Vitamin A and Carotene: Their Stability in Mixed Feeds and Effects of Dietary Lipids on Their Utilization

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PURDUE UNIVERSITY

THIS IS TO CERTIFY THAT THE THESIS PREPARED UNDER MY SUPERVISION

b y _____________

Moore J. Bums___________________________

ENTITLED Vitamin A and Carotenes

Their Stability in Mixed

Feeds and Effects of Dietary Lipids on Their Utilization.

COMPLIES WITH THE UNIVERSITY REGULATIONS ON GRADUATION THESES

AND IS APPROVED BY ME AS FULFILLING THIS PART OF THE REQUIREMENTS

FOR THE DEGREE OF

_______

_

Doctor of Philosophy

Professor

H

ear of

in

Cha rg e

T hesis

S o h q o i .o r D e p a r t m e n t

June__________ is 50

TO THE LIBRARIAN:------

JSC THIS THESIS IS NOT TO BE REGARDED AS CONFIDENTIAL.

C H A D . S C H O O L F O R M 0— 3-4 3— 1M

of

VITAMIN A AND CAROTENE:

THEIR STABILITY IN MIXED FEEDS

AND EFFECTS OF DIETARY LIPIDS ON THEIR UTILIZATION

A Thesis Submitted to the Faculty of

Purdue University

by

Moore J. Burns

In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

June, 1950

ProQuest N um ber: 27714107

All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is d e p e n d e n t upon the quality of the copy subm itted. In the unlikely e v e n t that the a u thor did not send a c o m p le te m anuscript and there are missing pages, these will be noted. Also, if m aterial had to be rem oved, a n o te will ind ica te the deletion.

uest ProQuest 27714107 Published by ProQuest LLC (2019). C opyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C o d e M icroform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106 - 1346

ACKNOWLEDGMENT

The author is indebted to Dr. F. W. Quackenbush for counsel in the planning and conduct of all of the research, to Dr. S. M. Hauge for counsel in the conduct of the animal experiments, and to Dr. S. R. Miles for assistance in design­ ing the animal experiments and statistical treatment of the data.

TABLE OF CONTENTS

Page ABSTRACT.

.................................................

i

INTRODUCTION...............................................

1

METHOD FOR VITAMIN A DETERMINATION....................

3

Introduction. .. . ....................................

3

Analytical Procedure.........

4

Results and Discussion..............................

7

Summary...............................................

12

STABILITY OF VITAMIN A CONCENTRATES......................

13

Introduction............ Experimental Procedure

13 ........

14

Results and Discussion..............................

15

Summary..........

15

EFFECT OF TOCOPHEROL, TWEEN, AND DIETARY FAT ON THE UTILIZATION OF VITAMIN A AND BETA -CAROT E NE ......

25'

Introduction..............

25

Experimental Procedure.......................

26

Results and Discussion. .......

30

Summary

........................................

32

UTILIZATION OF VITAMIN A AND BETA-CAROTENE IN THE PRESENCE OF SMALL AMOUNTS OF MINERAL OIL..................

33

Introduction.......................

33

Experimental Procedure...................

33

Results and Discussion.....................

35

Summary......

39

BIBLIOGRAPHY AND CITED REFERENCES........................

40

FIGURES Figure 1.

Page

Stability of Concentrate No. 1 Alone and When Mixed with Various Carriers.................

2.

Stability of Concentrate No. 2 Alone and When Mixed with Various Carriers.................

3.

..........

21

Stability of Concentrate No. 7 Alone and When Mixed with Various Carriers.......................

8.

20

Stability of Concentrate No. 6 Alone and When Mixed with Various Carriers.

7•

19

Stability of Concentrate No. 5 Alone and When Mixed with Various Carriers.................

6.

18

Stability of Concentrate No. 4 Alone and When Mixed with Various Carriers.................

5*

17

Stability of Concentrate No. 3 Alone and When Mixed with Various Carriers.................

4.

16

22

Effect of Mineral Oil on the Utilization of Vitamin A and Beta-Carotene

......

38

TABLES

Table 1.

PaSe

Efficiency of Acetone-Hexane Extraction of Vitamin A from Feeds under Various Conditions....

2.

Recovery of Vitamin A Esters in Mixed Feeds

8

and of

Vitamin A Acetate Added to Eluates from Mixed Feeds 3#

.... ••*•........................

