been noted by many careful workers. Kline and Eheart °* reported that the amount of ascorbic acid needed to reach 50% excretion in urine varied up to 4-fold in a group of 9 normal adult females. To reach this level, 6 subjects needed 1.4 to 1.8 mg/kg body weight; 1 needed only 0.6 mg/kg body weight, while 2 subjects required more than 2.2 mg/kg body weight. Stewart and

TABLE

ToTaL

Tissu—E

Ascorsic

7-Day Embryo

Brain Liver Heart Eyes

20.8 Del ila! 15.8

AciIp

2

(ug)

IN CHICK

EMBRYOS

14-Day Embryo

20-Day Embryo

62.1 16.8 3.0 Loy

iNBike? 54.5 8.3 19.9

Yew:

Biological Variation

453

Booth °° reported that by using the criterion of 25% excretion of an oral dose of ascorbic acid when none had been administered on the previous days, it took from 1 to 5 g to reach saturation within a group of 13 normal subjects. The total amount of excretion in 9 hours after oral dosage of 11 mg/kg body weight varied from 176 mg to 361 mg (mean 252 mg; S.D. 42 mg). Pauling et al.*° studied ascorbic acid excretion in normal and schizophrenic subjects and observed three different excretion patterns among the normal subjects. Of the 44 normal subjects, 17 were high excretors, 16 were medium excretors, and 11 were low excretors. When ascorbic acid is administered to individuals, oxalic acid is excreted in the urine. Briggs et al.2* studying this phenomenon found that the usual 24-hour increase of urinary oxalate of normal subjects on an oxalate-free diet receiving 4 g of ascorbic acid daily was about 12 mg. But the increase in one healthy young male volunteer was 584 mg and 420 mg during two separate courses of studies. Although Srikantia et al.?* concluded that maximal leukocyte ascorbic acid level can be reached by intake of 10 to 22 mg of ascorbic acid daily, their

TABLE LEVELS

OF

ASCORBIC

AND

Tissue Brain Liver

ACID

THE

3

IN TISSUES

MOTHER

OF

GUINEA

GUINEA

PIG

EMBRYOS

*

PIG

Ascorbic Acid of Embryos + (mg/100 g tissue )

Ascorbic Acid (ug)

Ascorbic Acid of Mother (mg/100 g tissue)

19.82.97 14533-3539

362 398

16.1 16.4

Total Tissue

;

Heart

(S525 077>5

37

7.4

Kidney

20.7+1.19

93

5.8

* Average weight=47 grams.

+ Values are the means

of 6 embryos followed

by standard deviations.

report showed variations in the maximal leukocyte ascorbic acid levels of the 15 normal subjects to be from 10 to 18 y»g/10* cells. Furthermore, when the volunteers were given diets containing 10 to 12 mg ascorbic acid daily, the period of time maintained at the initial level of leukocyte ascorbic acid concentration varied remarkably. For 3 subjects it was between 15 and 20 days, for 4 subjects it was 45 days, and for 2 subjects it was longer than 70 days. As for 4 other subjects, drastic decline in leukocyte ascorbic acid concentration was obvious within 10 days. When these 4 subjects were given ascorbic acid supplementations at 10 mg/day, tissue retention for each of the four was distinctive. The leukocyte ascorbic acid concentration of one subject remained very low throughout the 80 days of supplementation. For the three other subjects, it increased at different rates.

We have previously studied ascorbic acid intakes and lingual decolorization time in a group of healthy young adults.*° The levels of ascorbic acid supplementation needed for individuals to reach the shortest decolorization time varied from 0 to 4000 mg per day within the group of 47 volunteers. Individuals also

454

Annals

New

York Academy

of Sciences

varied in the shortest decolorization time obtainable in each case (from 7 to 15 seconds). Additional tests and analyses of the dietary survey showed that even individuals with similar levels of dietary ascorbic acid intake may differ significantly in degrees of tissue saturation (TABLE 4). Most authors have agreed that human infants need about 10 mg of ascorbic acid per day to prevent scurvy.” But DeJong et al.*° have reported than an infant with Hurler’s syndrome was maintained on a diet containing only 1.7 to 3.4 mg of ascorbic acid per day for a year without any symptoms of scurvy. On the other hand, Cochrane *! has shown two infants with scorbutic symptoms at intake of about 60 mg of ascorbic acid per day. Hamil and coworkers ** studied dietary ascorbic acid intake of 21 infants with mild scurvy and found 7 of them had consumed less than 9 mg of ascorbic acid/day, but 14 of them had consumed more than 9 mg ascorbic acid/day. They pointed out that there is a distinct variability in susceptibility of individual infants to scurvy that is determined by prenatal as well as postnatal factors. It is apparent not only that the ascorbic needs of normal, healthy human subjects may differ distinctively from individual to individual, but also that the

TABLE DISTRIBUTION ADULT VOLUNTEERS

4

OF LINGUAL DECOLORIZATION TIME OF 54 HEALTHY WHEN NO SUPPLEMENTATION OF ASCORBIC ACID WAS

we Sig : Decolorization Time (sec)

7-9 10-15 16-20 > 20 Total

Approximate Daily Diet Ascorbic Pee Reig ey eS 50 mg 50 to 100 mg

GIVEN

Acid Intak eee ee > 100 mg

3 3 3 5

1 5 1 4

3 15 4 {

14

11

29

range of variation in ascorbic acid needs may be extremely wide. The level of ascorbic acid need of an individual may further vary due to changes in the rate of metabolism, hormonal activities, and stages of development.

ACKNOWLEDGMENTS

I wish to express my appreciation to Prof. R. J. Williams for his continuing support and active interest in this work and to Mrs. Yvonne Lo for her technical assistance.

REFERENCES

1. BarTLey, W., H. A. Kress & J. R. P. O'BRIEN. 1953. Vitamin C requirement of human adults. Medical Research Council Special Report Series No. 280: 1-21.

2.

GoxpsmitH, G. A. clinical medicine.

1961. Human requirements for vitamin Ann. N. Y. Acad. Sci. 92: 230-245,

C and

its use in

Yew:

Biological Variation

455

CHERASKIN, E., W. M. Rincsporr, Jr., D. W. MICHAEL & B. S. Hicks. 1973. Daily vitamin C consumption and reported respiratory findings. Int. J. Vit. Nutr. Res. 43: 42-55. Kasuta, A. L. & M. M. Karz. 1960. Nilttitional factors in psychological test behavior. J. Genet. Psychol. 96: 343-352. PAULING, L. 1970. Vitamin C and the common cold. W. H. Freeman & Co.

.

San Francisco, Calif. SCHRAUZER, G. N. & W. J. RHEAD. 1973. Ascorbic acid abuse: Effect on long term ingestion of excessive amounts on blood levels and urinary excretion. Int. J. Vit. Nutr. Res. 43: 201-211. Hucues, R. E., R. J. HurLeEy & P. R. Jones. 1971. The retention of ascorbic acid by guinea pig tissues. Brit. J. Nutr. 26: 433-438. CHATTERJEE, I. B. 1973. Evolution and biosynthesis of ascorbic acid. Science 182: 1271-1272. PaTNAIK, B. K. & M. S. KANUNGO. 1966. Ascorbic acid and aging in the rat. (Uptake of ascorbic acid by teeth and concentration of various forms of ascorbic acid in different organs). Biochem. J. 100: 59-62. Dorr, P. E. & C. F. Nockers. 1971. Effect of aging and dietary ascorbic acid on tissue ascorbic acid in the domestic hen. Poult. Sci. 50: 1375-1382. PATNAIK, B. K. 1968. Change in the bound ascorbic acid content of muscle and liver of rats in relation to age. Nature 218: 393. MorenHousE,

A. L. & N. B. GUERRANT.

1952.

