The identification of novel steroid N-acetylglucosaminides in the urine of pregnant women

ft. Steroid Biochem. Molec. BioL Vol. 58, No. 5/6, pp. 585-598, 1996 Copyright © 1996 Elsevier Science Ltd. All fights reserved Printed in Great Britain PII: S0960-0760(96)00080-5 0960-0760/96 $15.00 + 0.00

Pergamon

T h e Identification of Novel Steroid N - A c e t y l g l u c o s a m i n i d e s in the U r i n e of Pregnant Women L. J. Meng, W. J. Griffiths and J. Sj6vall* Department of Medical Biochemistry and Biophysics, Karolinska Instituter, S-171 77, Stockholm, Sweden

A series o f p r e g n a n e d i o l s a n d p r e g n a n e t r i o l s d o u b l y c o n j u g a t e d with N - a c e t y l g l u c o s a m i n e a n d g l u c u r o n i c o r sulfuric acid has b e e n identified in u r i n e f r o m p r e g n a n t w o m e n . S t e r o i d conj uga te s w e r e s e p a r a t e d b y i o n - e x c h a n g e c h r o m a t o g r a p h y a n d the g l u c u r o n i d e a n d m o n o s u l f a t e f r a c t i o n s w e r e a n a l y s e d b y fast a t o m b o m b a r d m e n t m a s s s p e c t r o m e t r y . A ft er r e m o v a l o f t he aci d m o i e t y , t he n e u t r a l steroids wer e isolated, d e r i v a t i z e d , a n d a n a l y s e d by gas c h r o m a t o g r a p h y - m a s s s p e c t r o m e t r y ( G C - M S ) . T h e analyses r e v e a l e d t he p r e s e n c e o f steroids c o n j u g a t e d with N - a c e t y l h e x o s a m i n e b o t h in th e g l u c u r o n i d e a n d the m o n o s u l f a t e fractions. Following e n z y m e hydrolysis, t he s u g a r was identified b y G C - M S as N - a c e t y l g l u c o s a r n i n e (GIcNAc). T h e m a j o r s t e r o i d c o n j u g a t e d with GIcNAc b o t h in th e g i u c u r o n i d e a n d m o n o s u l f a t e f r a c t i o n s was identified as 5~t-pregnane-3~,20et-diol. 5li-Pregnane-3a,20a-diol was also p r e s e n t as a GIcNAc c o n j u g a t e in b o t h f r a c t i o n s w h e r e a s a GIcNAc c o n j u g a t e o f 5a-pregnane-3li,20et-diol was only f o u n d in t he sulfate fract i on. 5a-Pregnane-3~,20a,21triol was a d o u bl e c o n j u g a t e with GlcNAc in the sulfate f r a c t i o n w h e r e a s a p r e g n a n e - 2 , 3 , 2 0 - t r i o l was a d o u b le c o n j u g a t e !in t he g l u c u r o n i d e f r a c t i o n . T h e positions o f c o n j u g a t i o n w ere d e t e r m i n e d b y c o l l i s i o n - i n d u c e d dissociation o f t he p s e u d o m o l e c u l a r anions p r o d u c e d b y fast a t o m b o m b a r d m e n t ionization. T h e sulfate a n d g l u c u r o n i c acid m o i e t i e s w ere l o c a t e d at C-3 a n d N - a c e t y l g l u c o s a m i n e at C-20. An a l t e r n a t i v e localization o f GlcNAc at C-21 o f 5 a - p r e g n a n e - 3 a , 2 0 a , 2 1 - t r i o l c a n n o t be excluded. Ju d g in g f r o m t he e n z y m a t i c hydr ol ys i s o f the conjugates, the s u g a r was a t t a c h e d in [i-giycosidlc linkage. T h e m e a n e x c r e t i o n o f N - a c e t y l g l u c o s a m i n i d e s o f the p r e g n a n e d i o l s a n d p r e g n a n e t r i o l s was 32.2 Itmol/g creatin~ae (range 17.9--49.1 Itmol) in five h e a l t h y w o m e n in the 38th-39th week o f p r e g n a n c y . T h e m e a n e x c r e t i o n o f 5ll-pregnane-3~t,20a-diol g l u c u r o n i d e in t he s a m e w o m e n was 71 Itmol/g creatinine~. ( r a nge 27-127 ltmol). T hi s i ndi cat es t h a t c o n j u g a t i o n with N - a c e t y l g i u c o s a m i n e co n s titu tes a q u a n t i t a t i v e l y i m p o r t a n t p a t h w a y o f p r o g e s t e r o n e m e t a b o l i s m in h u m a n p r e g n a n c y . C o p y r i g h t © 1996 E l s e vi e r Science Ltd.

ft. Steroid Biochem. Molec. Biol., Vol. 58, No. 5/6, pp. 585-598, 1996

INTRODUCTION

N-acetylglucosamine (GlcNAc) [1-4]. These have usually involved estrogens [1, 2], but 5-pregnene313,20et-diol has also been found in human urine as a 3-sulfate, 20ct-13-N-acetylglucosaminide [3, 4]. Conjugation with GIcNAc seems to be limited to acidic steroids [ 1, 2], and recently, 713-hydroxylated bile acids were found to be selectively conjugated with GlcNAc at the 713-hydroxy group [5-7]. During pregnancy large amounts of pregnanolones, pregnanediols and pregnanetriols are formed as metabolites of progesterone and are excreted as glucuronides and sulfates (for references see [8]). However, comparisons of progesterone production rates with values for the

The metabolism of steroid hormones in humans includes the conjugation of metabolites with glucuronic and sulfuric acids. Other conjugation reactions have also been observed, e.I;. with glutathione, glucose and

*Correspondence to J. Sj6w~ll. (Received 6 Feb. 1996; accepted 27 Mar. 1996). Pregnanolone and pregnanediol are used as collective names for isomers of 3-hydroxypregnane-20-one and pregnane-3,20-diol, respectively. Pregnanetriol and pregnanediolone are used as collective terms for pregnanes carrying three hydroxyl groups, two hydroxyl groups and one oxo group, respectively. 585

L.J. Meng et al.

586

excretion of metabolites in urine and feces reveal a difference, indicating that all metabolites are not being measured by existing methods. In connection with studies of bile acid and steroid metabolism in w o m e n with intrahepatic cholestasis of pregnancy, we have combined previously developed methods for group separation of conjugates and analysis by gas chromatog r a p h y - m a s s spectrometry ( G C - M S ) [9] with fast atom b o m b a r d m e n t mass spectrometry (FABMS). W h e n applied to the analysis of urine of healthy pregnant women, F A B M S indicated the presence of GlcNAc conjugates of acidic steroid metabolites. This p a p e r describes their identification as novel N-acetylglucosaminides of pregnanediols and pregnanetriols.

