STEROIDS

VIII. A COLORIMETRIC METHOD FOR THE ESTIMATION OF REDUCING STEROIDS*

BY R. D. H. HEARD AND H. SOBEL

(From the Department of Biochemistry, McGill University, Montreal, Canada)

(Received for publication, June 8, 1946)

The pioneer investigations of Kendall, Reichstein, Wintersteiner, and collaborators (reviewed by Reichstein and Shoppee (2)) on the structure of the steroids of the adrenal cortex early revealed the strongly reducing sugar- like property of many members of this series (including all of the active hormones) which is imparted by the presence in the side chain of the prim- ary a-ketol grouping characteristic of fructose. To provide a simple chemical method for the estimation of small quantities of reducing steroids of this type, the reduction of phosphomolybdic acid to molybdenum blue has been quantitatively and satisfactorily standardized.

In preliminary qualitative experiments the possible use of 2,6-dichloro- phenol indophenol as oxidant was explored. While the dye is decolorized by desoxycorticosterone on being heated (sealed tube) at 100” in an inert atmosphere, the slow rate of reduction and the requisite anaerobic condi- tions precluded a rapid and simple technique. With phosphomolybdic acid (Folin-Wu reagent (3)) in a medium of acetic acid, reduction takes place rapidly and is not appreciably influenced by atmospheric oxygen. Accord- ingly the latter reaction was selected for standardization.

Development of the molybdenum blue color (Fig. l), as measured in the photoelectric calorimeter at 650 to 660 rnp, proceeds rapidly at 100” during the first 30 minutes, after which time the intensity gradient falls off suffi- ciently to permit reproducible results to be obtained at any arbitrarily chosen time interval thereafter. In practice, a period of heating of 1 hour is allowed for the development of the color. Under these conditions, a series of ten determinations on the same quantity of desoxycorticosterone showed agreement in the optical density (log lo/I) of the resulting color within f2 per cent. Verification of Beer’s law that the intensity of the color varies directly with the quantity of reducing substance was then es- established for desoxycorticosterone, 21-hydroxypregnenolone, and Ken- dall’s Compound E (Fig. 2). These standard curves may thus be applied to estimation of any reducing steroid or suitable biological extract in terms of the reducing equivalent of whichever pure compound as may be selected as standard of reference.

* Preliminary accounts have previously appeared (1).

687

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ESTIMATION OF REDUCING STEROIDS

FIG. 1. Rates of reduction of phosphomolybdic acid by 0.303 mM of a-ketolic steroids. The curve numbers correspond to the compound numbers listed in Table I.

Fro. 2. Relation in intensity of molybdenum blue color to quantity of reducing steroid.

From a survey of the reducing characteristics of a large number of steroids (Figs. 1, 3, and 4; Table I) the following general conclusions have been reached concerning the relation between chemical structure and power to

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R. D. H. HEARD AND H. SOBEL 689

cause reduction of phosphomolybdic acid. The reference numbers to the various compounds are those assigned in Table I.

0 HOURS 1 2 3

FIG. 3. Rates of reduction of phophomolybdic acid by 0.303 rnM of the acetates of cr-ketolic steroids. The curve numbers correspond to the compound numbers listed in Table I.

FIG. 4. Rates of reduction of phosphomolybdic acid by 0.303 mM of c+fl-unsatu- rated 3-keto steroids. The curve numbers correspond to the compound numbers listed in Table I.

Group A-The aliphatic primary a-ketol grouping

-C-C-CHzOH I

as in desoxycorticosterone and Compounds 2 to 6 exhibits the strongest reducing properties.

