Excretion of steroid hormones in adults steroids in urine from adults

79 WA 5106

EXCRETION STEROIDS

OF STEROID IN URINE

HORMONES

IN ADULTS

FROM ADULTS

SUMMARY

Gas chromato~aphy-m~s spectrometry has been used to study the excretion pattern of neutral steroids in urine from adult female and male subjects. The compounds were isolated as free steroids, steroid “glucuronides”, monosulphates and disulphates. The major part, 72% (female) and 76% (male) of the steroids isolated occurred as “glucuronides”, whereas the free, monosulphate and disulphate fractions contained 19% (female) and 12% (male), 6% (female) and 9% (male), 3% (female) and 3 Q/*(male) of the steroids isolated, respectively. The free steroid fractions contained IO steroids, and the glucuronide, monosulphate and disulphate fractions 17, 18 and 7 steroids, respectively. Of the total steroids identified 23 were C,, steroids and 13 were C,, steroids. g steroids were found only in urine from female subjects and 3 only in urine from male subjects. The following II compounds have not previously been found in urine from normal, non-pregnant adult subjects: 5-androstene-3cq7a-dio1, 3cx-hydroxy-5@,17a (and r7P)-pregnan-zo-one, 3a(and 3/?),&dihydroxy-$+androstan-r7-one, sa-androstane-3cc,rga,r78-triol, Sol(and $)-p reg nane-3cl,r6cc,zool-triol, 5-pregnene-3%,&z, zoc+triol, ga-pregnane-3/J,rr~,r7cz,2o~-tetrol and 3a,r6/%dihydroxy-ga-androstanr7-one. The results are discussed in relation to present knowledge of excretion of steroids in humans.

INTRODUCTIOrU’

In previous reports we have described the identification of a large number of C,, and C,, steroids in urine and faeces from pregnant women and in faeces from healthy adult female and male subjectP3. In these and other studieW the influence Trivial names and unusual abbreviations: Dehydroepiandrosterone, &hydroxy-5-androsten-r7one; androsterone, 3cc-hydroxy-gee-androstan-r7-one; etiocholanolone, 3whydroxy-$androstanx7-one; epi-androsterone, 3P-hydroxy-5cc-androstan-17-one. Silyl, trimethylsilyl. Retention times TV are calculated relative to ga-cholestane. CEip2.Ch6nz.A&,

.#I (1972) 7q-go

80

ERIKSSON.

GUSTAFSSON

of the intestinal microflora upon the steroid pattern in urine has been demonstrated. It was therefore of interest to investigate the possible occurrence of microbial metabolites of steroids in urine from healthy adult females and males. Several previous investigations have been carried out on total steroid content in urine after hydrolysis, but relatively little information is available concerning the mode of conjugation of steroids in urine. Recently, Janne” presented a study on some of the steroid sulphates in urine. Since sulphates only constitute between 9-12O/~ of the total steroid content in urine it was considered of interest to perform a more total analysis of urinary steroids. The present paper is a gas chromatographic-mass spectrometric study of the quantitatively predominant steroids in urine from adults. MATERIALS