9

Recovery of Vitamin A Acetate Added to Oils and Mixed Feeds..........

11

4.

Stability of Commercial Vitamin A Concentrates

23

5»

Basal Diets..........................................

28

6.

Balanced Incomplete Block Design for Division of Litters.........................

7*

29

Average Adjusted Gains of Vitamin A Depleted Rats Receiving Various Supplements...............

31

8. Growth of Vitamin A Depleted Rats Receiving Various Amounts of Mineral Oil....................

37

VITAMIN A AND CAROTENE :

THEIR STABILITY IN MIXED FEEDS

AND EFFECTS OF DIETARY LIPIDS ON THEIR UTILIZATION

ABSTRACT A chromatographic physico-chemical method is described for the estimation of vitamin A esters in oils and mixed feeds without saponification.

A hexane-acetone solution Is

chromatographed on magnesia and Hyflo Super-cel to separate the vitamin A esters from the oxidation products and the pigments of the feed.

The vitamin A esters are distinguished

from pigments, tocopherols and other constituents on the column by their fluorescence in ultraviolet light.

The

vitamin A in the eluate is measured colorimetrically after reacting the vitamin with antimony trichloride.

To correct

for the action of inhibitors of the -antimony trichloride reaction, a known amount of vitamin A acetate is added to a duplicate aliquot of the eluate. The above method was used to study the stability of vitamin A in commercial vitamin A products.

Seven different

dry vitamin A products were furnished by industry which con­ tained 150 to 1500 micrograms per gram of vitamin A or betacarotene.

They were subjected to a series of four storage

experiments as follows*

(1) stored as received without

mixing with a carrier; mixed with (2) ground whole yellow corn, (3 ) soybean oil meal (expeller), and (4) glucose (cerelose) In such proportions that the mixtures contained

il

approximately ten micrograms per gram of vitamin A.

Tlie

concentrates and mixtures were placed in screw-cap bottles and stored in the dark at room temperature.

The vitamin A

or carotene content of the concentrates and mixtures was determined at the time of storage and at monthly intervals thereafter for a period of six months.

The concentrates

retained 60 to 85 per cent of their initial content during the six-month storage period; the highest stability being shown by products which employed vegetable meals or the antioxidants which they contain.

Soybean oil meal improved

the stability of the vitamin in most of the products.

Ground

yellow corn showed a similar effect but to a lesser degree. Glucose (cerelose) diminished the stability in all cases. An experiment was conducted with the rat to determine -the' ©ffact of tocopherol and fat content of the diet upon the efficiency of vitamin A and beta-carotene in aqueous and nonaqueous media.

When 1.0 mg. of tocopherol was fed daily,

there was no significant difference between the potency of 1.0 microgram of vitamin A acetate and 1.0 microgram of betacarotene.

The optimum daily dose of tocopherol was 0.5

to

1.0 mg. for vitamin A acetate and 1.0 mg. for beta-carotene. 2.0 mg. of tocopherol daily diminished the efficiency of utilization of beta-carotene and to some extent that of vitamin A.

An aqueous medium diminished the efficiency of

utilization of 1.0 microgram daily doses of vitamin A acetate but slightly increased that of beta-carotene.

Vitamin A and

beta-carotene were more efficient when fed with a diet con-

ill

talning five per cent fat than with a diet which contained no fat but was supplemented with 0.1 g . of refined corn oil daily. An animal experiment was also conducted to determine the effects of small amounts of mineral oil upon the utilization of vitamin A and beta-carotene and to determine whether an increased percentage of digestible fat in the diet might aid in the absorption of these fat soluble vitamins in the pre­ sence of mineral oil.

As the amount of mineral oil increased,

there was a general decrease in the efficiency of utilization of beta-carotene ; the difference being significant at 0.08 per cent and greater.

The decrease in the effectiveness of

vitamin A was not as great as that of beta-carotene; however, the decrease in growth was significant for the 0.16 and 0.32 per cent levels.

The levels of fat used did not produce a

significant difference in the utilization of vitamin A or carotene.

Alfalfa leaf meal which contained five per cent

mineral oil supported only half as much growth as that which contained no mineral oil; there was no significant difference in the zero and two per cent levels.

Amounts of mineral

oil necessary to reduce dustiness in mineral mixtures or in alfalfa leaf meal were detrimental to the utilization of vitamin A and beta-carotene.