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690 ESTIMATION OF REDUCING STEROIDS

TABLE I Optical Density of Molybdenum Blue Produced by 0.303 rnx of Steroids after

Periods of Heating of 1 and S hours

Arranged in order of densitv at ;3 hours. -

w

I

fi

-

1 2

Common name

3 4

5

Desoxycorticosterone “ ace-

tate Dehydrocorticosterone 12.Ketodesoxycorticoster-

one acetate 21-Acetoxypregnenolone

6 21-Hydroxypregnenolone 7 Compound E (Kendall)

8 Methyltestosterone

9 None

10 “

11 12

13 14 15

“

Compound P (Reichstein) diacetate

None Progesterone None

16 “

17 Ethinyltestosterone

18 &-Testosterone 19 Androstenedione 20 Cholestenone 21 None

22 Testosterone 23 17-Hydroxypregnanolone 24 7-Ketocholesterol acetate

25 26 27

-

Pregnenolone None Pregnanedione

structure

A4-Pregnen-21-ol-3,20-dione ‘I ace-

tate A4-Pregnen-21-01-3, 11,20-trione A4-Pregnen-21-ol-3,12,20-trione

acetate AS-Pregnen-3@),21-diol-20-one

al-acetate A5-Pregnene-3(D), 21-diol-20-one A4-Pregnene-17@),21-diol-3, ll,-

20-trione 17-Methyl-A4-pregnen-17(a)-ol-

3-one 3((r), 12-Dihydroxy-ll-ketocho-

lanic acid Pregnane-3(a), 12,21-triol-20-

one al-acetate Cholestan-2(a)-ol-a-one acetate Allopregnane-3@),17@),21-triol-

20-one 3,21-diacetate Cholestan-2(p)-ol-3-one acetate A4-Pregnen-3, 20-dione Potassium 3(a), 11(p)-dihydroxy

12-ketocholanate 3(a), 11 (a) -Dihydroxy-12-keto-

cholanic acid 17-Ethinyl-Ad-androstene-17(a)-

ol-3-one A4-Androsten-17(P)-ol-3-one Ah-Androstene-3,17-dione A4-Cholesten-3-one 3(a)-Hydroxy-11,12-diketocho-

lanic acid methyl ester A4-Androsten-17(a)-ol-3-one Pregnane-3(a), 17-diol-20-one A4-Cholesten-3(p)-ol-7-one ace-

tate AS-Pregnen -3 (p) -ol-20-one Ah-Cholestene-3,6-dione Pregnane-3,20-dione

- I Log 9

1 hr. 3 hrs.

0.69 0.90 0.45 0.88

0.60 0.78 0.38 0.74

0.33 0.69

0.57 0.68 0.51 0.65

0.30 0.60

0.33 0.54

0.22 0.53

0.19 0.51 0.21 0.46

0.15 0.42 0.21 0.38 0.24 0.37

0.14 0.36

0.17 0.31

0.17 0.30 0.16 0.29 0.17 0.29 0.09 0.27

0.16 0.26 0.03 0.12 0.03 0.09

0.02 0.09 0.02 0.09 0.02 0.08

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- :: B &

V -

28 29 30 31 32

33

34

35

R. D. H. HEARD AND H. SOBEL

TABLE I-Concluded

691

Common name

cu-Estradiol Estrone Estriol Cholesterol Allocholesterol-isoallocho-

lesterol None

structure

Ars3+-Estratriene-3, 17(a)-diol A1~3+5-Estratrien-3-ol-17-one A1*3s6-Estratriene-3, 16,17, -trio1 AJ-Cholesten-3(p)-01 A4-Cholesten-3(a and p)-01

3(a)-Acetoxy-12-keto-A9*rr- cholenic acid

Cholestane-3(b), 7(u)-diold-one diacetate

Cholestane-3@), 7@)-diolS-one diacetate

-

_- \

,

1 hr. 3 hrs.

0.00 <0.04

We are grateful to Dr. T. F. Gallagher for specimens of Compounds 9,15,16, and 21, to Dr. E. C. Kendall for Compounds 3, 7, and 33, and to Dr. S. Lieberman for Compound 23. Compound 32 was prepared by the method of Schoenheimer and Evans (4) and Compound 26 by the method of Mauthner and Suida (5). In this laboratory our thanks are due to Miss D. Sainte-Marie and Mr. H. Falk for the preparation of Compounds 4 and 10 by a modification of the method of Fuchs and Reichstein (6), and to Miss J. Cohen for the isolation of Compound 12 from an ad- renal cortical fraction kindly provided by The Upjohn Company, through the courtesy of Dr. M. H. Kuieenga. The preparation of the cyclic cr-ketols, Compounds 11,13,34, and 35, will shortly be described by one of us (R. D. H. H.) in collaboration with Dr. B. K. Wasson; in each epimeric pair the B configuration has tentatively been assigned to the isomer with the lower melting point and the more negative rotation. For supplies of Compounds 2 and 5, we are grateful to Dr. C. R. Scholta, of Ciba Pharmaceutical Products, Inc., and to Dr. E. Schwenk, of the Schering Corporation.