Influence

of age and of sex on

hepatic ascorbic acid. J. Nutr. 46: 551-564. Stusss, D. W. 1973. The effects of age and sex on the biosynthesis, urinary excretion, and tissue concentration of L-ascorbic acid in rats. Tex. Rep. Biol.

.

Med. 31: 229-237. SALOMON, L. L. & D. W. Stusss. 1961. Some aspects of the metabolism of ascorbic acid in rats. Ann. N. Y. Acad. Sci. 92: 128-140. HucueEs, R. E. & P. R. Jones. 1971. The influence of sex and age on the deposition of L-xyloascorbic acid in tissues of guinea pigs. Brit. J. Nutr. 25: 77-83. WILLIAMS, R. J. & G. DEASON. 1967. Individuality in vitamin C needs. Proc. Nat. Acad. Sci. U.S.A. 57: 1638-1641. Yew, M. S. 1973. “Recommended daily allowances” for vitamin C. Proc. Nat. Acad. Sci. U.S.A. 70: 969-972. SALOMON, L. L. 1957. Ascorbic acid catabolism in guinea pigs. J. Biol. Chem. 228: 163-170. Opumosa, A. & C. W. M. Witson. 1971. Metabolic availability of ascorbic acid in female guinea pigs. Brit. J. Pharm. 42: 637-638. De Fasro, S. P. 1968. Gulono-lactone oxidase activity in guinea pig embryo.

C.R. Soc. Biol. (Paris) 162: 284-285. PELLETIER, O. 1968. biological material.

.

Determination of vitamin C in serum, urine, and other J. Lab. Clin. Med. 72: 674-679. Lou, H. S. 1972. The differences in the metabolism of ascorbic acid between the sexes at different ages. Int. J. Vit. Nutr. Res. 42: 86-90. Moraan, A. F., H. L. Grtum & R. I. WititaAMs. 1955. Nutritional status of the aging. III. Serum ascorbic acid and intake. J. Nutr. 55: 431-448. Kune, A. B. & M. S. EnEART. 1944. Variation in the ascorbic acid requirements for saturation of nine normal young women. J. Nutr. 28: 413-419. Stewart, J. S. & C. C. Boorn. 1964. Ascorbic acid absorption in malabsorption.

Clinvocie 272) 1522. PAULING,

ile

L., A. B. Ropinson,

S. S. OxLEY,

M. BERGESON,

A. Harris, P. Cary,

J. BLETHEN & I. T. KEAVENY. 1973. Results of a loading test of asocrbic acid, niacinamide, and pyridoxine in schizophrenic subjects and controls. In Orthomolecular Psychiatry. D. Hawkins & L. Pauling, Eds. : 18-24. W. H. Freeman & Co. San Francisco, Calif. Briccs, M. H., P. GarcrA-WEBB & P. Davies. 1973. Urinary oxalate and vitamin C supplements. Lancet 2: 201.

456 28.

29. 30.

Annals

New

York Academy

of Sciences

1970. Human requireSRIKANTIA, S. G., M. MOHANRAM & K. KrISHNASWAMY. ments of ascorbic acid. Amer. J. Clin. Nutr. 23: 59-62. Yew, M.S. & Y. Lo. 1973. Levels of optimal vitamin C intake in individuals as estimated by the lingual tests. Proc. Soc. Exp. Biol. Med. 144: 626-627. DeEJone, B. P., W. vAN B. RoBERTSON & I. A. SCHAFER. 1968. Failure to induce scurvy by ascorbic acid depletion in a patient with Hurler’s syndrome. Pediatrics 42: 889-903.

31.

COocHRANE, W. A.

32.

lem? J. Can. Med. Assn. 93: 893-899. Hamit, B. M., L. REYNALDs, M. W. PooLe & I. G. Macy. 1938. Minimal vitamin C requirements of artificially fed infants. Amer. J. Dis. Child. 56: 561-

1965.

Overnutrition in prenatal and neonatal life:

A prob-

583. 33.

WILLIAMS, R. J. 1956. New York, N.Y.

Biochemical

Individuality.

147-149.

John

Wiley, Inc.,

DISCUSSION

Dr. R. E. Hopces: I was troubled when you said that it requires at least 100 mg of ascorbic acid daily to cure the severe symptoms of scurvy. This is contrary to our experience.

Dr. YEw: I did not say that. Dr. Hopces:

I said 10 mg per day was required.

In any event, the subjects we studied were given 4, 8, 16, 32

or 64 mg of ascorbic acid in addition to the approximately 1.6 mg contained in their diet. We found that all of these doses alleviate scurvy. But from about the 32 mg level up, we found no difference in response: the subjects responded as quickly and as well to the lower doses as they did to the higher ones. The studies that we conducted involved nine subjects who varied in weight from almost 50 kg to almost 100 kg. Despite this, the ascorbic acid body pool sizes were remarkably similar, as were the daily rates of ascorbic acid catabolism and the responses to given doses. From this limited study we did not see evidence of the wide variability in the ascorbic acid requirement that you suggest. Dr. H. Sprince: Have you considered the effects of heavy drinking and heavy smoking in your studies of individual variability of ascorbic acid requirements? I bring that up because of the potential relationship to alcoholism and ethnic differences involving acetaldehyde. Dr. G, H. BEATON: You referred to 10 mg curing severe signs of scurvy, but up to 100 mg was required to cure, I think you used the phrase, “milder symptoms of scurvy.” Could you elaborate on this? What are the milder symptoms? Dr. YEW: Some findings on this are discussed in Dr. Williams’ book on biochemical individuality. Dr. C, G. KiNG: Have you done any work with animals at different intakes, for instance from 1—10 mg, and then given them what the chemists and pharmacologists would call an insult, such as an injection of toxic material, and seen what the response is in the tissue? Dr. YEW: No, not using “drugs.” I have done experiments with graded levels of intake of ascorbate using surgical stress as an insult. For optimal growth in guinea pigs, I found the ascorbate requirement to be about 5 mg per

Yew:

Biological Variation

457

100 g per day. To overcome surgical stress and anesthesia however, the requirement was about 10 times higher. Dr. I. B. CHATTERJEE: I think I can answer Dr. King’s question by saying that insulting guinea pigs with certain kinds of vaccines, drugs or nonspecific stresses like extra heat, cold, or even pregnancy, raises the ascorbic acid requirement to about five times the normal level.

ASCORBIC

ACID

AND

ATHLETIC

H. Howald Research

PERFORMANCE

and B. Segesser

Institute of Federal School of Physical Education Magglingen, Switzerland W.