MATERIALS AND METHODS

Chemicals and reagents

Subjects and samples Urine samples were collected five w o m e n who were 38-39 hospitalized for observation and delivery. T h e urine was stored at

in the morning from • weeks pregnant and subsequent caesarean - 20°C until analysed.

Analytical procedure A flow scheme of the procedure is shown in Fig. 1. Extraction and subfractionation. Urine, 20 ml, was passed through a column of Preparative Cls (1 × 0.8 cm). Following a wash with 10 ml water, the steroids were eluted with 10 ml 80% aqueous methanol. T h e column was then washed with 5 ml methanol and 10 ml water. T h e steroid eluate was evaporated to about 5 ml aqueous solution which was again passed through the column. After washing with 10 ml water, the steroids were eluted with 10 ml 80% methanol. T h e steroid eluate was applied to a

Solvents were of analytical reagent grade and were Urine distilled prior to use. I Unlabelled authentic steroids were from previous Prep.C 18 studies [8, 10]. [7~-3H]Testosterone glucuronide I TEAP-LH-20 was from the former N E N Chemicals ( D u p o n t N E N , D u Medical Scandinavia AB, Sollentuna, Sweden). I [4-~4C]Pregnenediol monosulfate was prepared by Glucuronides Monosulfates reduction of [4-~4C]pregnenolone (313-hydroxy-5pregnen-20-one; A m e r s h a m International plc, Little Chalfont, U.K.) with sodium borohydride to give a Enzyme hldrolysis Solvilysis mixture of the 20u- and 2013-epimers of 5-pregnene313,20-diol. This was sulfated [11] to give a mixture of Prep.C 18 Prep.C 18 the m o n o - and disulfates, which were separated by ion exchange chromatography. Helix pomatia juice was TEAP-LH-20 TEAP-LH-20 from M F Biotechnics, France (purchased from Kebo Lab, Sp~nga, Sweden). Immediately before use, 0.3 ml Neutral steroids Neutral steroids were added to 5 ml of 0.2 M sodium acetate buffer, p H 4 . 5 , and the solution was passed through a Lipidex-5000 Lipidex-5000 4 × 0.4 cm column of Preparative C18 [12] (see below). N-Acetyl-13-D-glucosaminidase from beef kidney was I I from Boehringer M a n n h e i m Scandinavia AB (Bromma, Sweden). Unconjugated Sugar-conjugated Unconjugated Sugar-conjugated Radioactivity was determined by liquid scintillation steroids steroids steroids steroids counting (1211 Minibeta; Wallac, Sollentuna, Sweden) using OptiPhase 'HiSafe' 2 (Wallac) as the scintillation Enzyme hydrolysis Enzyme hydrolysis liquid.

I

I

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Column packing materials Octadecylsilane-bonded silica (Preparative C18; Waters Associates Inc., Milford, MA), 0.2 or 0.3 g, was packed into glass columns to produce beds of 1 ×0.8 cm or 3 × 0 . 4 cm which were washed with 5 ml each of methanol, methanol/chloroform (1:1, v/v), methanol and water prior to use. Triethylaminohydroxy-propyl Sephadex L H - 2 0 ( T E A P - L H - 2 0 ) was synthesized as in Ref. 13, and Lipidex-5000 was from Packard Instruments Co. (Downers Grove, IL, U.S.A.). These Sephadex derivatives were washed prior to use [13].

I

I I Lipidex-5000 I

I

Prep.C 18

I

I

Prep.C18

I

Steroids
Steroids >tetra-OH

I

Derivatization

I

GLC and C-C/MS Fig. 1. Flow s c h e m e o f the analytical procedure employed.

N-Acetylglucosaminides in Pregnancy 1 2 x 0 . 4 cm column of T E A P - L H - 2 0 in its hydrogen carbonate ( H C O ; ) form packed in 80% methanol [13]. After collection of the effluent and rinses with 20 ml 85% methanol and 10 ml 0.1 M acetic acid in 85% methanol, steroid glucuronides were eluted with 30 ml 0.4 M formic acid in 85% methanol, and steroid monosulfates with 14 ml 0.3 M a m m o n i u m acetateacetic acid, p H 6.6, in 85% methanol. Glucuronide fraction. An aliquot (1/3) of the glucuronide fraction was analysed by F A B M S * after evaporation of the solvent. T h e remainder was taken to dryness in vacuo and the residue dissolved in 5 ml of the purified Helix pomatia enzyme solution. About 104 c p m of 7~-[3H]testosterone glucuronide was added to monitor the efficiency of the hydrolysis. Following incubation for 1 h at 62°C [13] the hydrolysed steroids were extracted with Preparative C~s (1 x 0 . 8 cm). One ml of water was added to the 10 ml methanol eluate which was then pa:ssed through a column of T E A P - L H - 2 0 in H C O ; form ( 6 × 0 . 4 cm, packed in 85% aqueous methanol). T h e effluent and a rinse with 10 ml 85% methanol contained the liberated neutral steroids and steroids conjugated with N-acetylglucosamine. T h e solvent was evaporated and the residue was subjected to normal-phase chromatography under gravity flow on a 4 x 0.4 cm column of Lipidex-5000 in chloroform/hexane (1:1, v/v) [14]. T h e sample was applied in 1 ml of chloroform/hexane (1:1, v/v) and rinsed onto the column~ with 6 ml of the same solvent. Neutral steroids containing four hydroxyl groups or less were eluted in the first fraction (A). Steroids conjugated with N-acetylglucosamine, estriols and bile alcohols were eluted with 5 ml methanol (B). T o an aliquot (1/25) of A 1.2 pg of n-dotriacontane (C32) was added as internal standard and to an aliquot (1/4) of B 1.4 pg of C36 was added. Steroids in these aliquots were converted to trimethylsilyl ( T M S ) ethers [5] for analysis by gas-liquid chromatography ( G L C ) and G C - M S . T h e remainder of B (3/4) was taken to dryness and dissolved in 25 lal of :methanol and hydrolysed with 1 U/ml of N-acetylglucosaminidase in 50 m M sodium citrate, p H 4.5, in a tol:al volume of 10 ml at 37°C for 16 h [6]. T h e reactiorL mixture was extracted with a Preparative C18 ( 3 × 0 . 4 cm) column. T h e methanol eluate was applied to a normal-phase chromatography colunm of Lipidex-5G,00 as above, to separate the liberated pregnanediols and -triols from the estriols and bile alcohols. Part (1/3) of the pregnanediols and -triols was analysed by G L C and G C - M S after conversion to T M S ethers. One tag of C32 was added as an internal standard prior to derivatization. Monosulfate fraction. The monosulfate fraction was diluted with 5 ml of 'water and evaporated to leave

* F a s t a t o m b o m b a r d m e n t i~s a f o r m o f l i q u i d s e c o n d a r y - i o n m a s s s p e c t r o m e t r y ( L S I M S ) . I n t h e p r e s e n t p a p e r w e will u s e t h e t e r m FAB to cover ionisation by bombardment with xenon atoms from a FAB gun and with Cs* ions from a Cs ÷ ion gun.

