Profiles of bile acids and progesterone metabolites in the urine and serum of women with...

Journal of Hepatology 1991; 21: 346-357 Printed in Denmark All rights reserved Munksgaard Copenhagen

Copyright 0 European Association /or the Study of the Liver 1997

Journal of Hepatology ISSN 0168-8278

Profiles of bile acids and progesterone metabolites in the urine and serum of women with intrahepatic cholestasis of pregnancy

Ling-Jie Meng’, Humberto Reyes*, Joaquin Palma *, Ismael Hernandez*, Jose Ribalta* and Jan Sjovall’

‘Department of Medical Biochemistry and Biophysics. Karolinska Instituter. Stockholm, Sweden and ‘Departments of Medicine and Experimental Medicine, Campus Oriente. Universiry of Chile, School of Medicine. Santiago. Chile

Background/Aims and Methods: The etiology of intrahepatic cholestasis of pregnancy (ICP) is unknown. We have performed comprehensive chromatographic and mass spectrometric analyses of progesterone metabolites and bile acids in serum and urine of six patients in order to characterize changes that might be of importance for the development of the disease. Results: Conjugated bile acids were increased in serum and urine of patients with ICP while the levels of unconjugated bile acids were similar in healthy pregnancies and ICI? Unconjugated and conjugated 7a,l2a-dihydroxy-3-oxo-4-cholenoic acid was excreted in urine both in healthy pregnancies and in ICP, possibly indicating a rate limitation of 3-oxo-d4-steroid 5/l-reductase in pregnancy. The serum levels and urinary excretion of total sulfated progesterone metabolites were increased in ICP while the glucuronides were unchanged or low. Confirming previous results, the fraction of metabolites with 3a-hydroxy-So(H)

I NTRAHEPATIC cholestasis of pregnancy (ICP) is char- acterized by pruritus and cholestasis that appear in

late pregnancy, persist until delivery, and disappear after parturition. The etiology of this disease is still unknown. In addition to the elevated levels of bile acids in serum (l-3) the excretion of bile acids in urine is lo-100 times higher in ICP than in normal pregnancies (4). Serum levels of sulfated progesterone metabolites are also markedly elevated in this disease and the pattern of progesterone metabolites differs significantly from that in normal pregnancy (5). Excretion of progesterone metabolites into bile appears impaired in ICP (6) and the fe-

Received 7 February: revised 10 March; accepted 12 March 1997

Correspondence: Dr Jan SjCivall, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S - 171 77 Stockholm, Sweden. Tel: 46-g-7287700. Fax: 46-8- 304452. e-mail: [email protected]

configuration was increased. The urinary excretion of 5a-pregnane-3a,20a-diol3-sulfate,20-N-acetylglucosaminide was greatly increased in ICP, as was that of 3a-hydroxy-5a-androstane-17/I-carboxylic acid, as- sumed to be a progesterone metabolite. Conclusions: The combined results of this and previous studies are compatible with a primary change in the reductive metabolism of progesterone in ICP, resulting in increased formation of metabolites with a 3a-hydroxy-5a(H) configuration and a larger fraction of sulfates. There also seems to be a selective defect in the biliary secretion of sulfated metabolites, particularly disulfates.

Key words: Bile acids; Chromatography; Glucuroni- des; Intrahepatic cholestasis of pregnancy; Mass spectrometry; N-acetylglucosaminides; Serum; Ster- oids; Sulfates; Urine.

cal excretion of neutral steroids is lower in ICP than in healthy pregnant women (7). The question arises whether all these changes are secondary to intrahepatic cholestasis or if some may precede it and play a role in the pathogenesis of the disease (5). An abnormal pattern of sulfated progesterone metabolites in plasma was observed in two patients in the third trimester of pregnancy before pruritus appeared and abnormal liver function tests were detected (5). However, no particular steroid metabolite or bile acid has yet been defined that could be related to the induction of cholestasis (7). Most of the previous studies were performed over two decades ago using methodologies available at that time. Conse- quently, compounds of pathophysiological importance may have escaped detection due to the presence of labile structures, or unknown forms of conjugation. Further- more, comprehensive investigations of bile acid and steroid profiles in serum and urine have not yet been done in

346

Steroids and bile acids in ICP

TABLE 1

Clinical data for six patients with ICP

Parameter= Patient

1 2 3 4 5 6

Gestational week 33 31 32 34 34 31 Severity of pruritus (o-4) 4 3 3 3 3 3 Bilirubin, total (mg/dl;< 1.1) 0.9 0.8 0.9 1.3 0.6 2.8 S-ALAT (U/d1;<40) 82 108 194 278 205 308 S-ALP (U/dl;< 117) 888 94 130 167 207 236 Cholesterol, total (mg/dl;<200) 255 - 309 268 229 352 Triglycerides (mg/dl;< 130) 306 - 264 - 261 411

B S-ALAT: serum alanine transaminase; S-ALP: serum total alkaline phosphatases. In parenthesis, units of measurement followed by the upper limit acceptable in fasting blood samples obtained from nonpregnant healthy women. in the comparable age range.

individual patients. The aim of the present study was to characterize the profiles of bile acids and progesterone metabolites in serum and urine of patients with ICP comparing them with normal pregnancies near term, to provide a basis for subsequent studies in patients treated with ursodeoxycholic acid, in whom pruritus was atten- uated and the biochemical cholestasis improved.