AND METHODS

Steroids 3P-Hydroxy-g- [7GH]androsten-r7-one 3-sulphate (specific activity 605 mC/ mmole) and 3P-hydroxy-5-[4-Xlpregnen-2o-one (specific activity 55.7 mC/mmole) were purchased from the Radiochemical Centre (Amersham, England). Most of the reference compounds used in this investigation were obtained from sources given in previous papers 132.Dr. L. Starka kindly donated 3P,7a-dihydroxy5-androsten-r7-one and 3P,7P-dihydroxy-5-androsten-r7-one. 3a,r8-Dihydroxy-gaandrostan-r7-one was a gift from Dr. D. K. Fukushima. Collectiofl of urine Urine was collected during 24 h from five healthy males (20-25 years of age) and four healthy females (20~25 years of age). The urine was pooled to a male urine pool (4700 ml) and a female urine pool (3800 ml). The urine was stored at -20’. Extraction and purification of urinary steroids 200 ml of urine was taken from each pool and was worked up principally as described in a previous publicationl: chromatography on Amberlite XAD-2, ion exchange chromatography on Amberlyst-15 in the sodium form and chromatography on Sephadex LH-20 in the solvent system chloroform-methanol (I: I, v/v) made 0.01 M with respect to NaCI. This procedure gave a free steroid+glucuronide fraction, a monosulphate fraction and a disulphate fraction. The free steroid+glucuronide fraction was partitioned between ethyl acetate and 8.4% (w/v) sodium bicarbonate in water. The ethyl acetate phase was washed with distilled water until neutral and evaporated to dryness (free steroid fraction). The sodium bicarbonate phase was made acid with I M HCI and extracted three times with equal volumes of ethyl acetate. The combined ethyl acetate phases were washed until neutral and were then evaporated to dryness (glucuronide fraction). Aliquots of the free steroid, glucuronide, monosulphate and disulphate fractions were analysed by thin-layer chromatography using the solvent system ethyl acetate-ethanol-15 M ammonium hydroxide (5 : 5 : I, by vol.)’ and were found to have mobilities as reference free steroids, glucuronides, monosulphates and disulphates, respectively. No “glucuronides” were overlapping into the monosulphate fraction. The final identification of the material in the steroid glucuronide fraction as glucuronides must await further characterization. The free steroid fraction was further purified by chromatography on a 10-g CZin. Chim.

Acta,

.+I (1972)

79-90

URINARY

81

STEROIDS IN ADULTS

Sephadex LR-zo column prepared in and eluted with chloroform-heptane-ethanol (5:5: I, by vol.) saturated with waters. Four fractions were collected: fraction A (0-30 ml of effluent), fraction B (30-50 ml), fraction C (50-90 ml) and fraction D (IOO ml of methanol). An aliquot of each fraction was taken for preparation of silyl ethers which were then analysed by gas-liquid chromatography and gas chromatographymass spectrometry9. Urine