587

about 5 ml of aqueous solution. Steroids were then extracted with a Preparative C.s (3 x 0 . 4 cm) column. An aliquot (1/3) of the eluate was analysed by F A B M S (see below) and [14C]pregnenediol monosulfate, 104 dpm, was added to the remainder to monitor the efficiency of solvolysis [15, 16]. T h e eluate was evaporated to dryness and the residue dissolved in 1 ml of tetrahydrofuran (freshly distilled)/methanol/trifluoroacetic acid 900:100:10 (by vol.). After 30 min the reaction mixture was neutralized with 0.1 M sodium hydrogen carbonate, diluted with 10 ml of distilled water and evaporated on a rotary evaporator for a short time to remove tetrahydrofuran. Steroids were then extracted with Preparative C~s and liberated neutral steroids and N-acetylglucosaminides were isolated and analysed as described for the glucuronide fraction.

Gas-liquid chromatography (GLC) G L C was carried out using a Carlo Erba G C 6000 gas chromatograph connected to a Spectra-Physics SP4270 integrator. An o n - c o l u m n injector system and a fused silica column ( 2 5 × 0 . 3 2 m m coated with a 0.25 lam layer of cross-linked methyl silicone (Quadrex Corp., N e w Haven, C T ) ) were used with a flame ionisation detector. T h e samples were injected as T M S ether derivatives in 0.3-0.8 pl hexane at 60°C. Helium was used as carrier gas at a flow rate of about 1 ml/min. T h e temperature was p r o g r a m m e d to rise from 60°C to 210°C at a rate of 30°C/min, remain at 210°C for 1.5 min, then rise to 320°C at a rate of 2.5°C/min, and remain at this temperature for 20 min.

Gas chromatography-mass spectrometry (GC-MS) G C - M S was carried out on a V G 7070E doublefocussing mass spectrometer with an electron-impact ion source, a Dani 3800 gas chromatograph and a V G 11-250 data system (VG Analytical, Manchester, U.K.). T h e capillary column was directly connected and extended into the ion source. An all-glass falling-needle system was used for the injection of the sample at 210°C. After 5 min the temperature was taken to 315°C at a rate of 2.5°C/min and maintained at this temperature for 25 min. T h e electron-impact energy was 70 eV and the trap current 200 mA. Spectra were recorded in the mass range of m/z 70-800 D a at a scan rate of 2 s per decade and a resolution of 1000 (5% valley).

Fast atom bombardment mass spectrometry (FABMS) Negative-ion FAB spectra were recorded on a V G 70-250 or a V G - A u t o S p e c - Q mass spectrometer (VG Analytical, Manchester, U.K.). Samples were prepared in aqueous methanol and dissolved in a drop of glycerol which coated the probe tip. T h e V G 70-250 spectrometer was fitted with a V G FAB ion source, and an I o n - T e c h a t o m gun (Teddington, U.K.). X e n o n atoms were used to b o m b a r d the sample, the ion gun conditions being

588

L.J. Meng et al.

typically 8 kV accelerating potential and 1 m A discharge current. T h e spectrometer accelerating potential was 6 kV. Collision-induced dissociation (CID) spectra were generated using helium as the collision gas in the first field-free region gas cell at a pressure which gave a reading of 2 x 10 -6 T o r r on the nearby analyser ion gauge. This pressure was sufficient to cause a 50% reduction in parent [ M - H ] - ion beam intensity. Daughter-ion linked scans at constant B / E were recorded on the steroid double conjugates. Scan 10~

durations were 20 s and 10-20 scans were collected in the multichannel analyser mode. Spectra were also recorded on the V G A u t o S p e c - Q mass spectrometer fitted with an L S I M S ion source. A cesium ion gun was operated at an anode potential of 25 kV. T h e spectrometer accelerating potential was 8 kV and ions were detected at the fourth field free region off-axis detector. Linked-scan C I D spectra were generated using helium as the collision gas in the first field-free region gas cell, at a pressure which gave a

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reading of 1.5x 1 0 -6 T o r r gauge. This was sufficient parent ion b e a m intensity. and spectra were recorded summed.

on the nearby analyser 1 ion to cause a 50% reduction in Scan rates were 10 s/decade in the continuous m o d e and

I~JESULTS

Analyses of conjugates ~y F A B M S and FAB/CID Spectra from both instruments were essentially similar, however the cross-section for steroid ionization was greater using Cs + :ion b o m b a r d m e n t than Xe a t o m b o m b a r d m e n t . T h e negative-ion spectra of the urine extracts from w o m e n in late pregnancy (Fig. 2) showed the expected ions at m/z 493, 495, 509 and 511, corresponding to anions of glucuronides of pregnanolones, pregnanediols, pregnanediolones and pregnanetriols. Peaks due to monosulfated steroids were m u c h less intense (i.e. m/z 397, 399, 413, 415). T h e pregnanetriol disulfates cannot be distinguished from the isobaric steroid glucuronides also at m/z 495. In addition to the expected ions, p r o m i n e n t peaks were seen at m/z 602 and 698 (Fig. 2), consistent with the presence of pregnanediols conjugated with both sulfuric acid or glucuronic acid and GIcNAc, respectively. G r o u p separation on T E A P - L H - 2 0 supported this assumption because the glucuronide fraction yielded peaks at m/z 698 (Fig. 2) and in some cases m/z 714 (double conjugate of pregnanetriol), and the monosulfate fraction peaks at m/z 602 and 618 (double