Materials and Methods Patients

In the nine pregnant individuals, urine samples were taken from a 12-h overnight collection and blood samples were obtained the same morning, at 8-9 a.m., after an overnight fast. Serum and urine samples were immediately frozen at -20°C and kept frozen until analysis. Shipment of samples to Stockholm was made in dry ice. The protocol for this study was approved by the local institutional ethics committee and informed consent was obtained from each subject.

Three healthy pregnant women and six patients with A detailed description of the analytical method is ICP were selected at the Department of Obstetrics, Hos- published separately (8). A solid-phase method was used pital de1 Salvador in Santiago, Chile. The three healthy to extract bile acids and steroids from 10 ml urine and pregnant women had been hospitalized during the last l-4 ml serum. Unconjugated bile acids and different weeks of normal pregnancies to perform ante-partum groups of conjugated bile acids and neutral steroids were follow-up because of pelvic anomalies or previous cesa- then separated by anion-exchange chromatography. The rean sections. They had no concomitant diseases, their groups of conjugated compounds were qualitatively liver function tests were normal and they were not given analyzed by fast-atom bombardment (FAB) mass spec- any drugs or progesterone. The six patients with ICP trometry (MS). After hydrolysis and solvolysis, the were selected among patients hospitalized with the pur- liberated bile acids and neutral steroids were purified on pose of monitoring fetal prognosis in late pregnancy. the ion exchanger. Bile acids were methylated with di- The diagnosis of ICP was based on widespread severe azomethane and all compounds were converted to tri- skin pruritus appearing between weeks 28 and 32 of an methylsilyl ethers (by heating in trimethylchlorosilane/ otherwise uneventful pregnancy, without any other hexamethyldisilazane/pyridine, 1:2:3 (by vol) at 60°C for possible explanation besides cholestasis of pregnancy. 30 min), before quantitative analysis by gas-liquid Simultaneously, a biochemical cholestasis was char- chromatography (GLC) on a capillary column coated acterized in them by an increase in fasting serum levels with a methyl silicone stationary phase. n-Triacontane of total bile salts (as determined by a routine 3a- (Cs,) and n-hexatriacontane (C,,) were added as inter- hydroxysteroid dehydrogenase assay), 335-fold above nal standards prior to derivatization. A GLC tempera- the upper normal levels in late pregnancy; serum alanine ture program was used that permitted detection of com- aminotransferase and/or aspartate aminotransferase pounds with retention indices (RI) between 2300 and were also mildly or moderately increased and two of 4500, i.e. covering a range from Cis steroids to sugar them had a mild hyperbilirubinemia. These and other conjugates of CZ1 steroids. The method of derivatization pertinent data are shown in Table 1. No other concomi- excluded several glucocorticoid metabolites since em- tant diseases were present, and after delivery pruritus phasis was put on the analysis of progesterone metabo- disappeared and liver function tests returned to normal. lites (8). The identification of steroids and bile acids was Two of the patients were multiparous and both had a based on mass spectra and retention times, as compared past history of ICP in their previous pregnancies. with those of reference compounds or data from the

Methods

347

L. J. Meng et al.

The spectrum of urine from the healthy subject showed ions at m/z 493,495, 509, 5 11, corresponding to anions of glucuronides of pregnanolones, pregnanediols, preg- nanediolones and pregnanetriols, respectively. Peaks due to monosulfated steroids were much less intense (i.e. m/z 397, 399, 413, 415). Peaks of disulfated pregnanediols and -trials were at m/z 479 and 495, the latter of which could not be distinguished from the isobaric pregnanediol glucuronides. Peaks at m/z 602 and 698 were due to sulfated and glucuronidated pregnanediol N-acetylglucosaminides, respectively (9).

The spectrum of urine from the patient with ICP showed more intense peaks of mono- and disulfated steroids and an additional peak at m/z 6 18 due to sulfated pregnanetriol N-acetylglucosaminide. The anions of glycine- and taurine-conjugated tri- and tetrahy- droxycholanoates were seen at m/z 464, 480, 514 and 530, respectively Sulfated glycine- and taurine-conjugated dihydroxycholanoates gave the peaks at m/z 528

literature. Quantitation was based on comparison of peak areas given by the individual compounds with the peak areas given by the internal standards. Highly accu- rate quantitation of individual steroids was not attempted and a mass response factor of 1.2 was used for calculations of the amounts of all steroids and bile acids except 3-0~0 bile acids for which the factor 1.5 was used.

Statistics

Data are presented as mean+SEM (standard error of the mean). The Wilcoxon’s test rank sum test was used to identify statistically significant differences between values for patients with ICP and the healthy pregnant women. ~~0.05 was considered statistically significant.

