the effect of zinc-hydrochloric acid hydrolysis on … · the effect of zinc-hydrochloric acid...

THE EFFECT OF ZINC-HYDROCHLORIC ACID HYDROLYSIS ON THE ESTROGENS IN HUMAN URINE*

BY BENJAMIN F. STIMMEL

(From the Laboratory of the Rees-Stealy Medical Research Fund, Ltd., San Diego, California)

(Received for publication, September 8, 1948)

Smith and Smith (1) reported that boiling of human female urine for 3 hours with 4 gm. per cent of zinc dust and 15 volumes per cent of hydrochloric acid resulted in a marked increase of estrogen potency over that acquired by optimum conditions of simple acid hydrolysis; namely, 10 minutes boiling with 15 volumes per cent of hydrochloric acid. They were unable to account for all of the increased estrogenic potency by conversion of estrone to a-estradiol and more complete hydrolysis and they therefore postulated the occurrence in human urine of certain non-estrogenic compounds closely related to the estrogens (estrone, a-estradiol, and estriol), which were rendered estrogenic by hydrogenation (2). Pincus and Pearl- man (3) have obtained evidence for the presence of an additional estrogen in human pregnancy urine which is ketonic and contains a hydroxyl group other than the usual phenolic one. The same type of estrogen recently was reported (4) as being present in human and rabbit blood. The synthesis of two more highly oxygenated estrogens related to estrone and a-estradiol has been accomplished (5), and the suggestion has been made (6) that these (16-ketoestrone and 16-keto-ar-estradiol) may be the compounds postulated by Smith and Smith. Since previous work (7) from this laboratory has given some chemical (calorimetric) confirmation to the hypothesis of Smith and Smith, it was felt that further information on the effects of the hydrolytic technique on the known natural estrogens and on the newly synthesized estrogens might further our knowledge concerning the possible presence of additional estrogens in the urine.

We now present data on the effects of the hydrolytic process as derived by our procedure for the fractionation and photometric estimation of urinary estrogens (8) which show that zinc-hydrochloric acid hydrolysis of the estrogens in human urine (a) converts the major portion of estrone to estradiol, (5) yields estradiol titers which are higher than can be accounted for by mere conversion of estrone to estradiol, and (c) yields estriol titers

* This investigation was aided by a grant from the American Cancer Society upon recommendation of the Committee on Growth of the National Research Council.

Part of the material in this paper was presented at the meeting of the Federation of American Societies for Experimental Biology at Atlantic City, March 16,1948 (Federation Proc., 7, 193 (1948)).

217

by guest on February 12, 2020http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

218 ZN-HCL HYDROLYSIS OF URINE ESTROGENS

which appear higher than can be accounted for by more complete hydrolysis. The effect of hydrolysis on known natural estrogens and on one of their conjugates (sodium estrone sulfate) does not satisfactorily account for these observations. The data derived from the addition of lG-keto- eatrone and 16-keto-a-estradiol to estrogen-free (male) urine before hydrolysis appear to offer a satisfactory explanation.

EXPERIMENTAL

Collection and Preservation of Urine-The human pregnancy urine was collected over a 24 hour period without preservative and was utilized on the following day. If pooled human pregnancy urine or pooled human male urine was required, collection and storage was made with toluene.

Hydrolysis and Extraction-The urine was extracted, chromatographed,

TABLE I

Estrogen Titer of Human Pregnancy Urine

E&one ................................... Estradiol................................. Estrone + estradiol....................... Estriol.................................... Estrone + estradiol + estriol. ............

