Inactivation of contraceptive steroid hormones by human intestinal clostridia

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JOURNALOFCLINICALMICROBIOLOGY, Sept. 1983,p.500-504 Vol. 18,No. 3 0095-1137/83/090500-05$02.00/0

Inactivation

of

Contraceptive

Steroid Hormones

by

Human

Intestinal

Clostridia

V. D.BOKKENHEUSER,l* J.

WINTER,'

B. I. COHEN,2 S.

O'ROURKE,'

AND E.H. MOSBACH2

Departmentof Microbiology, St. Luke's-Roosev,eltHospitalCenter, New! York, New York10025,1 andLipid

Research Laboratory, Department of Surgery, Beth Israel Medical Center, New York, New York100032

Received24March 1983/Accepted 27May 1983

Steroid hormones reduced in

ring-A

are devoid of hormonal activity. In metabolic experiments we found that human fecal flora reduced the Az4-3-keto structureof natural progestinsto3ot-hydroxy, 5A-steroid metabolites (3cx,5,B) and

of

synthetic progestins

to a mixture of 3cx,53 and 3I,5r compounds.

3cx,5r-Reductase was synthesized by Clostridium paraputrificum and had a strong

affinity for

natural

progestins

suchasprogesterone.

3r,513-Reductase

was

synthe-sized

by

Clostridium innocuium and had a stronger affinity for synthetic proges-tins. A third enzyme,

3r,5ct-reductase,

wassynthesized

by

St. Luke's strain 209 (Clostridium species "J-1") butwasonly observed when pure cultures were used.

Ring-A

reduction

of

synthetic progestins

was 3 to 10 times slower than that of

natural

progestins,

thus

explaining

the

pharmacological superiority

of

synthetic

progestins

over

naturally occurring analogs.

The

metabolism of synthetic progestins,

which are components of oral contraceptives, was extensively studied in the late 1960s and

early

1970s(5, 6, 9, 11, 12, 14, 16). The

observed

transformations, including oxidations

and

reduc-tions,

were attributed largely to hepatic

en-zymes.

The fact

that synthetic progestins, like other

steroid hormones, undergo

enterohepatic

circulation

(1) and therefore are exposed to

bacterial enzymes in the intestinal stream was not considered. In this study we describe the role of the human intestinal flora in the transfor-mation of synthetic progestins. Particular em-phasis is placed on the bacterial species

synthe-sizing

the enzyme responsible for the

metabolism.

MATERIALS ANDMETHODS

Abbreviations andtrivial names. The abbreviations and trivial names used in the text are as follows:

cortisol, 113,17c,21-trihydroxy-4-pregnene-3,20-dione; deoxycorticosterone, 21-hydroxy-4-pregnene-3,20-dione; 21-deoxycortisol,

11,B,17a-dihydroxy-4-pregnene-3,20-dione; dihydronorethindrone, 19-nor-17a-ethynyl-17,B-hydroxy-

5,f

-androstan-3-one;

dimethisterone,

19-nor-17a-methylethynyl-17f3-hy-droxy-6ot-methyl-4-androsten-3-one; 3ax-HSDH,

3cx-hydroxysteroid dehydrogenase; norethindrone, 19-nor-17a-ethynyl-17 -hydroxy -4-androsten -3-one; norgestrel,

13f,-ethyl-177a-ethynyl-17f3-hydroxy-gon-4-en-3-one; pregnanolone,

3oa-hydroxy-5p-pregnan-20-one;progesterone, 4-pregnene-3,20-dione;

tetrahydro-norethindrone,

19-nor-17a-ethynyl-3cx,17f-dihydroxy-53-androstane;

TLC, thin-layerchromatography.

Origin of bacteria. All three Clostridium species were isolated from human fecal flora: Clostridium

paraputrificumbydilutiontechnique (4)and Clostridi-urn innocuumandSt. Luke's strain 209bythe dilution

and replicate-plating techniquedescribedby

Macdon-ald et al. (13). The cultures were maintained lyophi-lizedorfrozen andwerepassagedtwice inprereduced mediumat 37°C beforeuse. A24-h culture was used fortheconversionexperiments.