conjugate of pregnanetriol) (Fig. 2). It should be pointed out that steroid disulfates were not included in this fraction. Although the FAB spectra of samples obtained from different w o m e n showed differences in the intensities of peaks, in general, there was a notable resemblance in the pattern of peaks. In order to obtain structural information, negative-ion F A B / C I D spectra were recorded on the ions at m/z 698 from the glucuronide fraction, and at m/z 602 and 618 from the monosulfate fraction. [ M - H ] - ions of fatty acids, bile acids, steroid sulfates and glucuronides have been shown to give charge-remote fragmentation (CRF) patterns on C I D [17-20], i.e. fragmentation induced in a region of the ion remote from the site of charge. It is believed that the mechanisms for these fragmentations are similar to those occurring in thermolytic or photochemical processes. A requirement for C R F is a stable site of charge such as a strong acid group. C R F patterns provide m u c h useful structural information and allow the elucidation of the structure of m a n y biomolecules. Monosulfate fraction. Shown in Fig. 3 and described in Table 1 is the F A B / C I D spectrum obtained from the major ion at m/z 602 from the steroid sulfate fraction (see Fig. 2). Prominent fragment ions at m/z 427 (M-H-175), 397 ( M - H - 2 0 5 ) and 381 ( M - H - 2 2 1 ) correspond to fragmentation through the sugar and loss of the GlcNAc moiety as shown in Scheme 1 and T a b l e 1. T h e remainder of the fragmentation pattern is consistent with a pregnane-3,20-diol 3-sulfate, 2 0 - N acetylglucosaminide structure. T o m e r and Gross [20]

590

L. J. Meng et al. 100% 95 FAB/CID m/z 602 90 8 keY, linked scan 85 80 75 70 65 60 55 50 45

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have made an extensive study of the fragmentation of sulfated steroids. Their spectrum of a pregnane-3,20diol 3-sulfate is similar to that presented here in the mass range m/z 240-370. Intense fragmentations are observed in ring D giving ions at m/z 313, 325 and 339. Less intense fragmentations also occur in ring C giving ions at 245 and 259. These ring fragmentations define the location of the sulfate ester group to ring A or B, and

together with fragment ions at m/z 353 resulting from the loss of the C-17 side chain, define the location of the N-acetylglucosamine on the side chain. T h e F A B / C I D spectrum from [ M - H ] - ions at m/z 618 is shown in Fig. 3 and described in Table 1. The spectrum is compatible with a pregnane-3,20,21-triol 3-sulfate, 20-N-acetylglucosamine structure. Fragmentations in the C ring are observed giving ions at m/z 245

N-Acetylglucosaminides in Pregnancy Table 1. Relative abundance of fragment ions in the CID spectra of[M-H]- ions of m/z 602, 618 and 698from the steroid double conjugates shown in Scheme 1 Mass/charge ratio and relative abundance (%)~ of Fragment ion

m/z 602 b

m/z 618 ~

m/z 698 d

[M-H-16] [M-H-18] [M-H-76] [M-H-120] [M-H-175] [M-H-205] [M-H-221] f-220 e-H d~2H d2 dt drl4 c: C1

586 584 526 482 427 397 381 367 353 339 327 313 299 259 245

602 600 542 498 443 413 397 367 353 339 327 313 299 259 245

682 (40) 680 (35) 578 (20) 523 (50) 493 (50) ~ 477 (100) ° 463 (20) 449 (15) 435 (20) 423 (10) 409 (45) 395 (15) 355 (15) --

(40) (40) (10) (15) (65) (40) ~ (1C,0)~ (10) (15) (25) (5) (45) (5) (5) (5)

(55) (55) (10) (10) (70) (35) ~ (100) ~ (20) (25) (30) (15) (95) (15) (20) (10)

alntensities to the nearest 5%. bOther significant fragment ions: 325(5)d2-2H. tOther significant fragment ions: 381 (85) [M-H-237]; 427(20) [M-H191]. dOther significant fragment ions: 495(60)[M-H-203]; 367(10)c3-2H; 287(20)bl-2H. °Accompanied by a fragment ion two mass units heavier.

and 259 and in the D :dng giving ions at m/z 313, 327 and 339. These fragment ions locate the sulfate ester group to ring A or B. Fragment ions at m/z 353 resulting from loss of the C-17 side chain indicates that the N-acetylglucosamine moiety and a free hydroxyl group must be attached to the C-17 side chain. However, the C I D spectra do not permit a differentiation between conjugation with GIcNAc at C-20 or C-21. Glucuronidefraction. T h e ion current at m/z 698 was 15-20% of that at m/z 495 (anion of pregnanediol

591

glucuronide) (Fig. 2). T h e CID spectrum of ions at m/z 698 is shown in Fig. 4. Although the signal-to-noise ratio is poor, fragment ions can still be picked out. As was the case with the pregnanediol sulfate conjugated with N-acetylglucosamine, fragmentations involving the sugar moiety M-H-221 (m/z 477), M-H-203 (m/z 495) and M-H-175 (m/z 523) are observed. Fragmentation also occurs in the steroid rings. T h e ion at m/z 287 corresponds to fragmentation in the B ring and locates the glucuronic acid moiety to ring A. This is supported by fragment ions at m/z 355 and 409 which correspond to fragmentations in the C and D rings, respectively, of a pregnane-3,20-diol 3-glucuronide, 20-N-acetylglucosaminide structure. T h e pseudomolecular anion corresponding to pregnanetriol doubly conjugated with glucuronic acid and GlcNAc (m/z 714) was observed in several urine samples, but was too weak to be analysed by FAB/CID and it may only be assumed that the GlcNAc moiety was located at C-20 (see below). It has recently been shown that bile acids derivatized with aminobenzenesulfonate give more informative CID spectra than their underivatized analogues [21]. T h e steroid glucuronide fraction was therefore derivatized with aminobenzenesulfonic acid [21]. T h e amino group of the sulfonate couples to the carboxylic acid group of the glucuronic acid moiety via an amide bond. Derivatized steroid glucuronides are shifted in mass by 155 Da from the underivatized parents. T h e FAB mass spectrum of the derivatized glucuronide fraction now showed a peak at m/z 853, corresponding to the aminobenzenesulfonate of the pregnanediol glucuronide-N-acetylglucosaminide. This ion was subjected to CID and the resultant spectrum confirmed the structure suggested above for the steroid double conjugate. Analyses by GLC and G C - M S after solvolysis/hydrolysis of conjugates

f

FABMS spectra of the glucuronide and monosulfate fractions (Fig. 2) showed that the subfractionation on T E A P - L H - 2 0 was satisfactory, with no overlap between the glucuronides and sulfates.

a3 m/z

602 618 698

b1

R1 SO4"SO4GIcA-

b 2 R2 H OH H

R3 GlcNAc GlcNAc GIcNAc

S c h e m e 1. F r a g m e n t a t i o n o f [ M - H ] - ions from steroids doubly conjugated w i t h N - a c e t y ] l g l u c o s a m i n e (GlcNAc) a n d sulfuric acid (SO~) or glucuronic acid (GlcA').