Amberlite

X4D-2

Amberlyst-15

Sephadex \-

chromatography

chromatography

LH-20 chromatography

CHCl~/Me~H

l:I,

0.01 M NaCi

Free steroid-t

Steroid

mono-

Steroid

steroid

glucc-

sulphate

frac-

sulphate

ronide

fractions

tion

I

fraction

di-

r--l’ Partition

between

NaHCO,

hydrolysis

and ethyl

Ia

I

tate

Steroid

Water

Water

Ethyl

Free

glucu-

phase

phase

acetate

steroid

ronide

fraction

fraction

4

ase

t

Solvolysis

phase

I Gas chromatography-Mass

-I d

Ethyl

I

i

1 Solvolysis

acetate

I

phase

spectrometry

(

82

ERIKSSON,

GUSTAFSSON

The steroid “glucuronides” were hydrolysed with Ketodasem as described beforel. The liberated steroids were purified by chromatography on Sephadex LH-zo in chloroform-heptane-ethanol (5:5: I, by vol.) as described above for the free steroid fraction. The steroids were analysed as silyl ethers by gas-liquid chromatography and by gas chromatography-mass spectrometry. Part of the monosulphate fraction was solvolyzed in ethyl acetate acidified with 2 M sulphuric acid for 18 h at 3g”. The ethyl acetate phase was then washed with 8.4% (w/v) sodium bicarbonate solution until alkaline and then with distilled water until neutral and finally taken to dryness under vacuum. An aliquot was silylated and analysed as described for the free steroid and glucuronide fractions. Another part of the monosulphate fraction was first hydrolysed with enzymes of the digestive juice from Helix pomatia at 37’ for 48 hlO. The incubation mixture was extracted three times with ethyl acetate. The combined ethyl acetate phases were washed with small volumes of distilled water, taken to dryness under vacuum and analysed as described above. The water phase remaining after Helix pomatia hydrolysis of the monosulphate fraction was made 2 M with respect to H,SO, and was extracted three times with 25 ml of ethyl acetate. The combined ethyl acetate phases were incubated over night at 39”. After solvolysis the ethyl acetate phase was washed with 8.4% (w/v) sodium bicarbonate in water until alkaline and then with water until neutral. The ethyl acetate phase was then evaporated to dryness under vacuum. The solvolysed steroids were analysed as described above. The steroid disulphate fraction was treated in the same way as the steroid monosulphate fraction. The principal procedure used in the purification of the urinary steroids is summarized in the flow sheet shown below. Semiquantitative analysis 50 ml of urine from the male and female urine pools was taken for semiquantitative analysis. 2 x 10~ counts/min of 3@-hydroxy-5-[4-Wlpregnen-zo-one and 2 x 105 counts/min of 3/?-hydroxy-5-[7&II]androsten-r7-one 3sulphate were added as tracers. The urine was worked up in the same way as described above for the quantitative analysis (using only the solvolytic procedure to liberate mono- and disulphurylated steroids). The recoveries of added 3P-hydroxy-5- [4-Wlpregnen-zo-one and 3/3hydroxy-5-[7@H]androsten-r7-one 3-sulphate were used to calculate the losses in the extractions and chromatographic procedures. Losses were about 20% of l4C and 50% of 3H. Most of the unrecovered 3H was lost during solvolysis. RESULTS

The C,, and C,, steroids identified in the free, glucuronide, mono- and disulphate fractions are listed in Table I. Gas-liquid chromatographic analyses of the steroids in some of these fractions are shown in Figs. 1-5. Mass spectrometric analyses of the silyl ethers of all of the steroids identified yielded mass spectra identical with those of the silyl ethers of the respective reference compounds. Certain identifications are discussed in further detail below. C,, steroids. Of the compounds identified, 8 were C,,O, and 15 were C,,O, steroids. C,,O, steroids. Three different isomers of 5-androstene-3,r7-diol were identified Clin.

Chim.

Acta,

41 (1972) 79-90

URINARY TARI,E

STEROIDS I

RELATIVE (F),

RETENTION

GLUCURONIDE

MALE

83

ITi ADULTS

AND

FEMALE

TIMES

(G),

(5~~ChOkStan’Z

(M)

MONOSULPHATE

SUBJECTS

AND

=

CrF SILYL

POUNDS -

Compound

Compound I 3ff-Hydroxy-5~-androstan-I7-*~~ Compound 2 5-Androstene-3a,17rdiol Compound 3 3a-Hydroxy-5B-androstan-x7-one Compound 4 $-Androstane-3 a, 17/%diol Compound 5 3fi-Hydroxy-5wandrostacr-/-one Compound 6 3cc-Hydroxy-5/?,17cc-pregnan-2o-one Compound 7 3P-Hydroxy-5-androsten-17-one Compound 8 5-Androstene-3~,17ff-diol Compound 9 3cc, 16ac-Dihydroxy-5-androsten-17-one Compound IO 3P,7cc-Dihydroxy-5-androsten-r7-one Compound I 1 5-Androstene-3p,17,f-diol Compound 12 3cc,r6cx-Dihydroxy-5wandrostan-r7-one Compound 13 3a-Hydroxy-5@,17@-pregnan-2o-one Compound 14 30(, 11~-D~ydroxy-5~-androstan-17-one Compound 15 3~,1S-Dihydroxy-s~-andros~n-~7-on~ Compound 16 ga-Androstane-3cc,15a,17/3-triol. Compound I 7 3@,7/%Dihydroxy-5-androsten-r7-one Compound I8 3a,rGfl-Dihydroxy-5cc-androstan-17-one i:ompound 19 38, r6a-Dihydroxy-5mandrostan-17..one Compound 20 38, I 6cc-Dihydroxy-5-androstenI 7-one Compound 21 58-Yrepnane-3~,zoB-diol Compound 22 3fi,18-Dihydroxy-5a-androstan-r7-one Compound 23 gcc-Androstane-3cr,16.z,17~-triol Compound 24 5oc-Pregnane-3cc,2owdiol Compound 25 3/?,17/3-Dihydroxy-5-androsten-16-one Compound 26 58-Pregnane-3cr,zoac-diol Compound 27 j-Androstene-3/3,16@,17oc-trio1 Compound 29