Strain 209,athinrodthatforms terminalsporesand

isnonsaccharolyticandnonproteolytic,wasidentified

as a member of Clostridium species "J-1'" (Virginia Polytechnic Institute). Ithas notbeenfrequently

iso-lated from human feces (L. V. Holdeman, personal communication).

Steroids. Steroids werepurchased from Steraloids,

Inc., Wilton, N.H. Synthetic progestins, norgestrel, and3-tetrahydronorgestrel isomers

(3a,5p;

3P,5p;

and

3f3,5ot)

were kindly donated by Wyeth Lab, Inc.,

Philadelphia, Pa.; norethindrone and norethindrone

acetate weredonatedby Parke,Davis&Co., Detroit, Mich., and Syntex Lab, Inc., Palo Alto, Calif.; and dimethisterone was donated by Mead-Johnson Co.,

Evansville, Ind.

Analysis. Conversion experiments and extraction andpurificationof metabolitesbyTLC and

identifica-tion by TLC, gas liquid chromatography, and gas

liquid chromatography-mass spectrometry have been describedpreviously (4, 19).

Enzymaticreaction for identification of3a-hydroxy steroids was carried out in solution as described by Yamaguchi (20).

Identification of metabolites. Ametabolite was con-sidered identical to agivenreference compound when

(i) it had a molecular weight and mass spectrometry

fragmentation pattern identical to those of the refer-encecompound; (ii) itsRJ value on TLC and relative

retentiontime to5a-cholestane on gasliquid

chroma-tography agreedwithin4% withthose of the reference

compound; and (iii) the reactivities of the unknown

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INACTIVATION OF STEROIDS BY CLOSTRIDIA 501

TABLE 1. Characteristics of reference compounds for natural steroidmetabolitesa

Steroid metabolite

Rfb

RRT' Reaction with_3a-HSDH

Series1

3a-Hydroxy-5S-pregnan-20-one 0.68 0.92 +

3,B-Hydroxy-5,B-pregnan-20-one

0.72 0.88

3P-Hydroxy-5a-pregnan-20-one

0.68 1.02

3a-Hydroxy-Sa-pregnan-20-one

0.72 0.90 +

Series2

50-Androstane-3a,110,17p-triol

0.25 0.87 +

5P-Androstane-3,B,11,,17p-triol

NAd NA NA

5a-Androstane-3a,11P,17,B-triol

0.34 0.88 +

5a-Androstane-31,1113,171-triol 0.34 1.03

Series 3

3a,110,17a,21-Tetrahydroxy-5S-pregnan-20-one

(tetrahydrocortisol) 0.28 1.44 + 3a,11,B,17a,21-Tetrahydroxy-5a-pregnan-20-one (allo-tetrahydrocortisol) 0.36 1.58 +

Series4

3a,11,17a-Trihydroxy-513-pregnan-20-one (tetrahydro-21-deoxycortisol) 0.46 1.04 +

Series S

3a,21-Dihydroxy-5S-pregnan-20-one (tetrahydrodeoxycorticosterone) 0.53 0.83 + a Mass spectrometry datafor each

series

indicateM'(molecular ion) and B+ (base peak). Series 1: M+ =419, B= 388. Series 2: M+ =524, B+ =434.Series 3: M+=683,B+=652. Series 4:M+ s 595, B+ =564. Series 5: M+=507, B+ = 358.

bTLC was done on Baker-flex silicagel 1B2-F, in the solvent system isooctane-ethyl acetate-acetic acid (15:75:0.6, vol/vol/vol).

cRRT, Retention timerelative to5a-cholestane on fused silica capillary column OV-101; oven temperature, 180to230°C; program rate,30per min. Steroids were analyzed as methoxime-trimethylsilyl ether derivatives.

dNA, Notavailable.

and the reference compounds with 3a-HSDH were identical(Tables 1 and2).

Identification of metabolites for which reference compoundswerenotavailablewasbasedon:(i)mass spectrometry, (ii) relative polarity as measured by

TLC and gas liquid chromatography, (iii) reactivity with3a-HSDH, and (iv) stereospecificity of enzymatic modificationofasimilar substrate forwhichreference

compoundswereavailable.