Solvolysis and enzyme hydrolysis. T h e extent of solvolysis was determined from recoveries of [t4C]pregnenediol that was added as monosulfate before solvolysis. T h e value was 9 4 . 8 _ 7.8% (mean _ SD). Recoveries of [3H]testosterone added as a glucuronide were 88.3 +_ 5.4% (mean _+ SD) after glucuronidase hydrolysis. T h e hydrolysis by N-acetylglucosaminidase to remove N-acetylhexosamines was checked by G L C . T h e peaks existing before hydrolysis disappeared from the retention time range of the N-acetylhexosaminides and were shifted to the range of pregnanediols and -triols. This showed that N-acetylhexosaminides were cleaved completely with N-acetylglucosaminidase.

592

L. J. Meng et al. lOO~

95-

4:

FAB/CID m/z 698

90- 8 keV linked scan 8 5~

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.95

602

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607

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~87

55 50 45

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409

495

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409

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250

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287

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.

381

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300

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350

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550

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600

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650

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m/z

Fig. 4. Daughter ions produced by CID of the [M-H] ion of m l z 698 given by the glucuronide fraction.

Separations on Lipidex-5000, used to isolate GlcNAc conjugates after solvolysis and hydrolysis with [3-glucuronidase, were monitored by G L C and G C - M S . T h e eluate with chloroform/hexane yielded no peaks of N-acetylhexosaminides upon G L C of the T M S ethers. T h e methanol eluate from the solvolysed sulfate fraction showed peaks of N-acetylhexosaminides. In the case of the hydrolysed glucuronide fraction, estriols, bile alcohols with more than four hydroxy groups and small amounts of pregnanediols were also present together with the N-acetylhexosaminides. T h e appearance of pregnanediols was due to tailing of the large amounts eluted in the preceding fraction. T h e polar unconjugated steroids were separated from the steroids conjugated with GlcNAc by rechromatography on Lipidex-5000 following hydrolysis with N-acetylglucosaminidase. Identification of the sugar moiety. The mass spectra and the complete cleavage by N-acetylglucosaminidase showed that the steroids were conjugated with N-acetylhexosamine. Because the enzyme also hydrolyses N-acetylgalactosaminides [22], the N-acetylhexosamine liberated by the hydrolysis was isolated and analysed by G L C of the T M S derivative as described [5]. It was identified as N-acetylglucosamine by comparison with authentic N-acetyLhexosamines.

Identification

of steroids conjugated with GlcNAc.

Examples of total ion current chromatograms obtained in the G C - M S analyses of the glucuronide and monosulfate fractions after removal of the acid moieties and isolation of the liberated neutral steroids and derivatization are shown in Fig. 5. T h e pattern of peaks was similar in all of the subjects studied, but the relative peak heights differed between the individuals. Peaks

appearing before C38 were from deconjugated steroids whereas those appearing after C40 were sugar conjugates and gave fragment ions typical of T M S ethers of pregnanediol (m/z 269, 284 and 374), pregnenediol (m/z 267, 282 and 372) and pregnanetriol (m/z 282, 372, 447 and 462). Fragment ions o f m / z 173 and 186 characteristic for T M S ether derivatives of N-acetylhexosaminides [23, 24] were present in all spectra. T h e G C - M S results are summarized in Table 2. T h e GlcNAc conjugate of the pregnanetriol from the monosulfate fraction (anion of the double conjugate at m/z 618, Fig. 2) was not detected with certainty. T M S ethers of pregnane-3,20,21-triols fragment with loss of C-21 and its trimethylsiloxy group to produce a very intense m/z 269 and very little m/z 282 or 372 (see below), which make them difficult to detect in a mixture with a pregnanediol T M S ether. It is possible that the peak with a retention index (RI) of 4201 (Table 2) represents the GIcNAc conjugates of both a pregnanediol and the pregnanetriol. Because FABMS does not detect neutral steroids, a search for steroids conjugated with GlcNAc alone was performed by G L C analysis of the neutral steroid fraction from the anion exchanger. No peaks were detected in the retention time range for T M S ethers of GlcNAc conjugates when analyses were performed in the same way as for steroids from which the sulfuric or glucuronic acid moieties had been removed. T h e neutral steroids obtained after removal of sulfuric and glucuronic acid comprised unconjugated steroids and N-acetylglucosaminides. These groups were separated by normal-phase chromatography on Lipidex-5000. T h e eluate with chloroform/hexane, 1:1 (v/v), contained unconjugated steroids, whereas the

N-Acetylglucosaminides in Pregnancy I

50 /

593

Glucuronidefraction

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Fig. 5. Total ion current c h r o m a t o g r a m s f r o m GC--MS a n a l y s e s o f steroid T M S e t h e r s o b t a i n e d a f t e r hydrolysis o f t h e g l u c u r o n i d e f r a c t i o n ( u p p e r c h r o m a t o g r a m ) and solvolysis o f t h e m o n o s u l f a t e f r a c t i o n (lower) f r o m t h e u r i n e o f a p r e g n a n t w o m a n . C32-C40, n - a l k a n e s a d d e d as i n t e r n a l standards.

methanol eluate contained N-acetylglucosaminides. In the case of the glucuronide fraction, deconjugated estriols and bile alcohols were also present in the methanol eluate. T h e GlcNAc conjugates were hydrolysed with N-acetylglucosaminidase and the liberated steroids analysed by G L C and G C - M S . T h e estriols and bile alcohols mentioned above were removed by a second chromatography on Lipidex5000. Examples of to~:al ion current chromatograms obtained in the G C - M S analyses of the hydrolysed and derivatized GIcNAc conjugates from the glucuronide and sulfate fractions are shown in Fig. 6. Table 3 lists the retention indices and structures for the eight major steroids which were originally conjugated with GIcNAc. T h e identifications are based on a comparison of the mass spectra and retention indices with those of reference compounds, and on the FAB/CID spectra. Authentic 5ct-pregnane-3~,20~,21-triol was not available, nor any pregnane-2,3,20-triol. In the former case comparisons were made with the endogenous 5at-pregnane-3ct,20~,21-triol that can be obtained from

the monosulfate fraction in plasma [8]. T h e partial identification of a pregnane-2,3,20-triol was based on a comparison of the spectrum of its T M S ether with that of the T M S ether of 5[3-pregnane-2[3,3~,20~-triol kindly provided by D r B. Nordn, Pharmacia, Stockholm. T h e spectra were very similar with molecular ions at m/z 552 and peaks at m/z 142 and 143, characteristic of T M S ethers of steroid 2,3-diols [25]. However, the stereochemistry at C-5 and of the hydroxyl group remain to be determined. T h e glucuronide fraction from several women also contained other pregnanetriols and pregnanetriolones conjugated with GlcNAc. Because of the small amounts and an inconsistent occurrence they were not further studied. In the chromatograms of the T M S derivatives of the GlcNAc conjugates, the peak with an RI of 4232 (4230) was much larger than that of the peak with an RI of 4203 (4201) (Fig. 5). Following removal of the GlcNAc moiety the peak of the pregnanediol T M S ether with an RI of 2772 (2770) was always much larger than that with an RI of 2787 (2786) (Fig. 6).