Results Analysis of urine extracts by FABMS

The high-mass regions of the negative-ion FAB mass spectra of the extracts of urine from a healthy pregnant woman in the 36th week of gestation and a patient with ICP in the 33rd week of gestation are shown in Fig. 1.

and 578, respectively; the ions by loss of SO3 from m/z 578.

at m/z 498 were formed

36 wk healthy pregnancy urine

602

60.

498 J

33 wk ICP urine

602

550 -Glo- 650 700

Fig. 1. High mass regions of the negative-ion FAB mass spectra of the extracts of urine from a healthy pregnant woman in the 36th week of gestation and a woman with ICP in the 33rd week of gestation. See Results, for explanation of the indicated

fragment ions.

348


TABLE 2

Concentrations of bile acids in serum of six women with ICP at 31-

34 weeks of pregnancy and three healthy women at 36-38 weeks of pregnancy

Structurea Concentration (mean?SEM’, PmoL’l)

ICP Healthy

glucuronidated and sulfated. In the previous study of normal pregnancy and ICP by Thomassen (4), only the three first-mentioned groups were analyzed. The present study confirmed and extended his results for these groups. The excretion of individual bile acids in the five different groups is exemplified with data for a pa-

Amidated 5/%B-3a, 12a-dial S/I-B-3a,7a-dial’ 5/?-B-3a,7a, 12n-triol’ 5P-B-1/?,3a,l2a-trio]‘ Total amidated’

0.920.2 0.3r0.02 5.2* 1.3 0.3+0.03 1926 0.2CO.08

0.3?0.04 25?7 0.8%0.1

tient with severe ICP and a healthy pregnant woman (Table 3). The distribution of total bile acids in urine according to mode of conjugation in six patients with ICP and three healthy pregnant women is shown in Fig. 2. Bile acids which are both amidated and glucu-

Unconjugated ~/?-B-~cx, 12a-diol S/&B-3a,7a-diol 5P-B-3a,7a.l2a-trioF CA5-3/?,7a-dial CAs-38-01 CA4-7a-ol-3-one Total unconjugated

0.05~0.03 0.2620.2 0.03~0.01 0.02?0.02 0.07?0.02 0.01~0.003 0.03~0.01 0.05?0.03 0.03~0.01 O.Ol+-0.01

0.07~0.01 0.06~0.02 0.28-cO.04 0.24t0.05

a Amidated, glycine- or taurine-conjugated; B, cholanoic acid; CA, cholestanoic acid; superscript indicates position of double bond; Greek letters denote configuration of hydroxyl groups and hydrogen at C-5.

b SEM, standard error of the mean. c Significantly increased in ICl?

ronidated were not analyzed. The relative intensities of ions at m/z 624 and 640 corresponding to glucuronides of di- and trihydroxycholanoylglycines, respectively, were much lower than those of corresponding amidates and sulfates in FAB spectra of urine extracts from ICP patients (exemplified in Fig. l), indicating that this mode of double conjugation is quantitatively less important in ICI? The analysis of FAB spectra revealed an even lower occurrence or absence of glucuronidated di- and trihydroxycholanoyltaurines.

Analyses by GLC and GUMS Urinary bile acids Bile acids in serum. Serum bile acids were analyzed in two groups: unconjugated and aminoacyl amidated. Healthy ICP

The levels of other groups, e.g. sulfates and glucuronides, were much lower and it was decided for practical reasons to exclude them from the analyses. The results of the analyses are shown in Table 2. In accordance with earlier reports (1,2), the bile acids in ICP were the 20%

same as in normal pregnancy except that small amounts of conjugated l/I,3lx,l2a-trihydroxy-5P-chol- IU G IT G IcA OS + S -G IT

anoic acid were found in ICI? The elevated level of amidated bile acids in ICP was 2527 pmol/l compared with 0.8kO.l Llmol/l in normal pregnancy. Cholic acid (CA) was the predominant serum bile acid in ICP and the ratios of CA: chenodeoxycholic acid (CDCA): deoxycholic acid (DCA) was 32:8:1 compared with 1.1: 1.6: 1 .O in healthy pregnancies.

In general, there were no significant differences in serum unconjugated bile acids between ICP and healthy pregnancies except for an increased CA: DCA +S -Gi-T

T ratio in ICI? When the level of amidated bile acids in ICP increased markedly, the unconjugated bile acids remained unchanged; 0.24kO.05 pmol/l in healthy, 0.28+0.04 ~molll in ICI? The concentrations of CZ7 bile acids were not affected by ICP and were similar to those in nonpregnant subjects (10).

Bile acids in urine. Five groups of urinary bile acids

A

Fig. 2. The distribution of total bile acids in urine according to mode of conjugation in the si.x patients with ICP and three healthy pregnant women. I=patient with ICP; H= healthy pregnant women; U=unconjugated; S+S-G/T=sul- &ated and both suljated and glycine- or taurine-conjugated,

were analyzed: unconjugated, aminoacyl amidated, sulfated, glucuronidated but not amidated, and both

GlcA=glucuronidated; G/T=glycine- or taurine-conjugated; S-GlcA is not shown due to its low percentuge.