Group B-The reducing power of the steroids of Group A is diminished slightly by the presence of a tertiary hydroxy group at C,; i.e., the

OH0 I II

-C-C-CH,OH I

side chain of Kendall’s Compound E (compare Compounds 7 and 3, and 12 and 6).

Group C-Acetylation of the primary alcoholic function of Groups A and B diminishes the rate of development of the blue color but not the intensity finally attained after a 3 hour period of heating (compare Figs. 3 and 1, and Compounds 2 and 1, and 5 and 6). Presumably hydrolysis of the acetate takes place slowly in the acid reaction medium to release the free ketol.

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692 ESTIMATION OF REDUCING STEROIDS

Group D-Substitution of a ketonic oxygen atom at Cl1 or CE adversely influences the reducing capacity of the ar-ketol side chain (compare Com- pounds 3 and 1,4 and 2, and 10 and 5).

Group E-A tertiary or-ketol (Compound 23) is essentially non-reducing. Group F-Cyclic secondary a-ketols in Ring A, as acetates (Compounds

11 and 13), and in Ring C, as free ketols (Compounds 9,15, and 16), exhibit reducing power of the same order as that of the aliphatic primary a-ketols. Ring B ketols (Compounds 34 and 35), as acetates, fail to reduce, but the saponification product of Compound 35 (m.p. 202203”) and the choles- tane-3(p) ,7(?)-diol-6-one (m.p. 179”) described by Heilbron, Jones, and Spring (7) are strongly reducing; the inertness of the acetates, Compounds 34 and 35, is accordingly ascribed to failure of the acid medium to effect hydrolytic removal of the 7-acetoxyl groups.

In the case of both epimeric pairs studied (Compounds 11 and 13, and 15 and 16), there is an appreciable difference between the rates of reduction shown by the cis and trans isomers, which is still more marked in the case of the free ketols presumably corresponding to the acetates, Compounds 34 and 35. These preliminary observations strongly indicate that relative reducing power may prove valuable in the establishment of the configura- tion of secondary hydroxyl groups vicinal to a ketone in the steroid ring system; investigations in this connection are being extended.

Group G-An cr,P-diketone (Compound 21) is moderately reducing. Group H--a!,/3-Unsaturated 3-keto steroids, void of a ketol grouping in

the molecule (Compounds 8, 14, 17, 18, 19, 20, and 22) show a reducing potential of the same order as the aliphatic and cyclic ketolic compounds not possessing the cr,/3-unsaturated 3-ketonic linkage. In the presence of both functions, the reducing power is approximately the sum of the two contributions.

Group I-Without reducing power are (a) a,~-unsaturated 7- and 12- keto steroids (Compounds 24 and 33), (b) A4- and AK-3-hydroxy compounds (Nos. 25, 31, and 32), and (c) the estrogens (Compounds 28,29, and 30).

Exceptions to the above generalizations are to be found in the behavior of methyltestosterone (Compound 8) and A4-cholestene-3, 6-dione (Com- pound 26). The former is much more strongly reducing than the other six cr,&unsaturated 3-ketones which were examined (Fig. 4), while the latter shows no appreciable reducing power. No explanation of the anomalous behavior of methyltestosterone is apparent. The non-reducing character of the doubly conjugated diketone (Compound 26) may be ascribed to the highly enolic nature of this system, which is evidenced by the ease with which enol esters are formed (5) and by the absorption spectrum data (Fig. 5). The main band in neutral ethanol (emaX. = 10,600 at 251.5 mr) is depressed and shifted toward the violet in the presence of alkali (emax. =

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R. D. H. HEARD AND H. SOBEL 693

10,000 at 259 mp). Contrariwise, with a simple cr,p-unsaturated 3-ketone such as cholestenone (Compound 20), which is moderately reducing, the position and intensity of the main resonance band (emaX. = 15,000 at 240 mp) are unaltered by the addition of alkali, but the secondary ketone band (Gn,. = 140 at 290 to 296 mp) is shifted and depressed (emrtx. = 127 at 302 mp). These facts are taken to indicate that Compound 20 exists in solu- tion mainly in the keto and Compound 26 mainly in the enol state.