F. Korner

Department of Vitamin and Nutrition Research F. Hoffmann-La Roche & Co., Ltd. Basel, Switzerland

INTRODUCTION

The question of whether nutrition improves an athlete’s performance is by no means a new one. As early as in the sixth century B.C. we can find special dietary recommendations given to the ancient olympic competitors. So it seems quite normal that in our time there has been a lot of discussion whether or not additional administration of vitamins—and especially vitamin C—would have a beneficial effect in sports. Reviewing the vitamin C literature on this question, one finds numerous papers, the earliest of them having been published in 1937.1. The conclusions, however, are very controversial, some studies postulating a marked performanceincreasing effect '® and others showing absolutely no effect.*° Most of the conclusions are based on parameters dealing with the respiratory and cardiovascular system, whereas the study of changes in metabolic processes during exercise has largely been neglected. We therefore concentrated our interest on possible influences of ascorbic acid administration on human muscle metabolism in a standard exercise test, especially as far as energy supply to the working muscle cell is concerned.

SUBJECTS

AND

TESTING

PROCEDURE

Thirteen athletes (TABLE 1) on continuous training of moderate intensity and showing fairly constant performance were examined after daily oral administration of placebo over a period of 14 days and again after a two-week intake of 1 g ascorbic acid per day (controlled trial). The performance test was carried out by bicycle ergometry starting at a work load of 30 watts and increasing stepwise by 40 watts every four minutes, until the subjects were exhausted.

RESULTS

AND

CONCLUSIONS

Performance

When comparing the totally performed work of the placebo and ascorbic acid series, we found a slight but not significant decrease after the two-week administration of vitamin C (TABLE 2). Looking at these results, one might

458

Howald

et al.:

TABLE AGES

N

AND

459

Performance

Athletic 1

DATA

ANTHROPOMETRIC

ON

VOLUNTEERS

Age

Weight

Size

Body-Surface *

(years)

(kg)

(cm)

(m”)

80.2

182.8

2.02

a1 }5)

Seale,

+0.04

AVS) 13

+0.9 T

* After Du Bois and Du Bois. + Standard error of the mean.

conclude that a daily intake of 1 g ascorbic tolerance in athletes. Working

Capacity

acid has no effect on exercise

at Submaximal

Heart

Rates

of When comparing, however, the physical working capacity at a heart rate in capacity ce performan of parameter current a is ch 170 beats/minute—whi C series exercise physiology—we found a significant improvement in the vitamin rate has (TaBLE 2). Further analysis of the exercise-induced increase of heart shown persistently lower values in the vitamin-C than in the placebo series 8 to throughout the testing procedure (FiGuRE 1). The differences averaged 9 beats per minute and were statistically significant. ed to A lower heart rate at a given submaximal work load is usually interpret during function heart economic more a and capacity work better a of sign a be effect on exercise. Evaluated by this parameter, vitamin C has a beneficial athletic performance.

TABLE PERFORMANCE

AND

2

PHYSICAL

Number

Totally performed work

(J-10?/m’)

13

Physical working capacity: (J/sec-m*)

13

* Wilcoxon matched-pairs signed-rank test. + Standard error of the mean.

WORKING

CAPACITY

Placebo

Ascorbic Acid

1204.3

1144.6

+74.2 +

+69.6

ie)

> 0.05

114.2

122.4

+4,3 +

SES) iui?

< 0.01

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300

400

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York Academy

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518 of Sciences

Coulehan

eft al.: Upper Respiratory Illness

S19

lated with those of Anderson et al., who noted 30% fewer days confined to home in adults taking 4 grams of vitamin C daily during illness, an effect that was most evident in syndromes with generalized, nonrespiratory symptoms.’ In a subsequent study, however, Anderson showed only minimal and not statistically significant therapeutic value of vitamin C.® He used a wide range of doses, up to 8 grams daily during the first four days of illness, and noted that

the small ameliorative effect appeared to be dose-related. Our hypotheses in the present study related to therapy rather than prophylaxis: specificity, value in secondary prevention, and relationship of plasma ascorbic acid levels to severity of symptoms. It was not practical, however, to give supplements only during illness in this population. Consequently, a 1-gram daily dose was given whether the child felt sick or well, and, in fact, supplementation during illness was less supervised that that during health if the child was sick enough to be absent from class. The surveillance method was used to detect primarily mild symptoms that caused neither absence nor referral to medical care. We found no meaningful differences in cold symptomatology between C and P children with this method, although younger children at Greasewood on C had 14% fewer sick days noted. Earache, a symptom that

could be related to a complication of colds (otitis media), was no less frequent among C children. On the other hand, those on C had significantly more side effects (diarrhea and abdominal discomfort) observed at Greasewood. How can the discrepancies between these findings and our previous study *

be explained? The surveillance method on sequential classroom visitation was the same as used previously, and many of the same personnel were involved. However, there are two indications that the data obtained by this technique are “softer” than we had supposed. First, individual symptoms were recorded up to 9 times more frequently at one school than at the other in this trial, with an overall factor of about three regardless of treatment group. This can be explained only by significant differences in child-observer interactions at the two schools, and results cannot be considered additively. Moreover, such large variations that apparently result from interview technique shed some doubt on the meaningfulness

of the 25-30%

intergroup differences we

reported earlier,

albeit only from one school with basically one observer.t Secondly, C children were more often absent from surveillance and more likely to acknowledge a neutral symptom (skin sores) than P children, at least at one school. In our

first study, P children were more frequently absent (TABLE 1). These inconsistencies at least suggest that children in the two groups may by chance have differing characteristics, unrelated to treatment regimen; for example, the C population in this study may contain more children with a high index of responsiveness to questioning than the P population. In any case, the conflicting results indicate that this method is not sensitive

enough to delineate a pharmacological action of ascorbic acid in a dose of 1 gram daily, if indeed such an action does exist. We anticipate that analysis of biochemical data and the clinical episode data, reflecting actual “illness” rather than more evanescent “symptoms,” may clarify the matter. A specific in symptomatic benefit, for example, could explain the conflicting findings at various studies in that methodology, population, and the syndromes prevalent the time of the study could all influence whether,

and to what extent,

benefit

vitawas observed in the given investigation. Nevertheless, this suggests that remedy. cold a as usefulness d min C is unlikely to have widesprea Some recent work is suggestive in proposing a site of action for ascorbic

520

Annals New

York Academy

of Sciences

acid in cold therapy, and in demonstrating physiological changes in leukocyte ascorbic acid levels during upper respiratory illness. Valic and Zuskin ® and Zuskin et al.8 have shown that aerosols of ascorbic acid have a definite effect in preventing or ameliorating histamine-induced bronchospasm in humans and experimental animals. This effect was evident in pulmonary function studies on a group of flax workers, some of whom had bysinnosis.° However, little is known about the functional aspects of acute airway responses during the common cold. It is clear, though, that a modest antihistaminic action could provide some relief for symptoms such as nasal congestion and cough, particularly in those persons with an allergic component to their cold syndrome. Leukocyte ascorbic acid levels have been shown to decrease in response to colds,’ 1° and to return to values near normal after about four days. Use of 1 to 6 grams of vitamin C daily during the illness can diminish or prevent this effect.1° However, a drop in leukocyte ascorbic acid may well be a nonspecific response to a variety of stresses, such as in the case of myocardial infarction.* There is no substantial evidence that preventing this physiologic correlate of colds has any clinical consequences. Our earlier work did, however, indicate that children with higher whole-blood ascorbic acid levels had fewer observed days of illness on surveillance than those with lower ascorbic acid levels. This was based on a single blood specimen, drawn approximately 24 hours after the last supplement was given, and on a small number of observations, both in terms of children (67) and days sick (30 versus 55). Analysis of biochemical data from the present study should shed further light on this association.

