Comparison between gravimetric and reduction methods for determination of pregnandiol glucuronidate

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COMPARISON BETWEEN GRAVIMETRIC AND REDUCTION METHODS FOR DETERMINATION OF PREGNANDIOL GLUCURONIDATE

A Thesis Presented to the Faculty of the Graduate School University of Southern California

in Partial Fulfillment of the Requirements for the Degree Master of Science

by Oscar Hechter June 1942

UMI Number: EP65500

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T h is thesis, w r itte n by

..........O S C A I L M C H T E R ....................

under the direction o f h.A.?. F a c u l t y Committee, a n d a p p r o v e d by a l l its m e m b e r s , has been presented to and accepted by the C o u n c i l on Graduate Study and Research in p a r t i a l f u l f i l l ­ m e n t o f the r e q u i r e m e n t s f o r the degree o f MASTER OF SCIENCE

Dean

Secretary Date.... JUNK,.19.42

Faculty Committee

Chairman

TABLE OF CONTENTS Page INTRODUCTION...........

1

HISTORICAL REVIEW

3

METHODS AND RESULTS

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

The development of the method for determination of sodium pregnandiol glucuronidate

.........

9

Comparison between gravimetric and reduction methods for determination of pregnandiol glucuronidate

• • • • • • • . • • • • • • • • « • • •

15

S U M M A R Y .......................................... . .

19

BIBLIOGRAPHY

20

LIST OF TABLES Page I*

Effect of Acid Concentration and Time of Boiling upon the Production of Reducing Activity from Pregnandiol Glucuronidate » » • • • • • • • • • • •

II#

11

Comparison of the Effects of Hydrolysis of Sodium Pregnandiol Glucuronidate in 50 per cent Acetone and in Water

III#

• • • • • • • # # • • • » • # • • • •

13

Comparison of Gravimetric and Chemical Determina­ tions of Pregnandiol Glucuronidate in Normal Women • • • • « • • « • • • • • • • • • • • • • • •

IV#

17

Comparison of Gravimetric and Chemical Determina­ tions of Pregnandiol Glucuronidate in Two Patients Failing to Show Luteal Activity as Evidenced by Vaginal Smears • • • • • • • • • » • •

18

LIST OP FIGURES Page 1.

Pathways of Progesterone Metabolism « • • • • • • • • •

2*

Light Absorption Plotted against Pregnandiol Glucuronidate Concentration

8

23

INTRODUCTION

There has long been need for an accurate clinical test for corpus luteum function#

First attempts to identify the active principle of the

corpus luteum, progesterone, in the body fluids of the human females were disappointing when various methods of bioassay were employed#

However,

recognition of pregnandiol glucuronidate as a metabolic derivative of progesterone, has led to the indirect evaluation of luteal activity in women by estimation of urinary pregnandiol glucuronidate

(1)*

The method

developed by Venning for determination of pregnandiol glucuronidate (2) and used thereafter by all workers in the field with little or no modifi­ cation consists of a four-fold extraction of pregnandiol glucuronidate from urine with butyl alcohol, and after purification sodium pregnandiol glucuronidate is precipitated from water with acetone and estimated gravimetrically*

Venning reported that after two precipitations the sodium

pregnandiol glucuronidate isolated is pure and may be estimated gravimetrically.

However, it is evident that Venning's modified gravimetric method

affords no absolute guarantee that the material obtained from urine by that procedure is actually pregnandiol glucuronidate*

The need for a quantitative

check of the purity of the pregnandiol glucuronidate isolated by the method of Venning has led to the development of a simple method of pregnandiol glucuronidate determination, which depends on the estimation of reducing activity obtained after acid hydrolysis of sodium pregnandiol glucuronidate* The results found with this method are compared with pregnandiol glucuroni­ date values obtained gravimetrically*

2

After this work had been completed and a summary sent for publication, the report of Allen and Viergiver appeared (3)*

These workers demonstrated

that the sodium salt of pregnandiol glucuronidate could be determined after acid hydrolysis by estimation of copper reducing activity, and improved Venning’s method at various points.

Although a different reducing method i

was used and different hydrolysis conditions were employed, it is. clear that our work furnishes independent confirmation for the general idea that the pregnandiol complex may be analyzed by reduction methods after acid hydrolysis.

HISTORICAL REVIEW

The first attempts to study corpus luteura function by assaying the blood and urine of pregnant women for progesterone, as determined by bioassay of progestational activity demonstrated that little or no proges­ terone was present in these sources.

Loewe and Voss (4) found only one

rabbit unit of biologically active material in 20 liters of urine from women during the secretory phase of the menstrual cycle, and Bloch (5) reported that 500 cc. of blood from a pregnant woman contained no proges­ tational activity.

It was further shown that the tissues responsible for

the production of progesterone, contained at most only minimal amounts of the hormone.

Thus, Adler and his co-workers (6) and Ehrhardt (?) reported

only small amounts of progestational activity*in human placentae, while Clauberg et al (8) and Pratt and his associates (9.) found questionable or only small amounts of activity in 50 to 60 grams of human corpora lutea* These findings dashed the hopes of many who believed that luteal evaluation might be accomplished by bioassay of the corpus luteum hormone in the urine and blood of patients, and posed the question as to the ultimate fate of progesterone in the body. In 1929, Marrian (10) isolated a steroid from the urine of pregnant women which he named pregnandiol.

Shortly thereafter, Dingemanse and co­

workers (11) and Butenandt (12) independently confirmed this finding. Butenandt and his associates (13) subsequently established the structural formula for this new substance, which is shown in Figure 1, and which may be compared to that of progesterone.

It will be seen that pregnandiol and

progesterone are closely related chemically.