Recently Talbot et al. (8) have applied to the estimation of certain adrenal steroids Nelson’s modification (9) of the classical Folin-Wu method

220 lnp. 240 260 280

FIG. 5. Absorption spectrum of z!,Qholestene-3,6-dione in neutral (Curve A) and alkaline (Curve B) ethanol. The scale of the ordinate represents the molecular ex- tinction coefficient.

for the determination of blood glucose. In this procedure, cupric ion serves as the primary oxidizing agent and the resulting cuprous ion in turn reduces arsenomolybdic acid to the blue chromophore. In order to compare the specificity of the two methods, it was found necessary to alter the com- position of the reagents used by Talbot et al. (8) because of the extreme insolubility of most steroids in the usual aqueous alkaline copper solution. Ethanol (50 per cent) served as solvent medium, and to prevent the pre- cipitation of sodium sulfate from the Nelson copper reagent, this salt was omitted; reduction of cupric ion was indicated by the formation of the usual blue color with phosphomolybdic acid. Under these conditions, the aliphatic primary a-ketol and the Ring A cyclic a-ketol groupings are strongly reducing, while a, fi-unsaturated S-ketones are not. Negative, or

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694 ESTIMATION OF REDUCING STEROIDS

doubtfully positive, results were observed with compounds containing a cyclic a-ketol grouping in Ring C; insolubility in the medium may limit the reaction in some of these cases.

EXPERIMENTAL

Reagents- Phosphomolyf&z acid solution, according to Folin and Wu (3). Glacial acetic acid, analytical reagent grade, distilled from chromic

anhydride. Phosphomolybdic acid reagent, a solution made up of equal volumes of

phosphomolybdic acid solution and glacial acetic acid (the precipitate formed on addition dissolves on mixing).

Method of Color Development and Estimation-The following procedure was 6nally adopted after preliminary investigations led to satisfactory methods of control of certain variable factors (discussed below).

A solution (0.10 ml.) of the appropriate steroid in glacial acetic acid, of such concentration that the stated volume contains the reducing equiva- lent of approximately 100 y of desoxycorticosterone, is introduced into the bottom of a micro test-tube (75 by 8 mm. internal diameter). The reagent (2.00 ml.) is then added, and, after mixture by shaking, the tube is placed in a boiling water bath (see below) for precisely 60 minutes. On removal, it is immediately cooled (30 seconds immersion in a beaker of cold water), and the contents are quantitatively transferred with 8.00 ml. of the reagent to one of a pair of optically matched Evelyn calorimeter tubes (internal diameter 2.0 cm.). On admixture, air bubbles form, which, because of the viscosity of the solution, require a period of 90 seconds to rise to the surface. The optical density is then read within the next 4 minutes at 650 to 660 rnlr (Filter 660 with the Evelyn calorimeter; Filter 650 with the Lumetron calorimeter).

A blank determination with 0.10 ml. of glacial acetic acid and 2.00 ml. of reagent is run simultaneously and in the same way, which serves as the con- trol for the adjustment of the calorimeter to zero optical density. In this a visible yellow coloration develops on heating, but it is not apparent after dilution to 10.1 ml. with the reagent. Compared to water, the blank solu- tion absorbs very slightly at 650 to 660 rnp, but the optical density remains constant regardless of the period of heating. Accordingly it is seemingly needless to carry out a control with every determination, but this is advis- able as a safeguard against the contamination of the reagent with any sub- stance which may reduce phosphomolybdic acid at the elevated tempera- ture.

The limit of error in the development and reading of the color is approxi- mately f2 per cent. Ten repeat estimations of aliquots (1017) of a solu-

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Time of heating

min.

15 30 45 60 90

120 180

R. D. H. HEARD AND H. SOBEL 695

tion of desoxycorticosterone gave optical densities of 0.67 (twice), 0.68 (six times), and 0.69 (twice).