ACKNOWLEDGMENTS We are indebted to Hoffmann-La Roche, Inc., Nutley, New Jersey, for materials used in this study, and for additional funding. In particular, we wish to thank Myron Brin for his assistance and encouragement. REFERENCES

1. CouLenan, J. L., K. S. REISINGER, K. D. Rocers & D. W. BRADLEY. 1974. Vitamin C prophylaxis in a boarding school. New Eng. J. Med. 290: 6-10. 2. ANDERSON, T. W., D. B. W. Rem & G. H. BEATON. 1972. Vitamin C and the common cold: a double-blind trial. Can. Med. Ass. J. 107: 503—508. 3. Witson, C. W. M. & H. S. Lon. 1973. Common cold and vitamin C. Lancet

1: 638-641. PAULING, L. C. 1970. Vitamin C and the Common Cold. W. H. Freeman & Company. San Francisco, Calif. 5. ANDERSON, T. W., G. SurRANYI & G. H. BEATON. 1974. The effect on winter illness of large doses of vitamin C. Can. Med. Ass. J. 111: 31-36. 6. Sasaki, Y., Y. Toco, H. N. WAGNER, Jr., R. B. Hornicx, A. R. SCHWARTZ & D. F. Procrer. 1973. Mucociliary function experimentally induced rhino4.

virus infection in man.

7.

Ann, Otol.

82(2):203-211.

ScHwartz, A. R., Y. Toco, R. B. Hornick, S. Tominaca & R. A, GLECKMAN. 1973. Evaluation of the efficacy of ascorbic acid in prophylaxis of induced rhinovirus 44 infection in man.

J. Infect Dis. 128(4): 500-505.

8. Zuskin, E., A. J. Lewis & A. Bounuys. 1973. Inhibition of histamine-induced airway constriction by ascorbic acid. J. Allergy Clin. Immunol. 51: 218-226. 9. Vatic, F. & E. Zuskin. 1973. Pharmacological prevention of acute ventilatory capacity reduction in flax dust exposure. Brit. J. Industr. Med. 30: 381-384.

Coulehan 10. 11.

Hume, R. common Hume, R., eer

et al.: Upper Respiratory Illness

& E. Wevers. cold. Scot Med. E. Wevyers, T. acid levels in

521

1973. Changes in leukocyte ascorbic acid during the J 18: 3-7. Rowan, D. S. Rew & W. S. Hituis. 1972. Leukocyte acute myocardial infarction. Brit. Heart J. 34: 238-

DISCUSSION

Ms. E. BARRETT: How often was ascorbic acid administered daily in both studies? Dr. CouLEHAN: It was given as 1 gram every morning after breakfast. Ms. BARRETT: What about 2 grams a day? Dr. CoULEHAN: The 2 grams a day were not given in the second study. This dosage had been given according to the same schedule as in the first study, once a day and in tablets. Ms. BARRETT: How did you get children under 10 years old to swallow four tablets in the morning? Dr. CouLEHAN: We did not have much trouble. There were 944 children at the start of the second study and only 870 completed it, giving about an 8% dropout rate. Much of this was because of inability to consistently take the tablets. However, we did not find this to be a major problem simply because the kids liked the tablets and in fact, had very little difficulty swallowing them. These children take tablets all the time, as they are sick frequently with respiratory illness. Dr. R. MarTIN (Mount Sinai Graduate School, New York, NEYO) saeco mentioned that the children with fewer colds had a higher blood level of vitamin C and those with more colds had a lower level, although the numbers were small. Is it possible that the number of colds affected the level and not the reverse? all Dr. CouLEHAN: We had two types of data. On one hand, we looked at of the children who were identified as having had an episode of illness because we they went to the clinic either by themselves or by referral. On the other, also did systematic regular examination. In the case of the systematic observasince tions it is obvious that our unit was limited to the day of observation and of that was every two weeks in a given child, we cannot relate these child-days of child-days in decrease a tly, Consequen observation to episodes of illness. well repreas equally might it but illnesses, fewer represent might n observatio data with the sent shorter illnesses. We were only able to correlate blood level were 30 days there think I data. n observatio of child-days or ce active surveillan consider the you When group. of illness in the one group and 55 in the other of 30 to 55 ratio the as well as children these in n observatio of possible days in either greater not was sick days sick, it would seem the total number of days group. time of Dr. E. Deckwitz: If you give ascorbic acid once a day, at what 6 to about is males in time Half-life levels? blood the the day do you measure 12 hours, so the level would depend on the time of measurement. It was impossible for us to give the tablets more than Dr. CouLEHAN: the first study once a day. The blood ascorbate levels that were measured in

ane

Annals

New

York

Academy

of Sciences

were determined 24 hours after the last tablets were administered. study included

samples

taken

at various

hours

after the dose

was

The second given,

but

these results are not available. Dr. K. E. SCHAEFER (Naval Submarine Medical Research Laboratory, Groton, Conn.): Since you were doing some blood chemistry determinations, do you have any serological evidence as to the type of infectious agents you were dealing with? Dr. COULEHAN: We were unable to do serological studies and the results of ear and pharyngeal smear studies are not available.

SAFETY

CONSIDERATIONS WITH HIGH ASCORBIC ACID DOSAGE Lewis

A. Barness

Department of Pediatrics University of South Florida College of Medicine Tampa, Florida 33620

When a new drug is found effective in the prevention or treatment of a disease, before use is recommended the toxicity of the drug must be weighed

against its beneficial effects. When the substance in question is a foodstuff, and especially a vitamin, it becomes difficult to clearly separate need, effect, and emotion. That any necessary substance, including water, can be toxic if taken in sufficient quantities is a truism. A level of intake of vitamin C must exist that is toxic to everyone and, at lesser levels, to many, a phenomenon related to biological variability of humans. The concept that humans have the capacity to regulate their intake of foods and toxic substances is outmoded. It is even excessive to claim that physicians, informed as they are, know their needs any better, whether of food or surgery,! than do laymen. Since properties other than the commonly acknowledged one of preventing scurvy have been attributed to large doses of vitamin C, and since a continuum of actions of ascorbate have been discussed for many years, definition of the toxic effects of vitamin C becomes important. Toxic effects of ascorbic acid that have been reported include acidosis, oxaluria, renal stones, glycosuria, renal tubular disease, gastrointestinal disturbances, sensitivity reactions, conditioned need, prothrombin and cholesterol disturbances, vitamin B,, destruction, fatigue and sterility; additionally, ascorbate ingestion has been noted to interfere with results of certain laboratory determinations. Probably the two most frightening

claims about the possible toxicity of ascorbic acid concern the development of acidosis, and the production of oxaluria.