RESULTS

Massspectraof metabolites ofsynthetic

proges-tins. The extracted metabolites were converted to the volatile methoxime-trimethylsilyl ether derivatives. The mass spectrum of the tetrahy-dro metabolite ofnorgestrel is shown in Fig. 1. The molecular weight of the derivatized mole-cule is 460. Loss ofmle = 31 (CH3-0)

indi-catesaketo function in themolecule;successive

losses of

mle

= 31 indicate the total number of

keto groups. Similarly, trimethylsilyl reagents reactedwithhydroxygroups,and the number of

fragments M+ - 90, M+ - (2 x 90), etc.,

indicatesthe number ofhydroxygroups. Figure 1 shows that thenorgestrel metabolite had two

hydroxy groups butnoketogroups. Moreover, the molecular weight was 4 mass units higher than the molecularweightof thesubstrate,

indi-cating

a

reduction

of both the keto

group and the

double

bond. The base peak of

M+ - 29

reflect-ed the

loss of the ethyl

group.

It

is noteworthy that the ring-A

isomers of a

given tetrahydro compound

showed the same

fragmentation

pattern.

Conversion of natural steroids.

Progesterone

(Fig.

2),

11i,17,-dihydroxy-4-androsten-3-one,

cortisol, 21-deoxycortisol, and

deoxycorticoste-rone were

all

reduced in ring-A by mixed human

TABLE 2. Characteristics of reference compounds forsyntheticprogestinsa

StandardbRI RRV

~~~Reaction

with

Standardb Rfc RRTd 3a-HSDH

3a,5p

0.14 0.88 +

3,5B, 0.32 0.89

3P,5a

0.21 0.95

3a,5a NAe NA NA

aMassspectrometry data indicateM+andB+ (see Table 1, footnotea). M+ = 460;B+ =431.

bRing-A reduced norgestrel:

3,17p-dihydroxy-13I3-ethyl-17a-ethynylgonane.

C TLCwasdoneonAnaltechalumina G-F

(fluores-centindicator) platesin the solvent system

benzene-acetone(80:20,vol/vol).

dSee Table1, footnotec.

eNA, Notavailable. VOL.18,1983

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502 BOKKENHEUSER ET AL.

I CH3

Si CH3

CH3

-C--CH

CH 0\\

CH3 -Si , Peak

CH3 , '

29=-CH2-CH3 15 -CH3 90= TMS-0

MW=460

Bt=4 31 (M-29)

at = 445(M-15) 370(M -90) 280(M-2x90)

370

280 (M-90)

CM 0)

1,

I 1 I .

300 350 400 450

m/e

FIG. 1. Massspectra oftetrahydronorgestrel (trimethylsilylderivative).

fecal flora andbypureculturesof C.

paraputrifi-cumto the

3cs,5p

compounds. In contrast, pure cultures of C. innocuumand Clostridiumspecies J-1 convertedthe substratesto31,51 and313,5ot compounds, respectively.

Conversion ofsynthetic progestins. Norgestrel anddimethisterone(Fig. 2)werereduced in ring-Abyfecalfloratoamixture of3(x,5,8and31,513 compounds. As suggested by the observations with natural steroids, C. paraputrificum synthe-sized the3ox,5,B-reductase and C. innocuum the 3,5,B-reductase. The

3P,5a-reductase

synthe-sized by Clostridium species J-1 metabolized synthetic progestins, although no trace of the metabolite was observed in experiments with mixedfecal flora.

Norethindrone acetate incubated with fecal florawasquickly hydrolyzedandconcomitantly reduced to the mixture of 3a,51 and 31,51 metabolites. It is noteworthy that fecal flora could be diluted to 109 and still perform the hydrolysis, whereas reduction of ring-Awasnot obtainedwithfecaldilutions above107, suggest-ing that different organisms are responsible for

thetworeactions. Thetransformation of noreth-indroneby fecal florawasadequately explained

by the presence of C. paraputrificum and C. innocuum. Clostridium species J-1 did not

ap-peartoplayanimportantrole in thisconversion.

Kinetics of ring-A reduction. The intestinal

flora produced similar alterations in ring-A of both natural andsynthetic progestins. However, orallyadministered synthetic progestins,in

con-trasttonaturalprogestins,arepotent contracep-tives. This difference in biological activity prompted us to compare the kinetics of ring-A