Table 2. Gas chromatographic and mass spectrometric characteristics of T M S ethers of steroid N-acerylhexosamine conjugates from pregnancy urine after removal of glucuronic and sulfuric acid moieties Relative intensities (%) of m/z Fraction from T E A P - L H - 2 0 Glucuronides

Monosul~tes

Retention index

173

186

204

217

4203 4232 4383 4201 4230 4308 4325

73 57 84 100 53 68 64

100 100 100 63 100 100 100

25 24 33 41 16 30 20

47 35 57 62 26 35 30

267

269

372

50 59 5

374

20 19 10 17 21

462

21

10

7 18

15 29 4

447

14

594

L. J. Meng et al. %

3a,~

8o

Glucuronide fraction

E

60

C26

40

/I 3a,58

2,3,20-triol

20

C36

o

i0:00

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20:00

25:00

30 :00 I . . . T. i m e

%

30,5a

80_

317,,5a

Sulfate fraction

6oi 4o:

C26

3a, 20a,21 -triol

20 0 Fig. 6. Total ion current chromatograms f r o m G C - M S a n a l y s e s o f steroid TMS ethers obtained after e n z y m e h y d r o l y s i s o f GlcNAc conjugates in the glucuronide (upper chromatogram) and monosulfate (lower) fractions f r o m t h e u r i n e o f a pregnant w o m a n . C26, C36, n-alkanes added as internal standards.

This indicates that the order of elution of the T M S ethers of these pregnanediol isomers (3~,5~ vs. 3~,5~) is reversed between the GlcNAc conjugates and the free steroids. Quantitative analysis of steroids conjugated with GlcNAc

T h e GIcNAc conjugates were quantified from the gas chromatograms of the T M S ethers of the steroids after removal of all conjugating groups. T h e peak areas in these chromatograms were about 60-80% of those in the chromatograms of the derivatized GIcNAc conjugates after removal of only glucuronic and sulfuric acid moieties. This difference may reflect losses in the procedure of hydrolysis of GlcNAc conjugates and indicates that the quantitative values are minimum figures. T o enable a comparison with well-known metabolites of progesterone in urine, the excretion of 513- and 5~-pregnane-3~,20ot-diols was quantified in the glucuronide and monosulfate fractions [26]. T h e total excretion of pregnanolones, pregnanediols, pregnanediolones and pregnanetriols was also estimated from the Table 3. Retention indices of the T M S ethers and structures of steroids conjugated with N-acetylglucosamine in pregnancy urine Fraction from TEAP-LH-20

Retention index

Glucuronides

2772 2787 2949 2770 2786 2846 2854 3002

Monosulfates

Steroid structure 5at-Pregnane-3a%20ct-diol 5]3-Pregnane-3ct,20ct-diol Pregnane-2,3,20-triol 5ct-Pregnane-3ct,20~-diol 5 ~-Pregnane-30%200c-diol 5-Pregnene-3 ~,20~-diol 5ct-Pregnane-3]3,20ct-diol 5 ~-Pregnane-30%200%21-triol

sum of peak areas of compounds giving mass spectra characteristic of such steroids. In this way it was also possible to compare the quantitative G L C results with the relative intensities of the peaks of the corresponding conjugates in the FAB spectra. T h e excretion of GIcNAc conjugates and of the classical pregnanediol conjugates in five pregnant women are given in Table 4. T h e average total excretion of GlcNAc conjugates of saturated steroids was 32 ~tmol/g creatinine, about equally distributed between the glucuronide and sulfate fractions, as compared to the mean of 71 pmol/g creatinine for 5[3-pregnane-3~,20~-diol glucuronide. T h e major steroid conjugated with GlcNAc was 5~-pregnane-3~,20~diol, both in the glucuronide and the sulfate fraction but 5~-pregnane-313,200~-diol GlcNAc was only found in the sulfate fraction and the pregnanetriols were different in the two fractions. T h e relative amounts of different groups of steroids in the glucuronide fraction are given in Table 5, which also shows the relative intensities of [M-H]- ions of glucuronides of the corresponding steroids. In view of the fact that FABMS is not a quantitative method and that an unsaturated alcohol and a saturated ketone have the same molecular masses, the agreement between the values is good. It is likely that the conjugation of all steroids with glucuronic acid and the absence of suppressor effects by stronger non-polar acids (removed by the ion-exchange chromatography) results in similar responses for similar steroids in the FABMS analysis. Table 6 shows the relative composition of groups of steroids in the monosulfate fraction. T h e most abundant steroid metabolites in this fraction are the pregnanediols doubly conjugated with sulfuric acid and GlcNAc. T h e agreement between G L C and FABMS

N-Acetylglucosaminides in Pregnancy

595

Table 4. Excretion of steroid N-acetylglucosaminides and two major pregnanediol isomers in pregnancy urine Fraction

Urinary excretion in five subjects (lamol/g creatinine)

steroidsa

AP

WN

BR

FM

JG

Mean

Glucuronides 5 ~-P-3%20~-diol 5~-P-3~,20~-diol 5t-P-3~,20~-diol-GlcNAc 5~x-P-3~,20cz-diol-GlcNAc P-2,3,20-triol-GlcNAc Total GlcNAc conjugates

62 12 3.4 13 1.6 18

110 22 3.4 19 2.8 25

28 10 1.5 10 1.4 13

127 26 0.5 7.5 1.2 9.2

27 7.5 0.5 9.1 0.4 10

71 16 1.9 12 1.5 15

Monosulfates 5 ~-P-3ct,20~-diol 5ct-P-3zq20ct-diol P5-3 ~,20ct-diol-GlcNAcb 5 [3-P-3ct,20ct-diol-GlcNAc 5 ct-P-3%20~t-diol-GlcNAc 5 ct-P-3 [3,20ct-diol-GlcNAc 5ct-P-3%20~t,21-triol-GlcNAc Total GlcNAc conjugates