349

L. J. Meng et al.

TABLE 3

Bile acids in urine of a patient with intrahepatic cholestasis in the 31st week of pregnancy and a healthy pregnant woman at 36 weeks of gestation @mol/g creatinine). Values for total excretion marked with an asterisk were significantly higher in all patients with ICP than in the healthy subjects

Structured Rib ICP - patientC Healthy pregnancy’

U G/T GlcA S+S- S- Total % U G/T GlcA S+S- S- Total %

G/T GlcA G/T GlcA

5a-A-3a-ol- 17b-carboxylic 2644 - 5p-A-3a-ol- 17p-carboxylic 2659

5a-A-3/?-ol-17P-carboxylic 2720

5/?-B-3P-al 3087 -

Nor-S/l-B-3a,7a.l2rr-o1 3096 2.5

5/?-B-3a-01 3103 -

5/?-B-3P, 12a-01 3145 -

5p-B-3a, 12p-01 3154 5a-B-3a, 12~01 3167

5/3-B-3a, 12a-01 & BS-3,&01 3174 tr

B-diol; B-trio1 3184 - 5p-B-3P,7a,12a-o1 3186 0.6 5/i’-B-3a,7a-ol 3199 tr

5/I-B-3a,7a, 12a-ol 3212 0.5

5/I-B-3a,6a-ol 3224 -

B-trio1 3226 tr 5a-B-3a,6a,7a,12a-o1 3226 ~

B5-3/?,12a-ol 3234 ~

5a-B-3P,12a-ol 3248 -

5P-B-3a,7b-o1 3249 -

5a-B-3a,7b,12a-ol 3255 B-trio1 3271

5b-B-3a,6a,12a-ol 3280 ~ SB-B-3a-ol-12-one 3289 - 5/?-B-l/J,3a, 12a-ol 3296 - 5D-B-3a,6a,7a-ol 3304

5/I-B-3a.6a.7a,l2a-o1 3305 0.4 5B-B-l/?,3a,7a,12a-o1 3320 - B-trio1 3329 - B4~6-12a-ol-3-oned 3335 - B-diol 3337 ~ B4-7a, 12a-ol-3-one 3346 0.6 B-trio1 3359 - Bd-diolone 3365 ~

5P-B-3a,12a-ol-7-one 3365 0.2 B-olone 3366 ~

5/J-B-3a,4/I,12a-ol 3390 - 5/?-B-3a,7a-ol-12-one 3391 tr B-tetrol 3397 5a-B-3a,4p,7a,12a-ale 3436 tr S/&B-2fi,3a,7a,12a-ol 3440 - B-tetrol 3454 5/&B-3a.4p,7a,12a-o1 3455 - B-trio1 3493 - Total 4.85

4.2 - 4.2* 0.9 - - _ 0.1 - 0.1 0.03 ~ - - _ 0.5 - 0.5* O.l- - - _ ~ <O.Ol to.01 - _ _