1.00)

OF SILYL

AND DISULPHATE ETHERS

(D)

OF TBE

ETHERS

OF STEROIDS

FRACTIONS

OF URINE

CORRESPONDING

State of conjugation of isolated conspound in male wine

female wine

G, M

-

-

G

G

G, M

G

G

-

M

G

G

M

F, Bt

D

D

G

p, G

-

F

M, D

G, D

M

G, M

G

G

G

G

M

X

G

G

M

G, M

D

-

M

M

M

M

G

G

-

IVL

M

M

-

F

M

M

-

D

I;, M

F, M, D

FREE COM-

SE-30

QF-I --

0.42

1.1:

0.3s 0.35 0.40% 0.39 0.40 o.g1

0.50 0.51 0.50” 0.53 0.53 0.52 0.55 0.53 0.63 0.60 0.61 0.61 0.63 0.61 0.66 0.65 0.67 0.67 0.71 0.71 0.74 0.71 0.71 0.73 0.71 0.73 0.72 0.70” 0.85 0.83” 0.85 0.84 0.89 0.89 0.92 o.g1= 0.95 0.93 0.91 0.93 o,95 0.94 0.97 0.96 I.02 1.02

F

THE

REFERENCE

0.51

-

IN

FROM NORMAL

1.11

Clin. Chiwz. Acta, 41

I.14

0.38 0.36a 1.18 1.18

0.45 0.46 1.49 1.47’ 1.39 1.40 1.34 1.33 0.50 0.48 1.04 I .o* 0.58 0.57 1.12 1.x* 1.61 I.63 I.58 1.58 I.66 I.68 I.90 1.89 1.32 1.30a 1.40 1.4~~ I .40 I.41 0.83 0.83 2.20 Z.I@ 0.72 0.72 0.82 0.85 1.79 1.80 0.92 0.93 0.72 0.75 0.88 (1972)

p-go

84

ERIKSSON,

Covnpound

State of conjugation of isolated compound in

5/j-Pregnane-3cc,16cc,zoa-triol Compound 29 j-Pregnene-3B,2occ-diol Compound 30 5-Pregnene-3cr,I6cc,2oa-triol Compound 31 5-Androstene-3/3,16c(, I7/3-trio1 Compound 32 jti-Pregnane-3a,I6c,2og-triol Compound 33 5B-Pregnane-3cc, 17cc,2owtriol Compound 34 3c4,z1-Dihydroxy-j@-pregnan-20-01~ Compound 35 j-Pregnene-3B,I7a,20cc-triol Compound 36 5E-P-3~,11~,r7c(,zo~-tetrol a Calculated

retention

male urine

female wine

M, D

M, D

G

G

M

M

SE-30

I.11

F, G

1.15 1.14 I.14 1.15&

F, G, M,

-

F

F, G, M, D

F, G, M, D G

time by using analogous

I

I

10

5

QF-r

0.86 0.96 0.98

1.20

0.92

I.20

0.92

1.22

0.84 0.85

1.22

F, G, M

GUSTAFSSON

I.32 1.31 I.33 I.35 =.57 I.57 1.65

0.97 0.96 2.00 2.01 1.87 1.88

isomers.