-

Aliquot A (400 ml.), 10

min. refluxing, 15 vol. per cent

HCl added

Y

340 210 550

3200 3750

Aliquot B :400 ml.), 3 hrs.

refluxing, 15 vol. per cent

HCI + 4 gm. per cent zinc dust added

Y

35 1180 1215 5800 7015

Aliquot B Aliquot A

2.20 1.81 1.87

and the titer determined as previously described (8), except that the butyl alcohol extraction was omitted and the hydrolyzed urine was extracted with an ethyl ether-toluene mixture (9: 1). This extract was concentrated to a convenient volume and separated into neutral and phenolic fractions (8). Wherever the L-520 rnp to L-420 rnp ratio of our Kober color product fell below 6.0, our color correction equation (9) was utilized.

RESULTS AND DISCUSSION

The typical colorometric estrogen data on human pregnancy urine as derived by simple hydrochloric acid hydrolysis and the zinc-hydrochloric acid technique of Smith and Smith are summarized in Table I. Each urine aliquot after hydrolysis was processed in identical fashion, so that any difference in estrogen titer must be attributed to the hydrolytic techniques under investigation. Zinc-hydrochloric acid hydrolysis of human


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

B. F. STIMMEL 219

pregnancy urine caused almost complete depletion of the estrogen in the estrone fraction and yielded an estrogen titer in the estradiol fraction (calculated as cY-estradiol) which was 2.2 times as much as the combined estrone and estradiol fractions by simple hydrolysis. The absence of estrone from the estradiol fraction was verified with the Zimmermann test (10). It is obvious that factors other than merely conversion of estrone to estradiol are involved when the zinc-hydrochloric acid hydrolytic technique of Smith and Smith is employed.

In order to test the hypothesis that more complete hydrolysis may account for the augmented estrogen titers obtained by zinc-hydrochloric acid hydrolysis, a number of experiments were performed in which the residual urine after simple hydrochloric acid hydrolysis and ether extraction was rehydrolyzed by the zinc-hydrochloric acid technique. Data

TABLE II Estrogen Titer after Butanol Extraction of 5’4 Hour Urine Specimen and

Hydrolysis of Aliquots of Extract -

I I Estrogen titer

Aliquot Conditions of hydrolysis -

Zstrone + estra-

diol

15 vol. ?Zo HCI + 4 y. zinc gm. 15 “ yo “ (10 min. refluxing) B1 rehydrolyzed with zinc Total estrogen titer, B1 + Bz BI + B, ---,% A

--

7 Y

220 1500 360 250 46 640

406 890

7 Y

1720 8700 610 5000 686 1000

1296 6000

76 69

Estriol

typical of these experiments are summarized in Table II. In this experiment, a preliminary butyl alcohol extraction of the unhydrolyzed urine was performed as described in a previous publication (8), and the hydrolytic techniques were performed on aliquots of the aqueous extract of the residue from evaporation of the butyl alcohol. The Zimmermann test on the estrone fraction of Aliquot A gave a titer of 96 y. The sig- nificantly higher titer of 220 y obtained with the Kober reagent would suggest that the estrone fraction is contaminated with estradiol, presumably due to the limitations of the chromatographic fractionation procedure, in the presence of relatively large amounts of estradiol. Rehydrolysis by the zinc-hydrochloric acid technique of the urine extract (Aliquot B) following simple hydrochloric acid hydrolysis and extraction yielded an estrogen titer in the combined estrone-estradiol fractions even larger than


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


those obtained by the initial simple hydrolysis. However, the combined estrogen titers derived by sequential hydrolysis (Aliquot B) were still only 76 per cent in the estrone-estradiol fractions and 69 per cent in the estriol fractions of the values obtained by the zinc-hydrochloric acid hydrolysis technique (Aliquot A).

That the marked increase in the estrogen titer obtained by the zinc- hydrochloric acid technique is not peculiar to human pregnancy urine is borne out by the typical hydrolytic data on the urinary estrogen excretion of a man following injection of 2.0 mg. of estrone. The increase of the ratio of estrogen excretion by the two hydrolytic techniques was even greater than that found in human pregnancy urine (Table III).