~ 0.9 - _ 9.4

_ 1.7 0.5 - 3.6 _ 0.1 0.2 _ 0.3 81 ~ 0.1 - ~ _

0.5 0.2 94 24 0.3 106

0.7 - ~ _ _

2.6 - _ 9.4

_ 0.09 nd tr

_ _ 0.6 - _

1.7 0.07 2.5 8 9.5 6.1 - _

~ 0.4 - 36 - _

25 - _ 0.2 1.1 - _

_ 0.7 - 16 _ 0.4 5.9 - _

_ _

0.2 - _ _ _ _

13 ~ _ _

9.5 3.2 0.5 - _

_ 0.7 ~ 154 10 317

3.4 0.7 1 ~ 0.1

9.4 1.9- - - 1.7 0.4 - - - 4.1 0.8 - 0.8 0.2

_ 0.3 0.07 - ~ 0.2 0.05 81* 17 tr 0.1 0.2

0.1 0.02 - 0.03 0.6 0.1 0.3 - -

0.1 95* 19 tr 0.07 0.05 0.1 131* 27 0.4 0.7 0.2

0.7 0.2 ~ 0.5

_ 0.4 - - 2.6* 0.5 - 0.6 -

9.4* 1.9- - - -. 0.09 0.02 - - 0.1 _ _ _ _

_ _ 0.4 - 0.6 0.1 - 0.1 - 4.3 0.9 0.2 0.2

co.01 18 3.7 - 2 - 6.1 1.3 0.3 0.3 -

_ 0.4 0.08 - - 0.8 36* 7.4 - 0.2 - 25* 5.1 - 0.5 -

0.2 0.03 ~ 0.3 _ 1.1 0.2 - 0.3 -

0.7 0.2 - - 0.7 _ 17* 3.5 2.1 1.7 -

0.4 0.07 - 0.5 _ 5.9* 1.2- - -

0.2 0.05 0.4 - -

0.2 0.04 - - 0.3 _ ~ _ _ _ _ _ 0.2 0.1 -

_ 13 2.7 ~ - _ 0.1 - - 9.5* 1.9 - 0.4 -

_ 3.2 0.7 ~ - _ 0.5 0.1 - 0.1 -

0.7 O.l- - 0.3 0.3 486* 100 5.1 8.0 4.7

0.2 _

1 0.05

0.1 3.9

0.5 0.2

0.3 0.3 _

0.1 1.4

_ _

_ _

_

_

7.7

_ _ _ _ 0.2 _ 1.1 0.05 1 _ 0.05 _ 1

0.3 0.06 4.3

0.03 _ 0.3 0.02 0.6 0.02 1,5

0.5 0.4

0.06 _

0.4 0.3

_ 0.4 0.1

_ 0.6 co.01 3.4

0.6 0.8 0.2

_ 0.5 _ 0.3

0.3 0.7

_ 3.8 _ 0.5 _

_ 0.4 0.3

0.03 0.03 0.3 _

_ 0.1 _ 0.4

_

0.02 0.1 _ 0.3 0.2 25.6

_ _ 0.8 4.3 4.1

0.2 3.8 1.3

17 0.1 1.1 2.5 5.9 2 1.5 0.2 _

1.6 1.1 1.5 0.4 2.3

13

2.1 3.0 0.9 1.8 1.0 1.1 2.7

15 2.1

1.4 1.1 0.1 1.3 _

0.5 1.5 _

0.5 0.1

100

a See footnote to Table 2. A=androstane; d, double bond in unknown position, b Retention index as methyl ester trimethylsilyl ether derivative. ’ U=unconjugated; G/T=aminoacyl amidated; GlcA=glucuronidated; S=sulfated; S-GlcA=both sulfated and glucuronidated; S-GiT=both sul-

fated and glycine- or taurine-conjugated. tr= trace amount; nd=not determined. d Possible analytical artefact, formed from B4-7a, 12a-ol-3-one. ’ Tentative structure.

The excretion of bile acids in urine of the healthy acids. As shown in Table 3, the main part of the uncon- pregnant women was about 30 pmol/g creatinine. jugated fraction consisted of trihydroxylated bile acids, About 12% of the total were unconjugated, 30% were the umidated fraction contained the 12/I isomer of amidated, 20% were glucuronidated, 37% were sulfated DCA, 12-oxolithocholic acid, l/3- or 6cr-hydroxy-DCA, and only 0.6% were both sulfated and glucuronidated. CA, and small amounts of several tetrahydroxychol- Each conjugate group had its typical pattern of bile anoates that were lp-, 2/I-, 6a- and 4/L hydroxylated

350


TABLE 4

Excretion of 7a,12a-dihydroxy-3-oxo-4-cholenoic acid and 12a-hydroxy-3-oxo-4,6-choladienoic acid in the urine of six patients with

ICP and three healthy pregnant women

Bile acid” Excretion pmol/g creatinine]

ICP Healthy

Mean+SEMb ‘%I” Mean?SEMb OX?

Unconjugated B4-7n, 12a-diol-3-one 1.1?0.3 19 1.2ZO.4 34

Amidated B4-7a,12a-diol-3-oned 1022 13 1.5?0.6 16 B4.6-12a-ol-3-onee 120.2 1.3 0.3+0.09 2.5

a.b See footnotes to Table 2. c Percentage in corresponding group of bile acids, see Table 3. d Significantly increased in ICF! ’ May be an analytical artefact formed from B4-7a,12a-diol-3-one.

derivatives of CA. The sulfated fraction was dominated by dihydroxycholanoates, lithocholic acid (LCA) and I2-0x0-LCA. The glucuronide fraction contained, in addition to 6-hydroxylated bile acids, CDCA and CA, four unknown cholanoates with longer retention times, RI:s 3329, 3337, 3359 and 3493, respectively They had two (3337) or three (3329 and 3359) hydroxyl groups or hydroxyl-oxo (3366) groups on the ABCD rings and presented regularly in both healthy pregnancy and ICP. The glucuronide fraction also contained three 3u@)- hydroxy-5a(P)-androstane- 17P-carboxylic acids which only appeared in the urine of patients with ICI? Sul- fated bile acid glucuronides were found in minute amounts in both healthy pregnancy and ICI? This fraction contained two 4-hydroxylated bile acids only found in healthy pregnancy.

During ICP urinary bile acid levels rose to a mean of 225 pmollg creatinine (Fig. 2). This was mainly due to increased excretion of sulfated and amidated bile acids. As exemplified in Table 3, the amidated fraction showed large increases of tetrahydroxy bile acids, CA and lp,3a,l2a-trihydroxy-S/I-cholanoate. Four common bile acids, i.e. CA, CDCA, DCA and LCA, were the main contributors to the large increase of the sulfated fraction. In addition, 3P-hydroxy-5-cholenoic and 3/I, 12a-dihydroxy-5-cholenoic acids increased in cholestasis. In contrast to these groups of bile acids, glucuronidated bile acids increased less markedly. Their con- tribution to the total urinary bile acids decreased from 20% in healthy pregnancies to 4.8% in ICI? The unconjugated bile acids in urine showed no significant changes.