TIMECmin) Fig. 1. Gas chromatographic analysis (SE-30) of silyl ethers of steroids in the free steroid fraction of urine from female subjects. The compounds were identified as 3/3-hydroxy-j-androsten-17-one (5), 3cc,I6wdihydroxy-5-androsten-r7-one (g), 3a,rIb-dihydroxy-jcc-androstan-17-one (14). 3arhydrosy-5P,I7/?-pregnan-2o-one (13), 5/3-pregnane-3cc,16cc,zowtriol (28).

(compounds z, 8 and II). One of these, 5-androstene-3cl,r7&-diol, found in the glucuronide fraction of urine from females, has not previously been isolated from urine from human subjects. The two other epimers, 5-androstene_3/2,17a(and r-//$diol, have previously been isolated as mono- and disulphates from urineil. In this study, 5-androstene-3/3,17/?-diol was also found in the glucuronide fraction of urine from females. The glucuronide fraction of urine from both female and male subjects contained $Landrostane-3a,r7p-diol (compound 1). Four different r7-keto C,,O, steroids were isolated (compounds I, 3, 5 and 7). ck$-‘LChi?‘?‘&n. ACta, 41

(1972)

79-90

UKISARY

STEROIDS IN ADULTS

lb

15

i

0

TIME (mid Fig. 2. Gas ~hromato~ap~c analysis (SE-30) of silyl ethers of steroids in the steroid glucuronide fraction of urine from female subjects. The compounds were identified as 5-androstene-3d,r7cc-diol (z), 3a-hydroxy-5B-androstan-17-one (3)‘ 5-androstene-3P,r7cc-diol (8). 3a,r6cc-dihydroxy-5-androsten-r7-one (9). 5-androstene-3B.r7P-diol (zI), 3cc,rrp-dihydroxy-gc-androstan-x7-one (14), $?-pregnane-3cc.2o/3-diol (zI), 5-pregnene-3cc,r6cc,2ow-triol (30), ga-pregnane-3cc,r6a,zocc_triol (32), $%pregnane-3a,r7a,zoc+triol (33) and 5-pregnene-3B,r7Lx,zoa-triol (35).

I

I

15

10

I

TIME(min)

5

Fig. 3. Gas chromatographic analysis (SE-30) of silyl ethers of steroids in the steroid monosulphate fraction of urine from female subjects. The compounds were identified as 3B-hydroxy-5-androstenr7-one (5), 3cc,r%dihydroxy-5oc-androstan-r7-one (15). 3fi.7/3-dihydroxy-5-androsten-r7-~ne (17). 3P,xGwdihydroxy-5-androsten-r7-one (zo), 5-androstene-3ie,16P,r7cr-triol (27), 5fi-pregnane-3fl, zowdiol (29) and 5-androstene-3@,r6cc,r7,k-triol (31).

Etiocholanolone and dehydroepiandrosterone were found in urine from both males and females whereas androsterone was only isolated from males and epkmdrosterone only from females. (For mass spectrometric data on these C,Q steroids, see ref. 13.)

ERIKSSON, GUSTAFSSON

86

I

I

I

I

I

I

30

25

20

15

10

5

0

TIME (mid Fig. 4. Gas chromatographic analysis (SE-30) of silyl ethers of steroids in the steroid glucuronide fraction of urine from male subjects. The compounds were identified as 3a-hydroxy-5P-androstanr7-one (3). 5/?-androstane-3cc,r7,!%diol(4). 3or-hydroxy-5/3,r7wpregnan-ao-one (6), 3cc,rbwdihydroxy-g-androsten-r7-one (g), 3a-hydroxy-5p,17/%pregnan-zo-one (13), 3t~,1ra-dihydroxy-gcc-androstan-r7-one (r4), 5a-androstane-3cr,15cc,r-/fi-triol(r6), 5P-pregnane-3cr,2op-diol(21), 5-pregnene3cr,r60(,2ocr-trio1(30) and 5,8pregnane-3cc,17or,20a-triol(33).