vH3

C=O

0

Progesterone

OH(Ac) --C _CH Progestins

0

Norethindrone(Ac)

a:MI

IOH --C--CH

0N s

Norgestrel

OH

--C-C-CH3

o~~~~~ 3

0

CH3 Dimethisterone

FIG. 2. Structure of progesterone and synthetic progestins.

100

431

I(M-29)

Ur)

c

a)

-460

0 _ 150

(445) M-15

200 250

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INACTIVATION OF STEROIDS BY CLOSTRIDIA 503

Time,days

FIG. 3. Comparative kinetics of the metabolism of progesterone and norethindrone. Concentration of substrate: 1 mg per 50 ml of converting medium. Inoculum: 0.25 ml of a 24-h culture of C. paraputrifi-cum.Converting medium: prereduced brain heart infu-sion broth supplemented with 0.05% cysteine-hydro-chloride obtained from Scott Laboratories, Inc., Fiskeville, R.I. Incubation: 37°C. TH, Tetrahydro derivative.

reduction

by

pure

cultures

of

steroid-converting

organisms.

C. paraputrificum, C.

innocuum,

and

Clos-tridium

species

J-1

all

quantitatively reduced

ring-A of natural

progestins.

Incontrast,

ring-A

reduction of

synthetic progestins

was

slow,

tak-ing

6 to 7

days

to

reduce about

70% of the

molecules

(Fig. 3).

C. paraputrificum

reduced the

synthetic

pro-gestins

in a two-step

reaction,

best observed

with

the

substrates norethindrone and

dimethis-terone

(Fig.

4). Within 18

to

24 h, the substrate

was

quantitatively reduced

to

dihydronorethin-drone,

which

over

the

next 6 to 7

days

was

partially

reduced

to

tetrahydronorethindrone.

Dihydro

intermediates

were not

observed in

metabolic

experiments

with fecal

flora

or

with

C. innocuum

and

Clostridium

species

J-1.

Under the

conditions

we

used, the maximum

amount

of natural

progestins converted in

24h

by the three Clostridium

species

was9mg per

50 ml of medium.

These

species

had a 3 to 10

times

lower

capacity

to

reduce

synthetic

progestins,

as

comnpared

with

the

natural

compounds.

The

re-duction

rate,

however,

varied with

the chemical

structure

of the

synthetic

progestins.

Substrate

competition

among

C.

paraputrifi-cum,

C. innocuum,

and

Clostridium

species

J-1.

As

observed

above, ring-A

reduction of

proges-tins

by

fecal

flora reflected the

enzymatic

activi-ty of C.

paraputrificum

and,

to alesser

degree,

of

C. innocuum.

Clostridium

species

J-1

activity

was not

expressed.

In

experiments

with mixed

cultures of

Clostridium

species (Table 3),

C.

paraputrificum

hadagreater

affinity

for

natural

progestins,

whereas C.innocuum and

Clostridi-um

species

J-1

were more

active

upon

synthetic

progestins.

In mixtures of C. innocuum and

Clostridium

species J-1,

the

enzymatic

expres-sion of

C.

innocuum,

i.e.,

the

formation

of the

3P,5P

isomer,

prevailed.

DISCUSSION

Origin of

bacterially

synthesized

steroid-inacti-vating enzymes. The

predominant and

perhaps the sole

effect of the

intestinal flora

on the synthetic

progestins studied

inthe present

inves-tigation is the

reduction of

the

A4-keto

structure

of

ring-A.

For

almost

10 years

it

has been known

that the humanintestinalflora

contains

bacteria

capable of

reducing ring-A and thereby

inacti-vating endogenous

progestins

(4).

Our

observa-tions show that

at

least three Clostridium

spe-cies

synthesize

ring-A active

enzymes

with

different affinities

and different

stereochemical

effects. The

enzyme

produced by

C.

paraputrifi-cum is

particularly active

upon

natural

proges-tins, whereas those produced by

C. innocuum

and

Clostridium

species J-1

are

relatively

more

active upon the

synthetic progestins used for

contraceptive

purposes.

Whether the observed