1.2 tr 2.6 0.5 12 7.8 2.9 23

2.3 tr 1.7 0.4 16 6.9 0.6 26

1.0 tr 0.6 0.2 4.7 5.6 1.3 12

1.6 tr 0.9 0.3 10 5.6 1.7 18

0.3 tr 0.5 0.2 5.0 2.3 0.4 7.9

1.3 tr 1.3 0.3 9.5 5.6 1.4 17

The determinations were based on the GLC peak areas of steroid T M S ethers after removal of all the conjugating group. 'P, pregnane; greek letters denote orientation of hydroxyl groups and the H at C-5; superscript indicates position of double bond. GlcNAc, N-acetylglucosamine. bNot included in total GlcNAc conjugates.

analyses is poor for the double conjugate of 5-pregnen313,20~-diol ([M-H]- at m/z 600). This is due to loss of Hz from the anions of the pregnanediol monosulfates which has not been corrected for, resulting in overestimation of an unsaturated sulfated steroid in the presence of the saturated one. T h e poor agreement for pregnanediolone sulfates ([M-H]- at m/z 413) is explained in part by the loss of Hz from ions at m/z 415 and in part by the presence of sulfated pregnenetriol (m/z 413) not included in the G L C analysis. Thus, there are no great discrepancies between the G L C and FABMS analyses indicating that sample workup and solvolysis/hydrolysis has not resulted in selective losses of any of the C2~Oz and CzlO3 steroids studied. DISCUSSION This study has shown that a n u m b e r of isomeric pregnanediols and pregnanetriols are excreted in the

urine of pregnant women in the form of double conjugates with N-acetylglucosamine and sulfuric acid or glucuronic acid. We have not found any previous report of the occurrence of saturated pregnane derivatives conjugated with GlcNAc. However, one of the steroids identified, 5-pregnen-3[3,20ct-diol, was characterized as a 3-sulfate,20-N-acetylglucosaminide almost 30 years ago following the administration of pregnenolone to healthy h u m a n subjects [3]. Double conjugates with GlcNAc and sulfuric acid have been shown to be major metabolites of estrogens in the rabbit [2] and such conjugates have also been found in h u m a n bile [27]. T h e structures of the steroids conjugated with GlcNAc were determined by G C - M S following removal of the conjugating moieties. T h e major pregnanediol was 5~-pregnane-3~,20~-diol, present as a double conjugate with GlcNAc and either sulfuric acid or glucuronic acid. Similarly, 513-pregnane-3~,20~-

Table 5. Relative amounts (%) of different groups of steroids in the glucuronide fraction as determined by GLC after removal of all conjugating groups Relative steroid composition in subjects Steroid group a Pregnanediols GlcNAc-conj. Pregnanolones Pregnanediolones Pregnanetriols GlcNAc-conj.

[M-H]- m/z 495 698 493 509 511 714

AP 100 20 42 40 31 2

(100) (9) (39) (38) (24) (1)

WN 100 16 27 40 49 2

(100) (10) (44) (43) (41) (3)

BR 100 28 40 53 42 3

(100) (20) (40) (46) (33) (3)

FM 100 7 43 56 45 1

(100) (5) (49) (44) (31) (1)

JG 100 10 45 53 27 1

T h e relative intensities of the [M-H]- ions in the FAB spectra of the corresponding intact conjugates are given in parenthesis. ~GlcNAc-conj., N-acetylglucosamine conjugates of pregnanediol (m/z 698) and pregnanetriol (m/z 714) glucuronides.

(100) (4) (47) (40) (22) (1)

596

L.J. Meng et al.

Table 6. Relative amounts (%) of different groups of steroids in the monosulfate fraction as determined by GLC after removal of all conjugating groups Relative steroid composition in subjects Steroid group ~ Pregnanediols GlcNAc-conj. Pregnenolones GlcNAc-coni. Pregnanediolones Pregnanetriols GIcNAc-coni. Pregnenetetrols

[M-H] 399 602 395 600 413 415 618 429

m/z

AP

WN

BR

FM

JG

8 (2) 100 (100) 8 (7) 19 (19) 9 (31) 34 (41) 22 (13) 5 (7)

12 (18) 100 (100) 13 (12) 9 (17) 10 (31) 30 (26) 3 (5) 26 (15)

12 (20) 100 (100) 8 (11) 6 (16) 14 (50) 25 (35) 12 (10) 10 (16)

13 (20) 100 (100) 16 (11) 6 (15) 21 (54) 34 (51) 11 (10) 16 (19)

7 (17) 100 (100) 6 (7) 9 (17) 31 (32) 28 (29) 7 (6) 31 (27)

The relative intensities of the [M-H] ions in the FAB spectra of the corresponding intact conjugates are given in parenthesis. "GlcNAc-conj., N-acetylglucosamine conjugates ofpregnanediol (rn/z 602), pregnenediol (m/z 600) and pregnanetriol (m/z 618) monosulfates.

diol occurred as both types of double conjugate, whereas 5~-pregnane-3~,20:t-diol and 5-pregnene3~,20Qt-diol were only found as double conjugates with sulfuric acid and GlcNAc. T h e major pregnanetriol conjugated with GlcNAc and sulfuric acid was 5~-pregnane-3~t,20cq21-triol, whereas a partially characterized pregnane-2,3,20-triol was conjugated with GlcNAc and glucuronic acid. Other pregnanetriols were present in much smaller amounts and were not studied further. T h e positions of conjugation were established by collision-induced dissociation of the anions of the intact conjugates produced by fast atom bombardment. T h e fragmentation of the pregnanediol conjugates clearly showed that the sulfate or glucuronide moieties were located at C-3 and the GlcNAc at C-20. It should be mentioned that the parent ions represented a mixture of the double conjugates of three (sulfate fraction) and two (glucuronide fraction) pregnanediol isomers, and that minor amounts of molecules with the GIcNAc moiety at C-3 might escape detection. However, the GlcNAc moiety of the double conjugate of 5-pregnene-313,20~diol was previously located to C-20 [3, 4]. In the case of pregnanetriols, only the double conjugate of 5~-pregnane-3¢q20~,21-triol was analysed by FAB/CID. T h e sulfate group was definitively localized at C-3, whereas the spectra did not permit differentiation between C-20 and C-21 as the site of conjugation with GlcNAc. Further studies by chemical degradation are needed to resolve this ambiguity. T h e nature of the N-acetylglucosaminyl transferase catalysing formation of the steroid conjugates identified has not been established. However, some conclusions concerning substrate specificity may be drawn from the structures of the conjugates formed in vivo. Early studies of the double conjugation of estrogens in rabbit liver showed that sulfation at C-3 was required for conjugation with GIcNAc at C-17 [2]. Estrogens sulfated at C-3 and carrying GlcNAc at C-15 have been found in human bile [27]. T h e double conjugates of the pregnane derivatives in the present and previous [3] studies have a conjugating acid at C-3 and the GlcNAc group in the side chain. This indicates a requirement for