10

5 TIME(min)

Fig. 5, Gas chromatographic analysis (SE-30) of silyl ethers of steroids in the steroid monosulphate fraction of urine from male subjects. The compounds were identified as 5-androstene-3/3,17@-diol (17). (II), 3cc,r8-dihydroxy-5cc-androstan-17-one (IS), 3/3,17p-dihydroxy-5-androsten-17-one 3P,17,B-dihydroxy-5-androsten-16-one (25), 5-androstene-3B,16cc,17p-triol (jr), 5@-pregnane-3% r7”,2owtriol (33) and 5-pregnene-3p,17cz,zowtriol (35). Clin. Chim. Acta,

41 (1972) 79-90

URINARY

STEROIDS

IN ADULTS

87

7-Hydroxylated C,O, steroids. Two 7-hydroxylated C,O, compounds were identified, 3,9,7a(and 78)-dihydroxy-5-androsten-r7-one (compounds IO and 17). The 7aepimer was only found in urine from females (free steroid fraction), while the 7/L epimer was excreted as a monosulphate both by males and females. For mass spectra of these steroids, see ref. 13. i-5-Hydro&&i C,,O, steroid. Compound 16 was isolated as a “glucuronide” from urine from female and male subjects. Gas chromatographic-mass spectrometric analysis of the silyl ether of this compound indicated a 3,r5,r7-tris(trimethylsiloxy)androstane structure14. The most likely 3,5configuration is 3a,5a (refs. 4,5). However, a 3a,5fl or 3p,5/3 structure cannot be excluded. m6-Oxygenated C,O, steroids. Five r6-hydroxylated r7-keto-C,,O, steroids were identified (compounds g, IZ, 18, rg and 20). Four of these, 3cc(and 3p), r6a-dihydroxy5-androsten-r7-one and ga(and 3/3), r6a-dihydroxy-5cc-androstan-r7-one, were found -principally as monosulphates-in urine from both females and males. ga,rb/J-Dihydroxy-5cr-androstan-r7-one was isolated as a monosulphate from urine from male subjects only. Three steroids with a r6,r7-dihydroxy configuration were isolated (compounds 23, 27 and 31).These steroids were identified as 5a-androstane-3a,r6a, r7/$trio1 and 5-androstene-3P,rGp(and 16a),r7&riol, respectively15. While 5a-androstane-3a,r6cc,r7p-triol and 5-androstene-3/?,16a,r7p-triol were only found in the monosulphate fraction, 5-androstene-3/3,rG/?,r7a-triol was also found as a free and as a disulphurylated steroid. Compound 25 in the monosulphate fraction of urine from both females and males was identified as 3,9,r7/3-dihydroxy-5-androsten-r6-one15. &Hy&oxyZated C,,O, steroids. The monosulphate fraction of urine from both males and females contained a steroid (compound IS), the silyl ether of which yielded a mass spectrum with a molecular ion at lnje 450 (M) and a prominent peak at m/e 420 (M-30). The mass spectrum indicated a 3,&bis(trimethylsiloxy)-androstan-r7one structurels. Compound 15 was identified as 3cc,r8-dihydroxy-5cc-androstan-r7-one. A similar mass spectrum was given by the silyl ether of compound 22 (found in the monosulphate fraction of urine from female subjects). This steroid was identified as 3b,r8-dihydroxy-5a-androstan-r7-one. C,, deroids. Thirteen C,, steroids were identified: six C,,O, steroids, six C,,O, steroids and one C,,O, steroid. The silyl ethers of compounds 6 and 13 gave mass spectra indicating a 3-hydroxy-pregnan-zo-one structure (see ref. 17). Retention time analysis on SE-30 and QF-I columns established the identities of the compounds as 3a-hydroxy-5j!I,r7a(and r7P)-pregnan-zo-one, respectively. Compounds ZI, 24, 26 and zg yielded mass spectra with a prominent base peak at ynje 117, typical of C,, steroids with a -CHOSi(CH,),CH, side chainls. The compounds were identified as 5P-pregnane-3a,zo/&diol, 5a-pregnane3a,zoa-dial, 5P-pregnane-3a,zoa-diol and 5-pregnene-3/?,zocc-diol, respectively. Only two of these compounds were found in urine from males, i.e. 5/3-pregnane-3a,zop-diol and 5-pregnene-3@,zoa-diol. Compound 34 isolated as a free steroid in urine from females was identified as 3a,zr-dihydroxy-5,&pregnan-zo-one. The free steroid fraction of urine from female subjects contained two compounds (compounds 28 and 32) the silyl ethers of which gave mass spectra with molecular ions at m/e 552 and prominent peaks at *a/e 117, 141, 156 and 157. These peaks are typical of steroids with a pregnane-3,r6,zo-trio1 structurelg. The steroids were idenClin. Chim.