Toxic

EFFECTS

OF

ASCORBIC

ACID

Acidosis

used as Acidosis seems to be a negligible problem. Ascorbic acid has been acid is a urinary acidifying agent for at least fifteen years. While ascorbic grams 3-6 of doses in agent, relatively ineffective alone as a urinary acidifying pH the lower can it , antibiotics or ine mandelam with on per day in combinati g/m? per 8 when Even pH.* blood the on effects negligible with urine the of day was given to normal subjects little effect was noted on blood pH.‘ Goldstein ed a crisis noted that a 40-year-old patient with sickle cell thalassemia experienc the crisis whether unclear is it acid; ascorbic of doses high of ingestion after was related to the ascorbate or the cold.°

SPE

524

Annals New

York Academy

of Sciences

Oxaluria-Oxalosis Studies on the sources of oxalic acid indicate that in the normal subject approximately 50% of the daily excretion of oxalic acid, about 50 mg, is derived from dietary ascorbate. The source of the other 50 mg varies. In a 9-year-old patient with oxalosis, 2 grams of ascorbic acid taken daily for five days resulted in significant increase in urinary oxalate. In a child with idiopathic urolithiasis and hypercalciuria, oxalic acid excretion was unchanged.® In adults, 4 grams or more daily produced increased oxalate excretion, which was unaffected by the administration of pyridoxine 100 mg, glycine 20 g, or sodium benzoate 20 g.* Enzymatic type of oxaluria apparently does not alter these effects. We could find no reports of oxalate stone formation in normal individuals who had no enzymatic defects.

Renal

Stones

One of the feared consequences of using a urinary acidifying agent is the possible development of renal stones or nephrocalcinosis.° No evidence of renal stone development could be found, except in a few instances. In those who are prone to renal stone formation, stones may form more easily. This includes persons with oxalosis, hyperuricemia, and cystinuria.t°: 11 While stone formation may be accelerated with the administration of large doses of ascorbate, this is difficult to quantitate. No reports of increase in symptoms of gout were found.

Hy perglycemia-Glycosuria

For many years, it has been known that ascorbate is a reducing agent that gives positive results on tests for urinary sugar. Many reports of hyperglycemia occurring after large doses of ascorbate ascribe diabetic symptoms or signs to ascorbate. Recently, some clarification has been afforded !* by the careful study of ascorbate in vitro. Not only does ascorbate reduce the usual agents used for testing urinary and blood reducing substances, but it also inactivates glucose oxidase, Therefore, any reports of glycosuria or hyperglycemia must include a specific test for glucose, e.g., chromatography. None of these has been found positive after administration of large doses of ascorbate.

Gastrointestinal

Disturbances

Gastrointestinal disturbances are perhaps the most consistent abnormalities noted following the ingestion of large quantities of ascorbic acid. Nausea, abdominal cramps, and diarrhea are frequently mentioned. These may be due to the ascorbic acid itself or to sensitization reactions such as hives, angioneurotic

edema,

and

skin rashes,

which

have

also been

these patients.'*:1* These effects may be ameliorated the ascorbic acid as a buffered salt or after meals.

reported

or eliminated

in some

of

by taking

Barness:

Safety Considerations Conditioned

with High Dosage

525

Need

A suggestion has been made that systemic conditioning can occur after prolonged intake of 2 to 3 grams of extradietary ascorbic acid. Case histories have been reported of several adults who may have developed scurvy following the cessation of the extra ascorbic acid.1° Even more alarming is the report by Cochrane, who attributed scurvy in two young infants receiving presumably adequate vitamins to prenatal conditioning in their mothers who were given large amounts of vitamin C during pregnancy.'® Prothrombin

An inhibitor of prothrombin, dicoumarol, was given to a patient with pulmonary embolism. Prothrombin time rose to 19 seconds, but fell to normal when vitamin C was given for a developing cold.‘7 A similar effect was noted in a patient with thrombophlebitis given 16 grams of ascorbic acid daily.‘* The earlier literature records toxic effects on platelets, particularly thrombocytopenia, but none was found recently. Serum

A rise in serum cholesterol receiving large doses of vitamin the atherosclerosis and suggests toxic effects be explored.1® A reported by others.*°

Cholesterol

was noted by Morin in atherosclerotic patients C. The author believes that this may aggravate that this hypercholesterolemic effect and other similar but less well defined effect has been

Vitamin

B,,

Herbert and Jacob 2! have recently noted that when ascorbic acid is ingested with food, substantial amounts of vitamin B,, are destroyed. They recommend regular evaluation of vitamin B,, status in anyone taking more than 0.5 grams of vitamin C daily. Laboratory

Errors

Mention has already been made of the double errors that may occur in the determination of glucose, especially urinary glucose but presumably also blood glucose. In addition, interference with autoanalyser estimation of serum transaminase and lactic dehydrogenase has been reported. Serum bilirubin concentrations are also significantly reduced, so that the presence of liver disease may be masked. Other Effects

Occasional effects such as fatigue have been noted in human subjects. Effects on zinc and copper in the body, especially in patients with deficiencies of these

526

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elements, may be beneficial or else the deficiency may be aggravated. one instance of sterility following ascorbate ingestion has been reported. SUMMARY

Many of the toxic effects of large amounts of vitamin C are insignificant, or rare, or troublesome but of little consequence. These include possible acidosis, gastrointestinal complaints, glycosuria, or sensitivity reactions. Effects on

prothrombin

are

rare,

and

laboratory

errors,

while

troublesome,

can

be

obviated by better understanding. Not so negligible, however, are effects that have been reported in a few instances. Especially threatening is the possibility of systemic conditioning, thus far reported in the rare patient, but also in the infants born of women receiving added ascorbate during pregnancy. The possibility of increasing serum cholesterol levels in atherosclerotic patients and of destruction of vitamin B,, requires further documentation. That patients with inborn errors of metabolism such as cystinuria, oxalosis, and hyperuricemia may be especially susceptible to difficulty when consuming large doses of vitamin C should be an especial warning. It is indeed rare that a vitamin should cause any increase of symptoms of metabolic diseases. Special caution must be exercised in infants or young, rapidly growing children, where adverse effects may be more than dose-related. If it is true that vitamin C in large doses is effective in certain disease states, treating it as a drug should have doubly beneficial effects. First, it puts users on warning that toxicity may exist. Second, users of this drug, like those of all other drugs, will take it irregularly or not at all, as they do with all other medicines. Preventive medicine, to be effective, must be given by a single injection or by several injections in infancy. Expecting a person to change his life style to prevent disease is tilting at windmills. REFERENCES

1, Bunker, J. P. & B. W. Brown. 1974. Physician-patient as an informed sumer of surgical services. New Eng. J. Med. 290: 1051-1054. 2.

3.

PAULING, L., 1970. Vitamin C and the Common Co. San Francisco, Calif. Murpuy, F. J., S. ZetMan, & W. Mav. 1965. acidifying agent. II. J. Urol. 94: 300-303.

Cold.

W.

Ascorbic

H.

Freeman

acid

and

a urinary

4.

Travis, L. B., W. F. Dopce, A. A. Mintz & M. AsseMi.

5.

cation with ascorbic acid. J. Pediat. 67: 1176-8. GoLpsTEIN, M. I. 1971. Letter to the editor. J.A.M.A. 216: 332-3. Hoesui, P. O., M. Just & H. VETTERLI-BUCHNER. 1959. Urol. Int.

6.

1965.

as

con-

Urinary acidifi-

(Basle)

8: 234-255. 7.

Briccs, M. H., P. GarcitA-Wess, & P. Davies. vitamin C supplements. Lancet 2: 201.

1973.