Interestingly, 7a, 12a-dihydroxy-3-oxo4-cholenoic acid was found in the unconjugated and amidated fractions in both healthy pregnancy and TCP (Table 4). In cholestasis, the absolute amount of this acid increased

in parallel with other bile acids in the amidated fraction where it constituted a mean of 16% and 13% in healthy pregnancy and ICP respectively It was a major unconjugated bile acid in healthy pregnancy, constituting 34% of this fraction. In ICP the levels were approximately the same and the relative amount decreased to 19% (all mean values) due to increased excretion of norcholic acid and CA in several ICP patients.

Correlation between bile acids in serum and urine. The total bile acid concentration in serum was positively correlated to the bile acid excretion in urine.

Progesterone metabolites in serum: The concentrations of steroid sulfates and glucuronides in the sera of six ICP patients and three healthy pregnant women are shown in Table 5. Since the samples from patients and healthy women were not from corresponding times of gestation and the levels of sulfated progesterone metabolites in serum increase with time, absolute concentrations cannot be compared between the two groups. However, taking the increasing levels into con- sideration, the results are in agreement with the previous study of serum steroids in ICP (5). Thus, the levels of the

TABLE 5

Concentrations of progesterone metabolites in serum of three healthy pregnant women at 3638 weeks of gestation and six women with ICP at 31-34 weeks of gestation tmean%SEM. Ltmol/l)”

Structureb RI’ ICP Healthy

Glucuronides 5a-P-3n-ol-20-one 5/?-P-3a-ol-ZO-one 5a-P-3a,20a-ol 5/&P-3a,2Ou-01 Total glucuronides

2622 0.1 -co.04 2637 120.4 2754 0.450.07 2768 4.721

6.1tl.3

Monosulfates 5a-P-3a-ol-20-one 5/I-P-3a-ol-20-one P5-3P-ol-20-one 5a-P-3/?-ol-20-one 5a-P-3a,20a-01 5/T-P-3a.20a-o1 5a-P-3a,16a-ol-20-one P5-3/?,20a-01 5a-P-3/?,20a-01 5a-P-3a, 16a,20a-ol 5a-P-3a,20a,21-01 Total monosulfates

2622 2.7~0.4

2637 0.5t0.08 2700 0.3?0.09 2705 0.5~0.1 2754 5.420.7 2768 2.920.7 2718 220.7 2828 0.2?0.01 2836 l.lt0.09 2894 0.5?0.16 2993 4.520.9

19z2.5

Disulfates 5a-P-3a,20a-01 5/&P-3a,20a-oI P5-3P,20a-01 5a-P-3/3,20a-01 5a-P-3a,20a,21-01 Total disulfates

2754 1223 2768 5.521.8 2828 1.320.3 2836 8.4~0.7 2993 1.9r0.3

3028

Total steroids 55212

0.2~0.01 0.720.3 0.4”0.03 4.924.5 6.220.3

3.921.2 0.620.4 0.5?0.08 1.820.3 5.1~2.3 1.6~0.3 1.4+0.6 0.6t0.2 3.720.8 0.1~0.03 2.92 1.6 2224

5.222.4 1.1*0.7 1.7?0.6 10?2.6

0.3”o.l 2027

48211

a.b See footnotes to Tables 2 and 3. P=pregnane. ’ Retention index as trimethylsilyl ether derivative.

351

L. J. Meng et al.

TABLE 6

Excretion of progesterone metabolites @mol/g creatinine) in urine of a woman with typical ICP in the 31st week and a healthy pregnant woman

in the 36th week of gestation

Structure” Rib ICF

MoS DiS

Healthy’

GlcA Total % MoS DiS GlcA Total %

5a-P-3a-ol-20-one 5P-P-3a-ol-20-one P-3,17-ol-20-one 5~-P-3~,2Ou-o1 5a-P-3a,20a-01 5/!-P-3a,6a-ol-20-one 5/?-P-3a,20n-01 5/?-P-3/?, 15ol-20-one 5a-P-3a, 16a-ol-20-one 5/3-P-3a, 16a-ol-20-one P-2,3-ol-20-one P5-3p,2001-01 5a-P-3/?,20a-01 5a-P-3~,15/16-ol-20-one P-trio1 5a-P-3a, 16a,20a-01

P-2,3,20-01 5a-P-3,17,20-01 5/?-P-3,17,20-01 5a-P-3a,2 1 -ol-20-one

P-2,3,20-01 P-1.3,16-ol-20-one P-trio] P-2,3,16-ol-20-one P-3,16,20-ol+Pj-3.17,20-1 5a-P-3a.20q2 l-01

Pd-triolone 511(-P-3a,20a,21-01 P5-3jj’,21-ol-20-one 5a-P-3B,21 -ol-20-one P-triolone+P-diolone

P5-38, 1 Sp, 17a,20a-01 5a-P-3/?, 16a,20a-01 P5-3b.20a,? l-01 5a-P-3/3,16n,208,2 l-01 5a-P-38,20a,21-01 P-triolone 5~-P-3a-ol-20a-GlcNAc’ 5a-P-3a-ol-20a-GlcNAc P5-3&ol-20~GlcNAc 5a-P-3/J-ol-20a-GlcNAc P-2,3,20-ol-GlcNAc Total