differences in their abilities

to

reduce

ring-A of

the substrates

are

due

to

differences between

synthesis of

theenzyme in

mixed

flora

or

differ-ences

in

enzymatic affinity for the substrates is

unknown. Presently, it is difficult

to

offer

a

convincing hypothesis for the existence of three

different

A4-3-keto

reductases,

namely,

3a,5,1-,

33,5p-,

and

3P,5a-reductases,

all

acting

upon

ring-A.

Preservation ofcontraceptive properties of syn-thetic

progestins.

Our

experiments

show that the

minimal loss

of

biological activity

of synthetic

progestins

in the intestinal

environment is

attrib-utable

to

the

ethynyl

group

which enhances the

resistance of ring-A

to

reducing

enzymes. It

is

more

difficult

to

explain the

rare

failure

of these

contraceptive

hormones in

women

receiving

antibiotic

therapy (3, 8, 17). Perhaps the

antibi-otics reduce the intestinal

population

of

deconju-gating bacteria and

thereby

promote a

substan-tial loss of

hepatically conjugated

hormones

through fecal excretion.

Alternatively,

it is

pos-sible that alteration

of the flora results

in a

relative increase in the

population

of

ring-A

C

0 L.

0 c

0

(.) al

100 80 60

40

20

0 0

I a /

-

IJ

"

I/l

!~~~~~~~~~~~~~~-|

'.-C)-

-/---/

2 4 6 8 14

Time, days

o---o Dihydronorethindrone

&---

Tetrohydronorethindrone

FIG. 4. Rate of conversion of norethindrone with

C. paraputrificum.

For

details,

see

legend

to

Fig.

3.

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504 BOKKENHEUSER ET AL.

TABLE 3. Substratecompetition between Clostridium species

Conversion in culture mixture with C.

paraputrificum(%)

Substrate Clostridium

species

J-1:

3ot,50

3,B,5

3a,5p

3,,5CY

Progesterone 80 20 80 20

Norethindrone 50 50 50 50

Dimethisterone 20 80 10 90

Norgestrela 10 40 15 35

a 50% of this substrate wasunconverted.

reducing bacteria, i.e., the reducing organism mightberesistanttotheadministeredantibiotics and multiply atthe expense ofsensitive intesti-nal strains.

Sitesofinactivation ofprogestins. Natural

pro-gestins are inactivated mainly in the liver through reduction of thedouble bonds (10, 15). However, the small proportion ofA4-3-keto ste-roidswhich enterstheenterohepatic circulation

undergoes hydrogenation by the intestinal flora

to form ring-A saturated hydroxy steroids. In contrast, the hepatic reduction of the A4-3-keto structure of synthetic progestins is slow and incomplete, allowing the majority ofmolecules

to enterthegeneralcirculationandperformtheir hormonal functions. The synthetic progestins undergoing enterohepatic circulation (1, 2) are

slowly reduced by bacterial enzymes as

de-scribed above.

For patients treated with norgestrel, Sisen-wineetal. (16)noted that allfour variants of the hydrogenated

M4-3-keto

function (ot,5o; 3ot,51;

3P,5ca;

and 3P,51) appeared in urineand feces. Evidencesuggests that the human liveris capa-ble of performing the

3ot,5p

reduction, but whether it synthesizes the other reductases is unknown. Ourresults showthatintestinal

Clos-tridium species synthesize at least three of the reductases active uponring-A. Ifit is

permissi-ble toapplytheresultsfromanimalexperiments (7, 18) to the human situation, it seems likely that ring-A reduction ofsynthetic progestins is

carried out both by the liver and by certain intestinal Clostridium species.

ACKNOWLEDGMENTS

Thisinvestigationwas supported inpartby Public Health

Servicegrants CA-25763 from theNational Cancer Institute and AM-25324fromthe Institute of Arthritis, Diabetes, and DigestiveandKidney DiseasestoSt. Luke's-Roosevelt

Insti-tute forHealth Sciences, and bygrant HL-24061 from the National Heart, Lung and Blood Institute to Beth Israel MedicalCenter.