Urinary

oxalate

and

8. ATKINS, G. L., B. M. DEAN, W. J. GrirFin, & R. W. E. Watts. 1964. Quantitative aspects of ascorbic acid metabolism in man. J. Biol. Chem. 239: 2975-2980. 9. GoLpsmiTH, G. A. 1971. Common cold: Prevention and treatment wtih ascorbic acid not effective. J.A.M.A. 216: 537. 10. Morpuy, F. J. & S. ZELMAN. 1965. Ascorbic acid as a urinary acidifying agent. I. J. Urol. 94: 297-9.

Barness: 1966.

with High Dosage

Gibt es eine C-Hypervitaminose?

11.

GrarzeL,

Free, H. M. & A. H. Free. 1973. Influence of ascorbic acid on urinary glucose tests. Clin. Chem. 19: 662. ANDERSON, T. W., D. B. W. Rew & G. H. BEATON. 1972. Vitamin C and the common cold: A double-blind trial. Can. Med. Assoc. J. 107: 503-8.

1945.

High dosage vitamin

C in allergy.

Med.

527

12.

13.

H.

Safety Considerations

Klin.

Amer.

61: 665-7.

14.

Ruskin, S. L.

15.

123528153113: ScHRAUZER, G. N. & W. J. RHEAD. 1973. Ascorbic acid abuse: effects of longterm ingestion of excessive amounts on blood levels and urinary excretion.

J. Digest Dis.

16.

CocHRANE, W. A.

Int. J. Vit. Nutr. Res. 43: 201-211. lem?

17. 18.

19. 20. 21.

22.

1965.

Overnutrition in prenatal and neonatal life:

A prob-

Can. Med. Assoc. J. 93: 893-899.

RosENTHAL, G. 1971. Interaction of ascorbic acid and warfarin. J.A.M.A. 215: 1671. Situ, E. C., R. J. Skatsk1, G. C. JoHNSON & G. V. Rossi. 1972. Interaction of ascorbic acid and warfaring. J.A.M.A. 221: 1166. Morin, R. J. 1972. Arterial cholesterol and vitamin C. Lancet 1: 594—5. ANDERSON, T. W., D. B. W. Rein & G. H. BEATOoN. 1972. Vitamin C and serum cholesterol. Lancet 2: 876-7. HeErsert, V. & E. Jacos. 1974. Destruction of vitamin Bi. by ascorbic acid. J.A.M.A. In press. Briccs, M. H., P. GarciA-WEBB & J. JOHNSON. 1973. Dangers of excess vitamin C. Med. J. Austral.

2: 48-9.

DISCUSSION

Ms. E. BARRETT: You mentioned that prothrombin times were a problem taking anticoagulants. Has anybody tried to give ascorbic acid patients in before the anticoagulant to see if it affected the prothrombin time? Dr. BARNESS: The only time we would use ascorbate therapeutically to normalize the prothrombin would be in those patients with liver disease, in those taking antibiotics, or in those who have ingested rat poisons. We see this in children who take warfarin and I would assume that this would be a good therapeutic measure. It brings the prothrombin down to normal levels, but not to levels lower than normal. 1 would like to Dr. M. BRIN (Roche Research Center, Nutley, N.J.): refer to the problem of “sickling.” Dr. Lubin at the University of California at Oakland has studied vitamin E levels in those with the sickle cell trait and found that they are very low, on the order of 0.2 mg %, and that persons with sickle cell trait do have pro-oxidative hemolysis correctable by vitamin E. Whether or not vitamin C would influence this in individuals who have the sickle cell trait but who have normal intakes of vitamin E has not been determined. Dr. D. Stusss: I would like to ask the speaker to comment on the therapeutic use of vitamin C in patients who have phosphate crystals in the urine. Dr. BARNEsS: The urine should not be acidified in the presence of phosphates. Patients with a high phosphate excretion level, at least such children, almost invariably have renal tubular acidosis, so that I would be concerned about acidification of the urine. There is the other aspect of collagen formation that makes this a more complicated question than I can answer. Dr. R. E. Honces: Dr. Barness, what do you feel about the reports relating

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to fetal developmental defects as a result apparently of taking very large doses of ascorbic acid during pregnancy? Now, the information that I am aware of is very flimsy and only suggests this. A Russian investigator reported spontaneous abortions in pregnant women as a result of ingesting large doses of ascorbic acid; she went on to report similar findings in experimental animals. There also were reports in the French literature, again not as well done as we would like, suggesting that large amounts of ascorbic acid given to experimental animals at the onset of pregnancy would or could cause either interruption of the pregnancy or birth defects. I do not offer these as factual evidence. I only mention them as a puzzling and often troublesome possibility. Do you have any more definitive information to either refute or support these reports? Dr. BARNESs: I do not have any information. At this conference we heard a paper where the opposite findings were reported, that is, that those on low doses of vitamin C aborted and those on high doses had a more successful pregnancy. Anything I would say in this area would be entirely speculative. Dr. G. H. BEATON: Is anyone doing long-term controlled studies with large numbers of guinea pigs for several generations and following that sequentially? I think that Dr. Rivers did so. Would you care to comment on that type of study from a toxicity standpoint? Dr. BARNEss: I think it is as good a toxicity trial as one can have for guinea pigs. One cannot do this kind of study with humans, however.

ASCORBIC

ACID

FUNCTION AND DURING COLDS C. W.

M.

METABOLISM

Wilson

Department of Pharmacology University of Dublin and The Allergy Clinic Mercer’s Hospital Dublin 2, Ireland

Investigations of the relationship between symptoms of the natural common cold and administration of supplementary vitamin C must record and take into account the following factors: 1. Definition of the cold syndrome in terms of incidence (number of colds per person in unit time, generally taken as duration of study); duration of syndrome (number of days during which a defined number of symptoms persist

in individual patients); and severity of symptoms (recorded subjectively on a daily basis for individual symptoms on a graded scale of which the lowest value records absence of the symptom). Days of absence from work provide a general measure arising from cold disability.1 Integrated morbidity * is defined as the product of incidence of colds, and severity of individual colds. The two values for incidence and integrated morbidity are not independent of one another. Integrated morbidity provides a general measure of intensity of the syndrome as opposed to the disability it produces. Total symptom intensity provides a similar measure to integrated morbidity. The Total Intensity Score is defined as the total severity of any symptoms of the cold syndrome present on each day of reporting, divided by the total number of days of reporting.® Variation in symptom quality can be evaluated by measuring the degree of association between symptom pairs.* If viral diagnostic tests, or antibody measurement, are not included in the investigation, a screening process is necessary to exclude upper respiratory allergic disease. Local respiratory symptoms of an allergic nature, those produced by mental stress in subjects prone to vasomotor rhinitis, and those arising from chronic infection of the nasal passages,” are eliminated from the data on the cold syndrome by excluding syndromes that exceeded 21 days in duration.* 2. Measurement of plasma and leukocyte ascorbic acid concentrations in the subjects. Correlation of these measurements with the cold symptomatology enables the relationship between tissue ascorbic acid and the cold syndrome to be measured directly. Administration of exogenous supplementary vitamin C during the cold syndrome only allows indirect assumptions to be made about the relationship between the cold syndrome and the effect associated with vitamin C administration. 3. The preliminary decision as to whether supplementary vitamin C will be administered on a prophylactic or a therapeutic basis. If it is decided that a prophylactic investigation will be carried out, it is essential that the period of administration of supplementary vitamin C is sufficiently long to ensure that tissue concentrations of ascorbic acid have been elevated in the subjects when they are included in the trial. Factors such as sex, age, dietary intake of vitamin C, and initial ascorbic acid status of the tissues can all have profound effects on