In these and other studies in which human male urine was used, the urine was divided into aliquots and the hydrolytic technique carried out directly

TABLE III Effect of Conditions of Hydrolysis on Urinary Estrogen Titer

Male subject, carcinoma of prostate; 2.0 mg. of estrone injected.

Experi-

72’

7 7

A Zinc-HCl; 3 hrs. refluxing 133 164 BX Simple HCl; 20 min. refluxing 19 52 BY ‘I rehydrolysis of B,; 3 hrs. refluxing 6 8

Bx + B, 25 60 A

Ratio, B, + BY 5 .f 2.7

-~ 7

26 ;07 14 5

6 0 20 5

T Conditions of hydrolysis

Estrogen titer

Est;one estra- dial

on the urine. Simple rehydrolysis of Aliquot B subsequent to preliminary simple hydrolysis and extraction yielded relatively insignificant estrogen titers, thereby indicating that the time of refluxing does not appear to account for the discrepancy in the estrogen titers obtained by the two hydrolytic techniques.

Since estriol has not been found in pregnant mare urine, the presence of intermediates which might play a r&e in the conversion of estrone to estriol would seem less likely than in the urine of the pregnant woman. Comparative studies of pregnant mare urine by the two hydrolytic techniques therefore might be expected to throw some light on the problem. Accordingly, simple hydrochloric acid and zinc-hydrochloric acid hydrolysis were carried out on aliquots of a specimen of pregnant mare urine.’

Sstriol

* It is recognized that there are present in pregnant mare urine more highly un- saturated estrogens which are not successfully fractionated by our chromatogram.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

B. F. STIMMEL 221

The data (Table IV) show that there were negligible amounts of unconjugated estrogens in our specimen and that simple hydrolysis yielded slightly higher total estrogen titers than zinc-hydrochloric acid hydrolysis. These data may be interpreted as offering some experimental support to the concept that there are present in the human pregnancy urine inter- mediate compounds which play a role in the conversion of estrone to estriol and which augment the estrogen titer in zinc-hydrochloric acid hydrolysis.2

TABLE IV E$ect of Conditions of Hydrolysis on Urinary Estrogen Titer of Pregnant Mare

Urine*

Chromatographic fraction

No hydrolysis Estrone Estradiolt Estriolt

Total......................................’...‘......

Simple hydrolysis, 15 vol. ‘% HCl; 20 min. refluxing

Estrone Estradiolt Estriolt

Total...............................................

Zinc-HCl hydrolysis Estrone Estradiolt Estriol

Total . . . .._._..................................._...

Estrogen ti;+y. per gm.

Kober z#iimemlann test test

0.014 No test 0.006 “ “ 0.004 “ “

0.024

0.449 0.450 0.123 0.168 0.051 0.000

0.623 0.618

0.003 0.022 0.485 0.000 0.091 0.000

0.579 0.022

* Spray-dried powder reconstituted to approximately normal specific gravity with distilled water.

t Calculated as estrone. $ Calculated as estradiol.

A comparison of the effects of the two hydrolytic techniques on essentially estrogen-free urine to which 16-ketoestrone and 16-keto-cY-estradiol

Equilin and equilenin are eluted with estrone and a-estradiol in our technique (un- published data). However, equilin occurs in relatively small amounts and equilenin does not react to produce the typical Kober color product.

* In the absence of more positive proof, we hesitate to attribute the relatively small amounts of material which appeared in our 30 per cent methanol-benzene (estriol) fraction to estriol; the diffuse nature of the trailing boundary of substances eluted principally in a preceding chromatographic fraction would seem a more probable explanation.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


have been added would aid in evaluating the capacity of these compounds to augment the estrogen titers by zinc-hydrochloric acid hydrolysis. It must be emphasized that 16-keto-ar-estradiol, in our Kober test, gave the typical pink color with about the same density at 520 rnp per microgram as did estrone, whereas pure 16-ketoestrone is practically non-chromogenic (11). 16-Keto-a-estradiol added to pooled male urine afterhydrolysisdistributed itself between the 5 per cent methanol-benzene and 30 per cent methanol-benzene filtrate fractions in the ratio of approximately 1: 1 (Table V). When added to pooled male urine previous to zinc-hydrochloric acid hydrolysis, 16-ketoestrone became chromogenic

TABLEV

Effect of Simple and Zinc-Hydrochloric Acid Hydrolysis on Titer of Pooled Male Urine

1 liter of male urine with 16-ketoestrone and 16-keto-cY-estradiol added.