Shortly thereafter, by fairly

4

simple chemical means Butenandt and Schmidt (14) were able to convert pregnandiol to progesterone*

This accumulated data, however, did not

immediately culminate in the recognition of the biological relationship between these steroids*

Several groups of investigators attempting to

demonstrate progesterone conversion to pregnandiol met difficulties in attempting to develop methods for pregnandiol, and thus their efforts in this connection proved of little value* In view of the observations of Cohen and his co-workers (15) that over 99 per cent of the estrogenic substances in the urine of pregnant women occur in an acid-hydrolyzable combined form, and those of Adler (16) that some of the androgenic principles excreted in the urine of males probably exist in a butyl alcohol-soluble acid-hydrolyzable combined form, it seemed reasonable to expect that pregnandiol is excreted in a similar combined form* In 1936, Yenning and Browne (17) were able to extract with butyl alcohol sodium pregnandiol glucuronidate from the urine of pregnant women.

Odell

end Marrian (18) reported almost simultaneously that they had determined in an "alkali washed butyl alcohol extract” of the urine of pregnant women, the presence of ”an ether solubile combined form” of pregnandiol*

These observa­

tions were followed with the development of a quantitative gravimetric method of pregnandiol glucuronidate determination by Venning (l, 19) described pre­ viously in this paper* Using the method of Venning as a tool, the Montreal group established that (a) sodium pregnandiol glucuronidate is excreted in the progestational phase of the menstrual cycle (20), (b) that sodium pregnandiol glucuronidate is excreted in the urine of human pregnancy in concentrations which become increasingly higher as gestation approaches term (21, 22), (c) parenteral

5

administration of progesterone to ovariectomized women and in those in whom no corpora lutea were thought to be present was followed by the onset of urinary excretion of sodium pregnandiol glucuronidate (23), (d) following administration of progesterone to hysterectomized women urinary pregnandiol complex was not recovered.

As a result of these findings, the Montreal

group (24) considered that progesterone, in common with other sex steroids, is excreted only in hydrosoluble conjugated form and is metabolized in the following manner:

progesterone is transformed into pregnandiol in the

uterus, and it is conjugated with glucuronic acid in the liver, whereupon the pregnandiol glucuronidate is excreted via the kidney;

when the uterus

has been removed progesterone is not metabolized completely and either is destroyed or is excreted by the kidneys in some form other than pregnandiol glucuronidate.

Subsequent work in other laboratories confirmed the essential

findings reported by Venning, Browne, et al, in that pregnandiol glucuronidate is solely excreted in the progestational phase of the menstrual cycle (25, 26, 27) and is increased progressively in pregnancy (28, 29, 30, 31).

Nevertheless

two principle findings consistently appeared which challenged the concepts advanced by the Montreal group.

First, the idea that the uterus (or the

endometrium) is necessary for the conversion of progesterone to pregnandiol was shown not to hold, when it was demonstrated by Buxton and Westphal (32) and immediately thereafter by Hamblen, Cuyler* and Hirst (33) that proges­ terone administered to males led to the appearance of urinary sodium preg­ nandiol glucuronidate.

Secondly, it was observed that progesterone adminis­

tered by injection to non-hysterectomized women was not recovered as urinary pregnandiol glucuronidate (25, 26, 27).

These data which gave rise to con­

flicting interpretations were reconciled when it was shown (35) that the

6

hysterectomized -women if given large doses of progesterone will indeed convert a small fraction of the luteal hormone to pregnandiol glucuronidate.

In like

fashion, the failure of some women with intact uteri to convert progesterone to pregnandiol was shown to be a question of hormone dosage*

If 5 mgm* doses

of the hormone are administered for many days (independent of the total amount given) pregnandiol glucuronidate does not appear in the urine;

however, if

20 mgm* or 40 mgm* of progesterone are given, pregnandiol complex can be regu­ larly recovered*

The percentage of recovery, however, of pregnandiol glucu­

ronidate never is quantitative under the most favorable circumstances (35)* The lack of quantitative recovery of administered progesterone in humans provides strong evidence for the existence of an alternative pathway of pro­ gesterone metabolism.

Some extra-pregnandiol route of progesterone metabolism

likewise exists in monkeys and other common laboratory animals since they fail to shoYr pregnandiol either in the free or conjugated form during pregnancy or following the administration of large doses of progesterone (36, 37)* The supposition that estimation of pregnandiol glucuronidate despite the shortcomings described, measures progesterone activity specifically, can no longer be held, since it has been shown that desoxycorticosterone administered to men leads to the appearance of urinary

pregnandiol complex (38).

The

present state of our knowledge concerning progesterone metabolism is illus­ trated in Figure 1*

It shows in summary form the conclusions derived from the

studies mentioned in the previous discussion. Examination of Figure 1 reveals that quantitative evaluation of luteal activity is not obtainable by estimation of pregnandiol glucuronidate in the absence of quantitative information regarding the efficiency of the extra-ovarian factors necessary for the excretion of pregnandiol complex.

Further, the

7

presence or absence of urinary pregnandiol glucuronidate may not serve as a qualitative test for luteal activity since it is possible to obtain zero values for pregnandiol glucuronidate in the presence of circulating pro­ gesterone.

Recognizing these difficulties, it is still possible to use

pregnandiol glucuronidate determinations for useful information.

The

presence of urinary pregnandiol complex may be taken as strong suggestive evidence for functional lutein tissue, and the quantity of pregnandiol glucu­ ronidate excreted represents a minimal estimate of the amount of metabolized progesterone.

8

FIGURE 1

c^cH*

Pregnandiol

Progesterone

Pathway of Progesterone Metabolism

progesterone

->???