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the outcome of the trial.© Administration of supplementary vitamin C during the cold syndrome alters tissue ascorbic acid levels, but the relationship between such therapeutic alterations and the prophylactic administration of supplementary vitamin C is unknown. 4. Control observations for comparison with the effect produced by supplementary vitamin C, whether administered on a prophylactic or a therapeutic basis. The control observations may compare the effect of the supplementary vitamin C with placebo tablets on the cold symptomatology, or examine the alterations produced by the vitamin C on blood and tissue concentrations of ascorbic acid and correlate these with the cold symptomatology. When the investigation consists of a therapeutic or prophylactic trial, it is essential to confirm that the medication is being taken by carrying out confirmatory measurement of blood ascorbic acid concentrations. 5. Information about the age, sex, dietary intake of vitamin C of the sample, and degree of tissue saturation with ascorbic acid at the beginning of the investigation, so that normal variations in ascorbic acid metabolism can be taken into account in evaluation of the results. Evidence about the effect of vitamin C on the symptoms of the common cold has provided results of questionable significance because these factors have been inadequately defined and measured in the past. Incomplete definition of symptoms results in imprecise evaluation of the effect of placebo or vitamin C supplementation. The pathophysiological alterations in ascorbic acid which are associated with the presence of cold symptoms‘ may be of sufficient magnitude to mask effects produced by administration of the supplementary vitamin C, which would affect tissue ascorbic acid concentrations and metabolism under normal circumstances. Differences between individuals, and samples, in age, sex, Vitamin C intake, tissue saturation, and other concurrent

illnesses, may

not

only alter cold symptomatology, but also introduce into the trial design unrecognized variations in ascorbic acid metabolism. The way in which these factors can influence and be used to interpret results of investigations on the relationship between cold symptoms and administration of supplementary vitamin C, are discussed below. PROPHYLACTIC

TRIALS

The common cold is not a precisely defined disease. To both the professional and the layman the term ‘“‘a cold” has a clearly understood meaning: a short mild illness in which the main local symptoms are found in the upper respiratory tract, and in which nasal symptoms predominate." Its occurrence is shown by the appearance of these symptoms at varying intervals, and with different degrees of severity, during the progress of the disease. Several investigations 1° have indicated that associations tend to occur between the individual symptoms of the common cold. The degree and extent of these associations have, however, been analyzed only recently,' when it was found that the symptomatology of the cold syndrome can be assessed in terms of symptom complexes. The cold syndrome comprises the following symptoms: Toxic

Complex

Catarrhal

Complex

1. Sore throat 4. Headache 6. Feverish

2. Cold in head 3. Cough 7. Nasal obstruction

9. Out of Sorts

8. Nasal

discharge

Wilson:

Ascorbic Acid During Colds

231

A score for the severity of each symptom when it occurs was obtained daily during prophylactic trials in which the effects of daily administration of vitamin C or placebo tablets were compared on the symptoms over a specified period in school children. The degree of association between pairs of symptoms was determined by the correlation values between individual pairs. These correlations measured the extent to which symptom pairs were reported together and extended over a range of 0 to 100%. All the symptoms were reported together to a varying degree in the whole common cold, the syndrome of the Wcomplex. This was demonstrated by the fact that there were no negative correlations among the symptom pairs. Toxic and catarrhal symptom complexes (Tand C-complexes) became separated at the value for the maximal correlation which linked a T-symptom and a C-symptom in the W-complex. Symptom-

Treatment procedure

Maximum Correlation Values in W- Complexes 4

Symptom - pair associations in

T- Complexes 9

Symptom - pair

associations in

C- Complexes

3

ox

| ‘

Figure 1. Maximal correlation values for the associations between symptom pairs making up the whole-cold complex (W-complex). Differentiation of the W-complex

into linked association pairs at values higher than the maximal correlation values for the W-complex to form separate T- and C-complexes. Walues and diagrams are shown for population samples of boys (B) and girls (G) receiving placebo tablets or different doses of vitamin C daily during 8% months. (From Wilson et al.* By permission of the European Journal of Clinical Pharmacology.)

pairs in the T- or C-complexes were associated together at higher correlation values than the values that linked T- and C-symptoms in the W-complex. It was found that symptoms 1, 4, 6 and 9 were associated together. They were therefore defined as the toxic complex. Symptoms 2, 3, 7 and 8 were similarly associated. They were defined as the catarrhal complex. Symptom complexes were evaluated in terms of the average values for the correlation coefficients which made up the individual symptom-pairs. The correlations of individual complexes could be related to the characteristics of the groups being investigated, and to the treatment being administered, during the trial. The symptom complexes were analyzed qualitatively by diagrams. These illustrate how individual complexes were made up of symptom-pair associations which were interlinked to varying degrees (FIGURE 1).

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Boys and girls treated with placebo tablets had high correlation values in their W-complexes in comparison to the boys and girls treated with 500 mg vitamin C daily. The average correlation within each of the complexes diminished when the dose of vitamin C was raised. Administration of 200 mg of vitamin C reduced the number of linking associations in the T-complexes in girls; 500 mg was required before the linking associations in the C-complexes in the girls became reduced. The average correlation for the W-complex in girls only became reduced when the numbers of linking associations in both T- and C-complexes had diminished. 500 Mg reduced the T-complex associations in boys in comparison with 200 mg, but this dose appeared to make their C-complexes more complicated. The average correlation for the W-complex became reduced with the smaller dose of vitamin C in boys. Administration of prophylactic vitamin C altered the intensity and quality of the complexes in relation to the dose administered, and in relation to the response of the sexes participating in the trial. Toxic and catarrhal complexes become dissociated, and can develop and disappear independently of each other, during administration of vitamin C. This suggests that the general disturbance of cellular metabolism and the localized mucosal inflammation are independent pathophysiological processes. This method of analysis of symptoms that together make up a disease syndrome is more sensitive than, and the results obtained from it may be independent of, evaluation of severity or duration of the syndrome in its entirety. This analytical method can detect changes in the syndrome produced by prophylactic and therapeutic procedures. The administration of prophylactic doses of 200 and 500 mg vitamin C to school children produced beneficial effects on the incidence, duration, severity and total intensity of symptoms when these are measured independently during the cold syndrome.* Supplementary vitamin C reduced catarrhal symptoms in the first instance. Larger doses were required in order to exert benefit on toxic symptoms. Vitamin C exerted more beneficial effect on total intensity and severity of catarrhal symptoms in girls than in boys. The larger dose of vitamin C appeared to enhance the severity of the catarrhal symptoms in boys in addition to maintaining the complexity of the C-syndrome. The whole-cold syndrome was reduced in intensity in girls. Its complexity was reduced only by the larger dose of vitamin C. The whole-cold syndrome was unaffected in total intensity in boys, although linking associations were reduced in their Wcomplexes. Unless a beneficial effect is produced on toxic and catarrhal symptoms, the subject is not prepared to acknowledge that any beneficial effect is occurring on the cold syndrome. It has been reported that vitamin C reduces catarrhal more than toxic symptoms" and also that toxic symptoms are reduced more than catarrhal symptoms.' The total intensity of the C-complex influences the patient’s subjective assessment of the T-complex when symptoms are being assessed throughout the duration of the syndrome. Analysis of total intensity and symptom-pair associations show that the T-complex is beneficially affected by a daily dose of 500 mg vitamin C in both sexes, and the C-complex is reduced in intensity in girls, Measurement of incidence and total intensity of the cold syndrome * provides values for measurement of effects of prophylactic treatment. It has been stated that 8-10% of the population are immune to the common cold.12._ By making use of this information, and combining it with the results obtained from the

cold trials in which the effects of 200 and 500 mg were compared with placebo

Wilson:

Ascorbic Acid During Colds

RSIS

therapy on cold symptomatology, a log-dose response line has been drawn that shows the effect of these prophylactic doses on cold incidence '* (FIGURE 2). The figure demonstrates that there is a difference between male and female response to prophylactic vitamin C therapy against the common cold. A daily dose of 500 mg provides protection against the common cold in 30-40% of the population of school girls, but has little protective effect in boys.