Experi. merit NO.

Per cent recovery of estrogen in chromatographic fractions

2 Conditions of experiment Pzeyhy

and- benzene

(estrone)

1.0 mg. 16-keto-or-estradiol added after simple 2 hydrolysis; 20 min. refiuxing

2.0 mg. 16-keto-c+estradiol added before Zn-HCl 0 hydrolysis; 3 hrs. refluxing

2.0 mg. 16-ketoestrone added before Zn-HCl hy- 0 drolysis; 3 hrs. refluxing

5.0 mg. 16-ketoestrone added before simple hy- 0 drolysis; 20 min. refluxing

Subsequent Zn-HCI hydrolysis; 3 hrs. refluxing 0.3

t

I

-

5 per cent

methanol- benzene

:estradiol)

40

10

8

0

1.66

30 per cent

Tl&baUOl- benzene (estriol)

41

3

4

1

0.58

with the Kober reagent to the extent of 12 per cent, calculated as 16- keto-a-estradiol, and appeared in the 5 per cent methanol-benzene and 30 per cent methanol-benzene filtrate fractions of our chromatogram in the ratio of about 2:l. Similarly, 16-keto-a-estradiol added to pooled male urine previous to zinc-hydrochloric acid hydrolysis was recovered to the extent of 10 per cent in the 5 per cent methanol-benzene fraction and 3 per cent in the 30 per cent methanol-benzene filtrate fraction. Since untreated male urine, upon zinc-hydrochloric acid hydrolysis, did not yield these results, we assume that 16-ketoestrone was reduced to 16- keto-ar-estradiol by the zinc-hydrochloric acid hydrolysis and thereby con- tributed the typical Kober color product. Reduction of 16-ketoestrone


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

B. F. STIMMEL 223

to 16-keto-Lu-estradiol with zinc dust and acid is in accord with Huffman and Lott’s data (5) on the synthesis of 16-keto-cY-estradiol.

Huffman and Lott (5) have commented on the high solubility of 16- ketoestrone in their acid reaction mixture. It occurred to us that the dis- crepancies in our simple and zinc-hydrochloric acid urine estrogen titers might be explained by the presence of a compound with such high aqueous solubility that it enters the organic phase of our extraction procedure only after partial reduction. Accordingly, 5 mg. of 16-ketoestrone were added to 1 liter of pooled male urine and simple hydrochloric acid hydrolysis and extraction followed by zinc-hydrochloric acid hydrolysis and extraction were carried out sequentially. Although there was some evidence of recovery of estrogens in the second extraction, in percentage figures the results are not very suggestive of the operation of such a mechanism in the case of 16-ketoestrone. In Experiment 4, the 0.3 per cent recovery of 16-ketoestrone as estrone presumably arises from 16-keto-cr-estradiol, which, as indicated in Experiment 1, occasionally appears in relatively small amounts in our estrone chromatographic fraction.