Temperature Control during Development of Color-The extreme impor- tance of maintaining a uniform thermal environment during the develop- ment of the color is well illustrated (Table II) by the significant differences in the intensity of the blue color observed on heating 0.303 mM of methyl- testosterone with 2.00 ml. of the reagent (a) in a moderately boiling water bath (standard conditions) and (b) in a gently boiling bath.

Satisfactory control of this variable was achieved by maintaining a con- stant heat input into a constant volume bath, and by immersing the tubes to the same depth on all occasions. A steam-heated bath was employed,

TABLE II Temperatur? Control during Development of Color*

Optical density

Moderately boiling bath (standard conditions) Gently boiling bath

0.08 0.07 0.17 0.14 0.24 0.20 0.30 0.27 0.39 0.36 0.48 0.46 0.60 0.56

* 0.303 mM of methyltestosterone; apart from the rate of boiling of the bath, all other conditions were maintained constant.

with the inlet needle valve always opened to the same aperture (main- tenance of a moderate degree of boiling). The water input regulator was adjusted to keep the level 1 inch below the top enclosure (concentric rings) of the vessel, and the test-tubes were suspended from the latter by means of spring clothes-pins clamped close by the lip of the tube, which insured immersion in the boiling water of the lower portion of the tube to a depth of l$ inches. Under these circumstances, numerous control estimations of desoxycorticosterone, made over a period of 2 years, consistently agreed wit,h the original standard curve within -13 per cent. Closer agreement, if desired, may probably be realized by the use of a thermostatically con- trolled, electrically heated oil bath maintained at 100”.

Because of the wide variation of color intensity with time, and particu- larly with temperature, it follows that the standard curves and rate of reduction curves obtained under slightly different conditions in other laboratories may depart appreciably from those recorded here.

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696 ESTIMATION OF REDUCING STEROIDS

Influence of Nature of Phosphomolybdic Acid Reagent and of Various Diluents on Development and Stability of Color-While the Folin-Wu phosphomolybdic acid solution alone is suitable for estimation of those crystalline steroids which are moderately soluble in water, its application to the neutral fraction of urine leads to a cloudy solution which must be cleared by extraction with ether before the intensity of the blue color may be determined. By the solvent action of the acetic acid contained in the phosphomolybdic acid reagent, this difficulty is obviated and a more intense blue color results.

Table III illustrates the effect of various diluents on the intensity and stability of the color produced on heating 117 y of desoxycorticosterone with 2.0 ml. of the phosphomolybdic acid reagent for 1 hour. Dilution with the same reagent is requisite to maximum color intensity and stability.

Construction of Standard Curves and of Rate of Reduction Curves-The standard curves (Fig. 2) were arrived at by ascertaining the optical density of the molybdenum blue color produced by the heating for 1 hour of the recorded amounts (each in 0.10 ml. of glacial acetic acid) of the appropriate steroid.

Each of the rate of reduction curves, illustrated in Figs. 1, 3, and 4 and recorded in Table I, was constructed from a series of seven determinations reached by heating for the periods of time indicated 0.303 mM of the appro- priate steroid (dissolved in 0.10 ml. of glacial acetic acid). This quantity represents the equivalent of 100 y of desoxycorticosterone.

Reference Standard-Desoxycorticosterone has been selected as the standard of reference because it is the most strongly reducing and most readily available adrenal cortical steroid.

Crystalline desoxycorticosterone, prepared by saponification of the ace- tate with KHC03 at room temperature (the method of Reichstein and von Euw (lo)), has a melting point of 141.5-142.5”. On storage for several months in a stoppered vial, the melting point falls (softens at 131“, flows at 13%141”), commensurate with which there is a diminution in reducing power of about 3 per cent. One recrystallization from ether yields a prod- uct identical with the freshly prepared material in these respects. In glacial acetic acid solution at room temperature, there is detectable loss of reducing capacity within 1 to 2 weeks; kept frozen and tightly stoppered in the refrigerator, the solution remains stable for many months.

Reduction of Cupric Ion-The following modification of the procedure of Talbot et al. (8) was adopted qualitatively to compare the behavior of the various reducing steroids in the two methods.