Acta,

41 (1972)

79-90

88

ERIKSSON.

GUSTAFSSON

tified as 5P-(and 5cc-pregnane-3a,Iba,aoa-triol. The corresponding 3P-hydroxy-ASsteroid was found in the glucuronide fraction of urine from both female and male subjects (compound 30, see Figs. z and 4). “Pregnanetriol” (5/3-pregnane-3cr,I7c,zocctriol) and 5-pregnene-3P,I7cc,zocr-triol were both found in the free, glucuronide and monosulphate fractions of urine from females and males (compounds 33 and 35), respectively. The unsaturated compound was also isolated as a disulphate. Compound 36 was found in the monosulphate fraction in urine from male subjects. After persilylation 2o this steroid gave a mass spectrum with a molecular ion at nzje 640 indicating a tetrakis (trimethylsiloxy)-pregnane structure. The base peak was at lnje 253. The mass spectrum was similar to that of persilylated 5-pregnene3~,II~,17~,zo~-tetrolzo except that the ions of the urinary steroid were two mass units heavier. Therefore, compound 36 is tentatively identified as 5%pregnane-36,116, 17~,2o&tetrol. Co&sol metabolites. Some of the fractions contained steroids that were metabolites of cortisol. The identification of these steroids is in progress and will be presented as a separate investigation. TABLE

II

QUAN’IWATW% REllATIONSHIPS OF AND

DISULPHATE

FRACTIONS

Fraction Free steroid fraction Glucuronide fraction Monosulphate fraction Disulphate fraction Total amount

TOTAL

OF URINE

STEROIDS

FROM

MALE

IN AND

THE

FREE,

FEMALE

GLUCURONIDE,

ADULT

MONOSULPHATE

HUMANS

Free steroid excreted i+z 24 h (mg) Male subjects

Female subjects

20 125 11 5.5 164.5

37 139 II

5.8 192.8

Quantitative relatiomhips. Table II shows the excreted amounts in the free, glucuronide, monosulphate and disulphate fractions of and female subjects. The quantitatively most important fractions ronide and free fractions. Only about 13 (males) and g (females) per amount of steroids were found in the monosulphate and disulphate

of total steroids urine from male were the glucucent of the total fractions.

DISCUSSION

In man the major pathway for elimination of steroid hormones and their metabolites is excretion in the urine21 and a large number of steroids have been identified in urine. In spite of the wealth of information available relatively few investigators have studied the conjugate pattern in urine and how the steroid structure influences the type of conjugate formed. In the present study the chromatographic behaviour of the urinary steroids on Sephadex LH-zo and their cleavage by solvolytic or hydrolytic procedures indicate that the compounds occur as free steroids, steroid and mono- and disulphates. In addition to these conjugates three “glucuronides”, other types of steroid conjugates have been described in urine by other authors, i.e. phosphoric acid conjugatezz, urea conjugates3 and z’-acetamido-z’-deoxy-cr-o-glucoside conjugatez4. Clin. Chim. Acta, 41 (1972) 79-90