The data in Experiments 1,2, and 3 suggest that 16-ketoestrone, if present in human urine in sufficiently large amounts, could account for some of the additional Kober color products obtained by the Smith zinc-hydrochloric acid hydrolysis technique. Study of the urine of human subjects treated with these compounds, for phenolic estrogens, would present a more favorable test of this concept.3

Although these data might seem to offer convincing arguments for the occurrence in human urine of more highly oxygenated estrogens which become chromogenic with the Kober reagent when subjected to zinc-hydrochloric acid hydrolysis, the alternative concept that these results are the consequence of varying relationships between the extent of simultane- ous hydrolysis and destruction of the labile estrogens already known to be present in human urine might seem equally valid. This latter concept can be evaluated only if the conjugated forms of the estrogens as they appear in human urine are available for experimentation. Since they are not generally available, only a partial solution to our problem can be at- tempted at this time. It must be emphasized, however, that current published data (1, 12) on the destructive effects of acid hydrolysis on the conjugated and unconjugated estrogens as derived by bioassay are of little help in evaluating similar phenomena obtained by calorimetric techniques, since the structural features which give rise to a physiological response are not necessarily the same as those which give rise to the calorimetric response. Production of color products with spectral characteristics typ-

3 A report on the urinary metabolites of 16-ketoestrone and 16-keto-cr-estradiol injected in men will appear in a subsequent publication.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


ical of those obtained with pure compounds is the only criterion of value in estimating recoveries by calorimetric techniques.

Table VI presents typical data on the recovery of the unconjugated natural estrogens after boiling with strong hydrochloric acid with and without the addition of zinc dust. 20 minutes refluxing with 15 volumes per cent of hydrochloric acid caused not more than 30 per cent destruction of the estrogens, as indicated by the Kober reagent, whereas 3 hours refluxing with 15 volumes per cent of hydrochloric acid and 4 gm. per cent of zinc dust resulted in almost complete destruction of estrogens. This is not surprising, since the amount of zinc dust which was added to the solution (4 gm. per cent) is stoichiometrically equivalent to approximately 10 volumes per cent of hydrochloric acid as used by us, and exerts its ac-

TABLE.VI Recovery of Added Estrogens from Distilled Water and Pooled Male Urine

After boiling for 3 hours with (a) 15 volumes per cent of concentrated HCl and (b) 15 volumes per cent of concentrated HCl and 4 gm. per cent of zinc dust.

Exper;? t Solution, 1 liter

Hz0 ‘I I‘

Pooled male urine “ ‘I I‘ “ “ ‘I “ “ “

Estrone cY-Estradiol Estriol None Estrone oc-Estradiol Estriol

Per cent recovery as shown by Kober test

(a)

70.4* 80.0* 92.0* 0.0

73.0 55.0 38.0

(b)

0 0 8 0

63.0t 86.0 48.0

* 20 minutes boiling substituted for 3 hours. t Recovered in estradiol fraction of chromatogram.

tion (evolution of hydrogen) actively only during the 1st hour of refluxing.

Moreover, the hydrogen ion concentration by glass electrode determination showed no detectable change; i.e., the pH was less than 0.2 over the 3 hour period.

Typical hydrolytic data obtained by the substitution of acidified pooled human male urine for simple hydrochloric acid solution of the estrogens indicated that the substitution of urine for aqueous solution provided con- siderable protection to the estrogens against the destructive forces oper- ating in hydrolysis in the presence of zinc dust (Table VI).4 Estriol ap-

4 Male urine hydrolyzed by this technique in 1 liter quantities consistently gave estrogen titers within the limits of error reported for our procedure; i.e., ~t7 y of estrone, f10 y of estradiol, and f5 y of estriol (9).


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

B. F. STIMMEL 225

peared to be the most labile of the three estrogens with both hydrolytic techniques. It was observed in experiments in which pooled male urine was used that the stoichiometric excess of hydrochloric acid became an actual excess of zinc, since from 6 to 8 gm. of zinc were recovered routinely at the end of the 3 hour hydrolysis. The titratable acidity at the beginning and the end of hydrolysis changed from an average of 1.5 N to 0.3 N (methyl orange indicator). However, the pH did not rise above 0.2.