2622 2637 2731 2750 2754 2758 2768 2778 2778 2790 2804 2828 2836 2856 2857 2894 2899

2907 2916 2928 2935 2955 2961 2970

01 2987 2993 2995 2999 3012 3023 3047 3058 3060 3086 3087 3091 3217 4201 4230 4308 4325 4383

_ _ 4

1 _

4.6 3.8 _ _

3.0 2.2 tr _

tr 2.0

1.6 _

0.8 1.9 2.2 _ _ _ _

2.5 _

0.6

15 31 0.4 0.7 _

76

_ _ _ 2

49 _

15 _ _ _ _

5.7 22

0.7 4.3 _

0.5

7.6

nd

5.2 25

_

0.2 1.3 2.3 1.5 _

0.8 nd

1.1

_ _ _ _ _

144

14 25

8

29 nd 87

_

13 13 12 5.9 _

16

33d

tr 11 _

8.1 7.9

5.2 12

_ _ _ _ _ _

6.1 1 5.6 _

0.4 324

14 2.6 25 4.6 12 2.2 2 0.4

79 14

nd nd 107 20

3.8 0.7 13 2.4 13 2.4 12 2.2 12 2.2 22 4 19 3.5 2.9 0.5

33d 6.1 _ _

0.5 0.1 13 2.4 7.6 1.4 8.1 1.5 7.9 1.5 1.6 0.3 5.2 1.0

18 3.3 27 4.9

2.2 0.4 0.2 0.03 1.3 0.2 3.2 0.6 1.5 0.3 2.5 0.4 0.8 0.1

nd nd 0.6 0.1 1.1 0.2 6.1 1.1

16 2.9 37 6.8

0.4 0.07 0.7 0.1 0.4 0.08

543 100

_ 2 _

0.7

2.9 2.7 _ _ _

2.1 1.8 4.5 _

tr 2.8 _ _ _

1.1

2.8 1.2 0.5

_

5.4 _ _

0.7

1.5 16 0.6 8.7 _

58

_ 0.5 7.7

1.0 _ _ _ _

11 9

tr 0.4 _

0.1

0.8 _

nd _

0.7 0.5

0.4 0.8 2.5 1.2 _

0.3 nd

0.1

_ _ _

_

37

20 39 20

25 5.5

112

33 20 10

8.3 _

25

24 6.0

tr 29

_

10 8.5 _

7.5 2.4 _ _

11 2.1

15 _

0.6 433

20 3.8 39 7.4 22 4.2

0.5 0.1 33 6.3

5.5 1 116 22

2.7 0.5 33 6.3 20 3.8 10 1.9 19 3.6 9 1.7

27 5.1 1.8 0.3

29 5.5 6.0 1.1 0.1 0.02

32 6.1 0.8 0.1

10 1.9 8.5 1.6 1.1 0.2 7.5 1.4 5.9 1.1 1.7 0.3 0.5 0.1 0.4 0.08 0.8 0.2 2.5 0.5 1.2 0.2 5.4 1.0 0.3 0.05 nd nd 0.7 0.1 0.1 0.02

11 2.1 3.7 0.7

31 5.9 0.6 0.1 8.7 1.6 0.1 0.1

528 100

a P=pregnane; A=androstane; GlcNAc=N-acetylglucosamine; superscript indicates position of double bonds, Greek letters denote configuration of hydroxyl groups and the hydrogen at C-5, d, double bond at unknown position.

b Retention index as trimethylsilyl ether derivative. c MoS=monosulfates; DiS=disulfates; GlcA=glucuronides; nd=not determined. d Mixture of 5uz-P-3a,16a,20a-triol and P-2,3,20-triol. e Probably a mixture with 5a-P-3a,21-01-20~-GlcNAc.

disulfates of 5a- and 5b-pregnane-3a,20a-diol were increased in the ICP patients. The ratio between 3a-hydroxy- and 3/Ghydroxysteroids was increased, and the level of 5a-pregnane-3/?, 20a-diol monosulfate was decreased. The 5a-pregnane-3a, 20a, 2 1 -trio1 previously shown to be markedly increased in ICP (5) was also increased in the present patients, particularly as a disulfate. The levels of another pregnanetriol, Sa-pregnane-

3a, 16a,20a-triol, were higher than in the previous study and were elevated in the patients. The patterns of glucuronidated steroids were similar in the patients with ICP and the healthy women. The relative concentration of glucuronides was lower in the patients.

Progesterone metabolites in urine: Three groups of steroids were analyzed according to their mode of conjugation: monosulfates, disulfates and glucuronides.

352


Table 6 shows a direct comparison between the excretion of individual steroids in urine from a patient with severe cholestasis and a healthy pregnant woman. The distribution of total steroids in urine according to mode of conjugation in six patients and three healthy pregnant women is shown in Fig. 3.

The mean excretion of steroids in the disulfate fraction was three times higher in ICP than in the healthy women (Fig. 3 and exemplified in Table 6). Sol(P)-Pre- gnane-3a(P),20a-diol, 5a-pregnane-3c@),2 1 -dial-20- one and 5a-pregnane-3a,20cc,21-triol dominated this fraction in patients with ICI?