URINARY

STEROIDS IN ADULTS

89

The major part (about 72-76%) of the steroids identified in the present study were found in the glucuronide fraction. The total amount of sulphates constituted of the st eroid content, whereas Iz--Ig”/o were excreted as free steroids. In an investigation on the steroid content in urine from a pregnant woman, approximately the same pattern of conjugates was found as in the present studyl. This indicates that generally the quantitatively predominant fractions in urine from human subjects are the free steroid and the steroid glucuronide fractions. The total amount of steroids excreted in urine from adult females and males during 24 h was rgz mg and 165 mg, respectively (uncorrected figures). Between 17-19 mg of these represented steroid sulphates. In a quantitative study of the neutral steroid mono- and disulphates in human urine, Janne reported6 a daily mean excretion of 6-7 mg, with the range 4-13 mg. The distribution of steroids between the four fractions isolated was to some extent related to the structure of the steroids. The major part of the steroids in the free steroid and steroid mono- and disulphate fractions had a 3,!I,A5 structure, whereas the g-12%

steroids with a 3x,5/3 or 3a,5a structure were predominantly found in the glucuronide fraction. These findings are in good agreement with what has previously been reported on the relationship between type of steroid conjugate and stereochemistry of conjugated steroid: steroids with a 3B,A5 structure, e.g. 2r-hydroxypregnenolone, dehydroepiandrosterone and 5-pregnene-3/3,17a,2r-triol, are chiefly excreted as sulphates (25-29) whereas steroids with a 3x,5/3 structure, e.g. $Lpregnane-3a,zoa-diol and are excreted mainly as glucuronides30-35. These genQ-pregnane-3a,r7a,zoa-triol, eral rules are true for neutral steroids in plasma 36, bile3? and faeces3 from humans. Several of the steroid metabolites isolated from urine from female subjects were not found in urine from male subjects, whereas the latter excreted three compounds that could not be identified in urine from females (see Table I). No significant sex differences were found in the pattern of steroid conjugates. This is in contrast to the great differences in steroid metabolism in female and male rats4>5. Whereas female rats excrete approximately 50% of the steroids in urine as sulphates, male rats only excrete unconjugated steroids in urine. The glucuronide fraction from both male and female subjects contained one r7cc-pregnane steroid, 3a-hydroxy-3P,r7cc-pregnan-zo-one. This steroid was previously isolated from faeces and urine from a pregnant woman1 and has been shown to be Although it cannot be excluded that a metabolite of r6a-hydroxyprogesterone38. r7cl-pregnane compounds may be formed in extra-intestinal tissues, studies on germfree and conventional rats have demonstrated the formation of r7a-pregnane steroids from IGa-hydroxylated precursors via 16,17unsaturated intermediates17339. It seems quite likely that at least part of the 3a-hydroxy-5a,r7”-pregnan-zo-one isolated in urine from adults is of microbial origin indicating the existence of an enterohepatic circulation of progesterone metabolites. Most of the steroid metabolites identified in this study have been described by other authors, but several new compounds have been identified, e.g. sa-androstane3a,r5cc,r7/?-triol, 3/3,r8-dihydroxy-3a-androstan-r7-one, 3cc,r6/3-dihydroxy+x-androStan-r7-one and 5-pregnene-3cl.,r6cc,zoa-triol. One new compound, the stereochemistry of which was not established, had the general structure pregnane-3,rr,r7,zo-tetrol. Several very polar compounds were also isolated. These steroids were cortisol metabolites and have not been included in this study. Methods are presently being develC&z. Chim. Acta, 41 (1972)

79-90

ERIKSSON,

90 oped to make a qualitative possible.

and quantitative

GUSTAFSSON

analysis of these polar steroid metabolites

ACKNOWLEDGEMENTS

This work was supported by grants from the Swedish Medical Research (project 13X-2819) and from “Syskonen WessCns Stiftelse”.

Council

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