Data on the destruction of unconjugated estrogens by the hydrolytic process can provide only a partial explanation of estrogen losses during hydrolysis. As Gallagher (13) has emphasized, it is necessary to investi- gate the water-soluble forms of the phenolic steroids and to devise more reliable methods than are now available for the hydrolytic process based

TABLE VII Recovery of Estrogen from Essentially Estrogen-Free (Pooled Male) Urine

2.0 mg. of sodium estrone sulfate added to 1 liter of urine previous to hydrolysis. - Experi. malt No.

Conditions of hydrolysis

1 15 vol. $ZO HCI + 4 gm. 70 zinc; 3 hrs. refluxing 2 15 ‘I y. “ + 4 ‘( y. (6 ; 3 (I ‘C 3 15 “ y. “ ; 5 min. refluxing 4 15 ‘I y. ‘I ; 10 “ “ 5 5 “ y0 concentrated H&O,; 10 min. refluxing 6 15 “ y0 HCl; 20 min. refluxing 7 15 “ y. “ ; 30 “ “ 8 15 I‘ y?* ‘( ; 40 ‘I I‘ 9 15 “ y. “ ; 60 “ ‘I

IgEZ test

m. 0.563* 0.588* 1.116 1.262 1.234 0.883 0.741 0.715 0.726

- Per cent

40 42 80 90 88 63 53 51 52

* Calculated as cr-estradiol.

upon the known composition of the conjugates. Some data on this point were provided by the hydrolysis of sodium estrone sulfate added to pooled male urine (Table VII) which show that simple hydrochloric acid hydrolysis yields even greater estrogen recovery than zinc-hydrochloric acid hydrolysis from sodium estrone sulfate. Typical recovery data reported in Table VII represent total estrogen recovered in all three of our chromatographic fractions. In Experiments 1 and 2, less than 10 per cent of the recovered estrogen appeared in the chromatographic estrone and estriol fractions, whereas in Experiments 3 through 9 less than 10 per cent of the recovered estrogen appeared in the estradiol and estriol fractions. The optimum time of refluxing for simple hydrolysis with 15 volumes per cent of hydrochloric acid appeared to be 10 minutes, which yielded 90 per cent


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


of the theoretical estrone content, whereas the zinc-hydrochloric acid technique of Smith and Smith with 3 hours refluxing gave approximately 40 to 42 per cent recovery. If sulfate is the only form of conjugation for human estrone excretion,6 then these data offer strong support for the postulate of Smith and Smith; i.e., that conversion of estrone to estradiol and more complete hydrolysis do not account for all of the increased estro- genie potency obtained by the addition of zinc during hydrochloric acid hydrolysis of human female urine.

Unfortunately, sodium estriol glucuronidate was not available to us at the time these studies were made. However, Venning et al. (14) have reported quantitative recovery of estriol from sodium estriol glucuronidate added to urine before hydrolysis by their method (approximately 2 volumes per cent of hydrochloric acid and 3 hours autoclaving at 15 pounds pres- sure). Van Bruggen (12) also has reported quantitative recovery of estriol from sodium estriol glucuronidate in aqueous solution following 10 minutes refluxing in an atmosphere of nitrogen gas with 15 volumes per cent of concentrated hydrochloric acid and 0.125 gm. per cent of protective agent (1-amino-2-naphthol-4-sulfonic acid). Our recovery data of estrone from sodium estrone sulfate together with similar estriol recovery data from sodium estriol glucuronidate, of Van Bruggen, offer strong support for the thesis that sodium estrone sulfate and sodium estriol glucuronidate cannot account for the increased estrogen titers obtained on addition of zinc dust during hydrolysis. The problem therefore resolves itself into a search for (a) other conjugate forms of these known natural estrogens or (5) discovery of additional estrogen degradation products which are rendered chromogenic with the Kober reagent by the addition of zinc during hydrolysis with hydrochloric acid.