VITAMIN A AND CAROTENE:

THEIR STABILITY IN MIXED FEEDS

AND EFFECTS OF DIETARY LIPIDS ON THEIR UTILIZATION

INTRODUCTION Vitamin A is one of the most important vitamins in the nutrition of farm animals since a dietary source is required by all.

It is also one of the most unstable of the essential

dietary factors.

It does not occur as such in plants but

rather as yellow pigments which may be converted to vitamin A in the animal body.

Beta-carotene is the chief provitamin

and is the only one that is capable of producing two mole­ cules of vitamin A. These yellow pigments are very important sources of vitamin A for farm animals; however, they are also easily oxidized.

Yellow corn may lose 60 per cent of its vitamin

A activity after seven months in storage (l).

The vitamin

A value of legumes decreases after the bloom stage and Russel (2) found that alfalfa may lose 80 per cent of its carotene content during the first 24 hours of the curing process.

Hauge (3) concluded that enzyme activity was re­

sponsible for the large initial loss of carotene in alfalfa following the cutting process. The richest sources of vitamin A are the fish oils but these are stable only when kept cold or in the absence of oxygen.

God liver oil may lose all of its vitamin A activity

within a few weeks when mixed with a feed and stored at room temperature (l).

2

The absorption of vitamin A and carotene is affected by a number of factors.

Mineral oil, emulsifying agents,

vitamin E, and the fat content of the diet are among the known factors (4). This work was designed to obtain more information con­ cerning the influence of certain factors on the ultimate biological effectiveness of vitamin A and beta-carotene. Since their effectiveness depends upon how well they are protected from oxidation in vitro and in vivo and upon the efficiency of their absorption, a reliable method for vitamin A determination wasneeded to study the stability of vitamin A in vitro and a series of animal experiments was required to study effects of dietary factors on their utilization. Each phase of this work has a separate introduction.

3

MET HOP FOR VITAMIN A DETERMINATION Introduction A reliable physical or chemical method Is needed for the determination of vitamin A in feed and food materials. 3io-assays with chicks and rats are quite reliable but are too costly and time consuming for extensive routine use. The best available methods for the colorimetric deter­ mination of vitamin A are based on the Oarr-Price antimony trichloride test (1-6).

This color reaction produced by

vitamin A with antimony trichloride offers the best approach to the determination of vitamin A because of its high sensi­ tivity to" small amounts of the vitamin.

Spectrophotometric

methods seem to work well with high-potency oils and a method has been prescribed by the Association of Official Agricul­ tural Chemists for use with oils.

However, low-potency oils

and feeds contain appreciable amountsof materials which absorb in the same region which is used for vitamin A and high

measurement of

values often result.

Vitamin A occurs in the fat-soluble fraction of feeds together with substances that interfere with the antimony trichloride reaction.

These interfering substances must be

separated to obtain accurate determinations of the amounts of vitamin A present.

Considerable difficulty has been en­

countered in attempting to separate vitamin A alcohol from other substances that appear In the unsaponifiable fraction of certain feeds.

4

Im mttemptlag to separate vitamin A from interfering substances, it was found that vitamin A esters could be separated more easily tban tbe alcohol; and since the vitamin occurs almost completely in the ester form, a simplified procedure for the determination of vitamin A esters has been tried which eliminates saponification and subsequent ex­ traction. In the procedure devised, a known amount of vitamin A acetate is added to a duplicate aliquot of the eluate to correct for the inhibition of the antimony trichloride reaction. Analytical Procedure Materials and Apparatus.

Ohloroform - Analytical Reagent

Grade. Antimony Trichloride Reagent - Prepare by dissolving 20 g . of fresh antimony trichloride in sufficient chloroform to make 100 ml.

Allow to stand for 24 h o ur s, filter and

store in the dark at room temperature. Hexane - Purify commercial hexane, boiling range 65-670G . (The Virginia Gas and Oil Go.) by percolating through silica gel.

(The Davidson Chemical G o . , No. 659 >528 - 2000).

Adsorbent - Prepare by thoroughly mixing one part by weight of Hyflo Super-cel (John-Mansville Go.) and one part of activated, magnesia (Westvaco Chemical Corp., No. 2641). Ultraviolet Light Source - A 100 watt G-E Mazda mercury arc bulb (B-H4).

5

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Added

Oils

and Mixed

Feeds