Wethank L. V.Holdeman, Anaerobic Laboratory, Virginia Polytechnic Institute, Blacksburg, Va., for the identification

of theClostridiumspecies, supported by project 2022820from the Commonwealth of Virginia.

LITERATURE CITED

1. Adlercreutz, H., F. Martin, P.Jarvenpaa, and T. Fotsis. 1980. Steroid absorption and enterohepatic recycling. Contraception 20:201-223.

2. Back, D. J., A. M. Breckenridge, F. E. Crawford, K. J. Cross, M. L'E. Orme, A. Percival, and P. H. Rowe. 1980. Reduction of the enterohepatic circulation of norethister-one by antibiotics in the rat: correlation with changesin the gut flora.J. Steroid Biochem. 13:95-100.

3. Bacon, J. F., and G. M. Shenfield. 1980. Pregnancy attrib-utable to interaction between tetracycline and oral contra-ceptives. Br.Med. J.iv:293.

4. Bokkenheuser, V. D., J. Winter, P. Dehazya, 0.DeLeon, and W. G. Kelly. 1976. Formation and metabolism of tetrahydrodeoxycorticosterone by human fecal flora. J. Steroid Biochem. 7:837-843.

5. DeJongh, D. C., and J. D. Hribar. 1968. The identification of some human metabolites of norgestrel, a new progesta-tional agent. Steroids 11:649-666.

6. Fotherby, K. 1974. Metabolism of synthetic steroids by animals and man. Meeting on pharmacological models to assess toxicity and side effects of fertility regulating agents. Acta Endocrinol. 75(Suppl. 185):119-147. 7. Freudenthal,R. I., C. E. Cook, M. Twine, R. Rosenfeld,

and M. E. Wall.1971.Metabolism ofnorethynodrelby rat liver. Biochem. Pharmacol.20:1507-1512.

8. Friedman,C. I., A. L. Huneke, M. H. Kim, and J. Powell. 1980. Theeffectof ampicillin on oral contraceptive effec-tiveness. Obstet. Gynecol. 55:33-37.

9. Kamyab,S., P.Littleton,and K. Fotherby. 1967. Metabo-lismandtissue distribution of norethindrone and norges-trel in rabbits.J. Endocrinol. 39:423-435.

10. Laatikainen, T. 1970. Excretion of neutral steroid hor-mones in humanbile.Ann. Clin. Res. 2(Suppl. 5):1-28. 11. Layne, D. S., T.Golab, K. Arai, and G. Pincus. 1963. The

metabolic fate of orally administered 3H-norethynodrel and 3H-norethindrone in humans. Clin. Pharmacol. 12:905-911.

12. Littleton,P., K.Fotherby,and K. J. Dennis. 1968. Metab-olism of("4C)norgestrel in man. J. Endocrinol. 42:591-598.

13. Macdonald, I. A., Y.P. Rochon, D. M. Hutchison, and L. V. Holdeman. 1982. Formation of ursodeoxycholic acidfrom chenodeoxycholicacid by a7f-hydroxysteroid dehydrogenase-elaborating Euibacte-ium aerofaciens straincoculturedwith7a-hydroxysteroid dehydrogenase-elaborating organisms. Appl. Environ. Microbiol. 44:1187-1195.

14. Martin, F., P. Jarvenpaa, K. Kosunen, C. Somers, B. Lindstrom,and H. Adlercreutz.1980.Ring-Areductionof medroxyprogesterone acetate in biological systems. J. SteroidBiochem.12:491-497.

15. Migeon, C. J., A. A. Sandberg, H. A. Decker, D. F. Smith, A. C. Paul, and L. T. Samuels.1956. Metabolism of4-'4C-cortisol in man. Body distributionand rates of conjugation.J. Clin. Endocrinol. Metab. 16:1137-1150. 16. Sisenwine, S. F., H. B. Kimmel, A. L. Liu, and H. W.

Ruelius. 1973. Urinary metabolites ofDL-norgestrel in women.ActaEndocrinol. 73:91-104.

17. Skolnick,J. L., B. S. Stoler, D. B. Katz, and W. H. An-derson.1976.Rifampin.oralcontraceptives, and pregnan-cy. J. Am. Med.Assoc. 236:1382.

18. Taylor,W. 1971.Theexcretion of steroidhormone metab-olites in bile andfeces.Vitam. Horm.29:201-285. 19. Winter,J., A. C. McLernon, S. O'Rourke, L. Ponticorvo,

and V. D. Bokkenheuser. 1982. Formation of 20,-dihy-drosteroidsby anaerobic bacteria. J. Steroid Biochem. 17:661-667.

20. Yamaguchi, Y. 1980. Enzymatic color development of urinary3a-hydroxysteroidsonthinlayerchromatograms. Clin.Chem. 26:491-493.

J.CLIN. MICROBIOL.