SUMMARY

Comparative calorimetric estimations of estrogens in human pregnancy urine by simple hydrochloric acid and by zinc-hydrochloric acid hydrolysis support the observation of Smith and Smith based on bioassay, that estrone is converted to estradiol by the presence of zinc and that markedly increased estrogen titers are obtained.

Pregnant mare urine does not show augmented titers by the zinc-hydrochloric acid hydrolysis technique.

Recovery of estrone from sodium estrone sulfate added to pooled human male urine prior to zinc-hydrochloric acid hydrolysis was not of high enough order to account for the increased estrogen titers obtained in human pregnancy urine.

‘ Hydrolytic studiee on human pregnancy urine to be reported later seem to indi- cate otherwise.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

B. F. STIMMEL 227

16-Ketoestrone added to pooled human male urine reacts to zinc-hydrochloric acid hydrolysis in such a way that its presence in urine could account for some of the augmented Kober color products found by this hydrolytic procedure.

We wish to acknowledge the technical assistance of William M. Corm in this work. We also wish to express our appreciation to Dr. Max Huff- man of the Southwestern Medical College, Dallas, Texas, for the 16-ketoestrone and 16-keto-a-estradiol used in this study; to Dr. Herman Cohen of E. R. Squibb and Sons, New Brunswick, New Jersey, for the pregnant mare urine; and to Dr. J. Murray Scott of Ayerst, McKenna and Harrison, Ltd., New York, for the sodium estrone sulfate (synthetic).

BIBLIOGRAPFIY

1. Smith, G. V. S., and Smith, 0. W., Proc. Sot. Exp. Biol. and Med., 36,460 (1937). 2. Smith, 0. W., and Smith, G. V. S., Endocrinology, 28,740 (1941). 3. Pincue, G., and Pearlman, W. H., in Harris, R. S., and Thimann, K. V., Vitamins

and hormones, New York, 1,334 (1943). 4. Werthessen, N. T., Baker, C. F., and Broci, B., Science, 107,64 (1948). 5. Huffman, M. N., and Lott, M. H., J. Biol. Chem., 172,325 (1948). 6. Huffman, M. N., and Grollman, A., Endocrinology, 41, 12 (1947). 7. Stimmel, B. F., J. Clin. Endocrinol., 7, 364 (1947). 8. Stimmel, B. F., J. Biol. Chem., 162, 99 (1946). 9. Stimmel, B. F., J. Biol. Chem., 166, 73 (1946).

10. Zimmermann, W., 2. physiol. Chem., 246, 47 (1936-37). 11. Marlow, H. W., Endocrinology, 42,479 (1948). 12. Van Bruggen, J. T., J. Lab. and Clin. Med., 33, ‘297 (1948). 13. Gallagher, T. F., The chemistry and physiology of hormones, Washington, 186

(1944). 14. Venning, E. H., Evelyn, K. A., Harkness, E. V., and Browne, J. S. L., J. Biol.

Chem., 120, 225 (1937).


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

Benjamin F. StimmelHUMAN URINE

HYDROLYSIS ON THE ESTROGENS INZINC-HYDROCHLORIC ACID

THE EFFECT OF

1949, 178:217-227.J. Biol. Chem.

http://www.jbc.org/content/178/1/217.citation

Access the most updated version of this article at

Alerts:

When a correction for this article is posted•

When this article is cited•

alerts to choose from all of JBC's e-mailClick here

tml#ref-list-1

http://www.jbc.org/content/178/1/217.citation.full.haccessed free atThis article cites 0 references, 0 of which can be by guest on February 12, 2020

http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/content/178/1/217.citation

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;178/1/217&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/178/1/217.citation

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=178/1/217&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/178/1/217.citation

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/content/178/1/217.citation.full.html#ref-list-1

http://www.jbc.org/content/178/1/217.citation.full.html#ref-list-1

http://www.jbc.org/

the effect of zinc-hydrochloric acid hydrolysis on … · the effect of zinc-hydrochloric acid...

Documents