an anionic substituent at an appropriate distance from the site of glycosylation. This is also supported by the fact that neutral steroids conjugated with GlcNAc only could not be detected. In the case of the pregnane derivatives, there seems to be a preference for the sulfate ester anion because the double conjugates with sulfuric acid predominated greatly over the monosulfates, while the double conjugates with glucuronic acid only corresponded to a minor fraction of the total amount of monoglucuronides. Although these ratios are also determined by renal and biliary [27] clearances and binding to plasma proteins, the importance of the anionic sulfate group is supported by the fact that the urinary excretion of sulfate-GlcNAc double conjugates increases in pregnant women with intrahepatic cholestasis, in contrast to the glucuronic acid-GlcNAc conjugates (to be published). T h e stereochemistry of the A/B rings also seems to influence conjugation with GlcNAc. T h e ratio between the GlcNAc conjugates of 5~- and 5~-pregnane3~,20~-diol glucuronides was thus about 30 times higher than the ratio between the pregnanediol monoglucuronides. T h e planar A/B ring fusion predominates among sulfated steroids (see Refs [8, 10]) which may also contribute to the predominance of sulfate-GlcNAc double conjugates. H u m a n liver and kidney tissues have been shown to possess N-acetylglucosaminyl transferase activity [2, 28]. Recently, microsome preparations from these organs were shown to catalyse N-acetylglucosaminidation of 713-hydroxylated bile acids [6, 29] at the 7~3-hydroxyl group [7]. This reaction is quite selective, and neither 7~- nor 3-hydroxyl groups are conjugated in this way. T h e bile acids have an anionic function in the side chain and conjugation with glycine or taurine at this position results in less reactive substrates [6]. This requirement for an anionic site at an appropriate distance from the hydroxyl group to be glycosylated may indicate a relationship to the conjugation of pregnane and estrogen derivatives with GlcNAc. T h e physiological function of the conjugation with GlcNAc is not known. T h e water solubility is sufficient for excretion of the anionic substrate steroids in urine

N-Acetylglucosaminides in Pregnancy and bile without conjugation with GIcNAc. Also, the s u b s t r a t e s are n o t k n o w n t o p o s s e s s a n y t o x i c i t y . O n t h e c o n t r a r y , 7 [ 3 - h y d r o x y l a t e d b i l e a c i d s , e.g. u r s o d e o x y c h o l i c acid, h a v e a b e n e f i c i a l e f f e c t o n c h o l e s t a t i c liver d i s e a s e s s u c h as p r i m a r y b i l i a r y c i r r h o s i s (see Ref. [6]). T h e r e l a t i o n s h i p s b e t w e e n this p h a r m a c o l o g i c a l e f f e c t and the conjugation with GlcNAc have not been clarified. It is also p o s s i b l e t h a t t h e r e a c t i o n is c a t a l y s e d by an N-acetylglucosaminyl-transferase with a different f u n c t i o n , e.g. in p r o t e i n o r l i p i d g l y c o s y l a t i o n , a n d t h a t t h e c o n j u g a t i o n o f a n i o n i c s t e r o i d s a n d bile a c i d s is f o r t u i t o u s . I s o l a t i o n o f t h e e n z y m e ( s ) r e s p o n s i b l e will b e r e q u i r e d to clarify t h e s e q u e s t i o n s . P r e g n a n e d i o l s u l f a t e s a n d g l u c u r o n i d e s are m e t a b olites o f p r o g e s t e r o n e . 5 ~ - P r e g n a n e - 3 ~ , 2 0 ~ , 2 1 - t r i o l s u l f a t e is a n e x c l u s i v e m e t a b o l i t e o f 5Gt-pregnane3 ~ , 2 0 u - d i o l s u l f a t e [8]. T h e s t e r o i d m o n o s u l f a t e s c a n u n d e r g o f u r t h e r s u l f a t i o n to d i s u l f a t e s , w h i c h are e n d p r o d u c t s in t h e m e t a b o l i s m [8, 10]. T h i s s t u d y i n d i c a t e s t h a t d o u b l e c o n j u g a t e s 'with G l c N A c are a l t e r n a t i v e e n d p r o d u c t s . T h e p r o d u c t i o n r a t e o f p r o g e s t e r o n e is a b o u t 2 5 0 - 3 0 0 m g / 2 4 h d u r i n g l a t e p r e g n a n c y [30]. M e a s u r e m e n t s o f k n o w n m e t a b o l i t e s in u r i n e a n d f e c e s [31, 32] and determinations of production rates of different isomers of pregnanolone and pregnanediol sulfates p r e s e n t in p l a s m a [8, 10] h a v e n o t g i v e n v a l u e s o f this m a g n i t u d e . T h i s d i s c r e p a n c y m a y in p a r t b e d u e to formation of unknown metabolites not measured by existing methods. The GlcNAc conjugates represent one such group of metabolites. Their urinary excretion was about 45% of that of 513-pregnane-30t,20~-diol g l u c u r o n i d e . B e c a u s e r2ae l a t t e r is e s t i m a t e d to a c c o u n t for 1 0 - 3 0 % o f t h e p r o g e s t e r o n e m e t a b o l i s m [30], t h e GIcNAc conjugates may represent 5-15% of the p r o g e s t e r o n e m e t a b o l i s m in p r e g n a n c y . A n a c c u r a t e e v a l u a t i o n o f t h e i r q u a n t i t a t i v e i m p o r t a n c e will r e q u i r e d e t a i l e d a n a l y s e s o f p r o g e s t e r o n e m e t a b o l i t e s in u r i n e , b i l e a n d feces. [26] Acknowledgements--This work was supported by grants from the Swedish Medical Research Council (no. 03X-219) and the Karolinska Institute. We thank Dr Gunvor Ekman-Ordeberg, Department of Women and Child Health, Karolinska Hospital, for providing urine samples from pregnant women, Mr R Reimendal for mass spectrometric analyse~, and Dr Bengt Norrn, Pharmacia, Stockholm for a reference spectrum.

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