Resistance 9], Cold Common the to °

200

500mg DAILY

5-0

1-0

DOSE OF ASCORBIC

Wg

ACID

and Figure 2. Relationship between percentage resistance to the common cold vitamin administration of prophylactic daily doses of 200 and 500 mg supplementary C to male

(@) and female

(X) school children.

(From

Loh et al.”

By permission of

Clinical Pharmacology and Therapeutics.) THERAPEUTIC

TRIALS

The attainment of a beneficial therapeutic effect on the common cold can be defined as the production of a significant decrease in the duration and tary severity of some or all of the symptoms by administration of supplemen be can action c therapeuti Its syndrome. the of progress the vitamin C during evaluated by comparing the effect of vitamin C or placebo administration from . the time when the first symptoms appear until the last symptom disappears ic and Anderson et al.1:#4 have carried out long-term combined prophylact in therapeutic trials in which vitamin C was found to exert beneficial effects c therapeuti and ic prophylact Short-term therapy. placebo with n compariso in colds induced artificially and trials have been carried out 2% 2° on natural Significant which doses of 1 g or more were administered daily to the subjects. colds, benefit was produced by vitamin C therapy in the naturally produced subjected were who subjects of number smaller the in whereas it was ineffective to nasally instilled viral infection.

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Another method for evaluating the therapeutic effectiveness of vitamin C in the common

cold is to measure

alterations

in ascorbic

acid

metabolism,

and

relate such changes to the symptoms during the cold, and to ascorbic acid blood levels after disappearance of the cold symptoms. Such investigations must take account of the fact that plasma and leukocyte ascorbic acid concentrations are reduced in individual patients while cold symptoms are present.1*:?* Ascorbic acid shows great lability in its passage to and from the plasma into the tissues.*® In consequence rapid changes in plasma levels may occur in response to metabolic demand from particular tissues which are not reflected by concurrent changes in leukocyte concentrations, Single loading doses of vitamin C in the range 500-2000 mg have been administered during colds, and plasma and leukocyte ascorbic acid concentrations measured during the following four hours. These ascorbic acid blood response curves (AABRC 500-2000) evaluate changes in ascorbic acid metabolism during the colds, and their determination is repeated after disappearance of the symptoms in the same subjects.*°°? Changes in the plasma-leukocyte (P/L) correlations and regression angles between successive AABRC’s enable the rate and direction of transfer of ascorbic acid between plasma and leukocytes to be evaluated.22 The occurrence of positive correlation and regression coefficients, together with rising plasma and leukocyte concentrations, indicate a positive accumulation of vitamin C in the tissues for metabolism or storage. This is associated with uptake of ascorbic acid from the plasma into the leukocytes. Negative coefficients together with a rising plasma and low leukocyte ascorbic acid concentrations, indicate a negative metabolic balance. This is attributable to increased for, or inadequate dietary intake of, the vitamin.

metabolic

demand

During colds, and after recovery from cold symptoms, plasma levels were significantly raised four hours after administration of vitamin C. The rise was greater after 2,000 mg than after 500 mg of vitamin C (TABLE 1). The resting leukocyte values and levels after supplementation of ascorbic acid were lower in males than in females during colds. There was a considerable increase in leukocyte uptake of ascorbic acid in females, but the increase in male concentrations was slight after the loading dose of 2,000 mg. The loading dose of 500 mg was not associated with elevation of leukocyte ascorbic acid during colds in either sex, but was associated with raised ascorbic acid values in the postcold test. During colds the mean P/L regression coefficients were similar in males to their post-cold regressions. This indicates that there was a similar flow of ascorbic acid into the plasma after supplementation during cold and postcold periods. Inadequate ascorbic acid was available for storage in both tests. There were relatively high plasma and low leukocyte ascorbic acid concentrations. This suggests that male ascorbic acid metabolism had not been restored to normal at the time of the postcold test. In the females, the mean regression coefficient in the postcold test was higher than in the cold test. This indicates that the uptake of ascorbic acid into the leukocytes occurred simultaneously with the rise in plasma concentration after supplementation, and was associated with accumulation of ascorbic acid in the tissues. The following metabolic factors appear to determine leukocyte uptake of ascorbic acid during colds: (1) Metabolic requirements for ascorbic acid in the leukocytes may be greater during colds in females than in males. (2) Requirements in females for ascorbic acid in the inflamed and toxic tissues are less, and so more ascorbic acid is available for storage in the leukocytes during colds. In either case it

appears that the level of ascorbic

acid saturation in the leukocytes is raised

535 Ascorbic Acid During Colds Wilson:

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during colds in females and is lowered again after recovery from the colds. If the saturation level is raised in males during colds, it does not appear that a dose of 2,000 mg is adequate to increase the uptake in males (TABLE 1). The relationship between plasma and leukocyte ascorbic acid concentrations, and the occurrence of toxic and catarrhal symptoms has been examined following loading doses of 500 and 2,000 mg vitamin C during colds.*°:*! To take into account concurrent changes in ascorbic acid metabolism and cold symptomatology, P/L regressions at the time of the AABRC were correlated with ratio of toxic and catarrhal symptoms (T/C ratio) during the progress of the cold. It was found that a significant positive correlation existed between these relationships with a loading dose of 500 mg vitamin C. This indicated that a flow of ascorbic acid from the plasma into the leukocytes was associated with colds predominantly of the toxic variety. C-colds were correlated with a reverse flow of ascorbic acid associated with high plasma and reduced leukocyte ascorbic acid concentrations. The correlation between total symptom score and P/L regression was not significant. When a loading dose of 2,000 mg was administered, a significant relationship was found to exist between severity of catarrhal symptoms and P/L regression particularly in females. This suggests that the larger dose was affecting predominantly the catarrhal symptoms, presumably because leukocyte and general tissue uptake was sufficient to meet metabolic requirements for dealing with toxic symptoms. During these colds the toxic symptoms were considerably less severe than catarrhal symptoms, and were less marked in males than in females. Plasma concentrations of ascorbic acid have been compared with salivary and lingual concentrations in the same individuals during and after recovery from colds

(FiGuRE

3).

During colds the concentrations

were

60-70%

of the

values after recovery from the cold symptoms; the concentrations in plasma and tongue were significantly lower during the colds. The reduction in leukocyte ascorbic acid levels during colds are reflected by corresponding changes in ascorbic acid concentrations adjacent to the infected areas. It can be assumed that supplementary vitamin C passes into these tissues from the plasma during colds. P

0-O5