The material (0.1 mg. in 1.0 ml. of redistilled ethanol) was introduced in a Folin-Wu tube and 1.0 ml. of alkaline copper reagent was added. The latter was prepared as described by Nelson (9), except for the inclusion of

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R. D. H. HEARD AND H. SOBEL 697

sodium sulfate. After mixing, the content,s of the tube were heated in the boiling bath for 20 minutes, cooled, treated with 2.0 ml. of phosphomolybdic acid reagent, and immediately diluted with ethanol (50 per cent) to 25 ml.

TABLE III Effect of Diluent on Intensity and Stability a

Diluent (8.0 ml.)

Phosphomolybdic acid reagent

Phosphomolybdic acid solution

Acetic acid (50%)

Water

- I Time after dilution* Optical density

min.

0 1 2 3 4 5 6

10 0 1 2 3 4 5 7

10 0 1 3 5 7

10 0 1 3 5 7

10

0.81 0.81 0.81 0.81 0.81 0.81 0.81 0.81 0.70 0.68 0.68 0.68 0.68 0.70 0.70 0.74 0.75 0.75 0.75 0.74 0.73 0.72 0.57 0.56 0.55 0.55 0.54 0.52

Color

* Time zero is read after 90 seconds have elapsed for the clearance of air bubbles formed on admixture after dilution.

The blue color developed was then compared in the usual way against the reagent blank, similarly treated.

Compounds 1, 2, 5, 6, and 13 rapidly and strongly reduced cupric ion. Progesterone failed to react, and with Compounds 9, 15, and 16 essentially negative results were observed, but in these instances limited solubility in the medium renders the interpretation doubtful.

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698 ESTIMATION OF REDUCING STEROIDS

In support of the investigation we are grateful to the Committee on Re- search in Endocrinology of the National Research Council (Washington), the Associate Committee on Medical Research of the National Research Council (Ottawa), and the Faculty of Medicine of McGill University (Cooper Fund). Our thanks are due to Miss L. Groth for skilful technical assistance.

SUMMARY

1. The reduction of phosphomolybdic acid to molybdenum blue has been applied to the quantitative estimation of small quantities (10 to 100 7) of reducing steroids of the adrenal cortical hormone class.

2. The reaction is given by steroids containing a primary or secondary (but not tertiary) cu-ketol function, an ar,@-unsaturated 3-ketone group, or both.

BIBLIOGRAPHY

1. Heard, R. D. H., and Sobel, H., Conference on metabolic aspects of convalescence including bone and wound healing, New York, June 15-16, 1945, under the auspices of the Josiah Macy, Jr., Foundation. Heard, R. D. H., and Sobel, H., Proc. Canad. Physiol. Sot., Canad. Med. Awn. J., 64, 69 (1945).

2. Reichstein, T., and Shoppee, C. W., in Harris, R. S., and Thimann, K. V., Vita- mins and hormones, New York, 1, 345 (1943).

3. Folin, O., and Wu, H., J. Biol. Chem., 41,367 (1920). 4. Schoenheimer, R., and Evans, E. A., Jr., J. Biol. Chem., 114,567 (1936). 5. Mauthner, J., and Suida, W., Monatsh. Chem., 16,362 (1894). 6. Fuchs, H. G., and Reichstein, T., Hetv. chim. acta, 26,511 (1943). 7. Heilbron, I. M., Jones, E. R. H., and Spring, F. S., J. Chem. Sot., 801 (1937). 8. Talbot, N. B., Saltzman, A. H., and Wixom, R. L., Conference on metabolic

aspects of convalescence including bone and wound healing, New York, June 1616, 1945, under the auspices of the Josiah Macy, Jr., Foundation. Talbot, N. G., Saltzman, A. H., Wixom, R. L., and Wolfe, J. K., J. Biol. Chem., 160, 535 (1945) .

9. Nelson, N., J. Biol. Chem., 153,375 (1944). 10. Reichstein, T., and von Euw, J., Helv. chim. acta, 21, 1181 (1938).

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R. D. H. Heard and H. SobelREDUCING STEROIDS

METHOD FOR THE ESTIMATION OF STEROIDS: VIII. A COLORIMETRIC

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