As expected, glucuronides constituted the major part of the steroids in urine. In addition to 5P-pregnane-3a,20a-diol, the major glucuronides in urine included 5c@)-pregnane-3a-ol-20-one, Sa-pregnane- 3a,20a-dial, Saw)-pregnane-3a(P), 16-diol-20-one and 5/?-pregnane-3,17,20-triol, the sum of which in most cases exceeded the value for 5P-pregnane-3a,20a-diol.

Urinary progesterone metabolites

Healthy ICP

4% 6% GO/. 2% 19% ., ,” - ._

6%

10%

63% 6%

79%

BMDiS IMOS- GICNAC AMOS UGICA HGICA- GICNAC

Fig. 3. The distribution of total progesterone metabolites in urine according to mode of conjugation in the six patients with ICP and three healthy pregnant women. I=patient with ICP; H=healthyJ pregnant women; GlcA=glucuronidated; MoS=monosul$ated; DiS=disulfated; GlcA- GlcNAc=both glucuronidated and N-acet_ylglucosaminidat- ed; MoS-GlcNAc=both monosulfated and N-acetylglucos- aminidated; MoS and GlcA do not include their corresponding N-acetylglucosaminides.

TABLE I

Urinary excretion of steroids conjugated with N-acetylglucosamine in six patients with ICP at 31-34 weeks of gestation and three normal pregnant women at 36-38 weeks of gestation (mean?SEM, pmollg creatinine)

Steroidsb ICP Healthy

Monosulfates 5B-P-3a-ol-20~GlcNAc’ 5a-P-3a-ol-20a-GlcNAcd P5-3B-ol-20~GlcNAc 5a-P-3p-ol-20a-GlcNAc Total Percentage in fraction

Glucuronides 5/J-P-3a-ol-20~GlcNAc 5a-P-3n-ol-20a-GIcNAcd P-2,3,20-ol-GlcNAc TotaId Percentage in fraction

151-8 321 3425 1422.4

0.920.3 0.9kO.4 2.1~0.8 7.5kl.2 52213 2524.4 62f5 4525

1.920.5 8.1 t-O.8 0.7t0.1 llZl.3

3.220.4

220.2 18+3

0.9?0.2 2123

520.4

a See footnote to Table 2. h For abbreviations, see Table 6. ’ Probably a mixture with 5a-P-3n,21-diol-2Oa-GlcNAc. d Significant difference between ICP and healthy subjects.

Compared with corresponding values in urine of healthy pregnant women, the concentration of steroid glucuronides in ICP tended to be lower (Fig. 3). In general, there were no significant changes in the profile of the glucuronide fraction caused by ICP (Table 6).

Both the monosulfate and glucuronide fractions contained steroids additionally conjugated with N- acetylglucosamine (GlcNAc) (9). They were quantified from the areas of the peaks of the derivatized N-acetylglucosaminides. Such double conjugates dominated the monosulfate fraction both in urine from healthy pregnant women (45% of the monosulfates) and women with ICP (62% of the monosulfates) (Table 7). The excretion of GlcNAc conjugates with a 3a-hydroxy configuration in the monosulfate fraction was greatly increased in ICP, whereas the excretion of double conjugates with glucuronic acid and GlcNAc was significantly decreased (Table 7).

Correlation between steroid sulfates in urine and

serum. The ICP-induced changes of the steroid profiles in serum were reflected in the urinary profiles. The urinary excretion of steroid sulfates was positively correlated to the levels of steroid sulfates in serum (Fig. 4). The ratio of sulfated to glucuronidated steroid metabolites was increased in both serum and urine. The ratio of 3a-hydroxy- to 3/3-hydroxy-steroids was significantly increased in both serum (5) and urine, in the disulfated as well as the monosulfated fraction (Table 8). The largest increase occurred among the sulfated N-acetylglucosaminides in urine. The ratios between sulfated 5a- and 5P-steroids were lower in ICP than in the

353

L. J. Meng et al

.

. .

0.

.

.

.

OC I

0 20 40 60 80 100

Concentration in serum [pmolll]

Fig. 4. The positive correlation between total concentration of sulfuted progesterone metabolites in serum and the urinary excretion. The individuals are six patients with ICP and three healthy pregnant women. The correlation coef- jicient r=O. 7043 and ~~0.05.

healthy subjects, but the differences were not statistically significant (Table 8).

Discussion This is the first study in which profiles of bile acids and progesterone metabolites have been simultaneously analyzed in both serum and urine of patients with ICI? The results confirm and extend previous results obtained in analyses of either bile acids (l-4) or steroids

(5-7) in either serum (l-3,5) or urine (4,6,7), using less comprehensive and older methods.

Bile acids

In agreement with previous results (l-3), the concentration of aminoacyl amidated bile acids in serum was increased in ICI? The CACDCA ratio was greatly elevated, probably a sign of early cholestasis. The level of DCA was not increased because of the impairment of the enterohepatic circulation. The excretion of bile acids in urine was also increased in ICI? Different forms were predominant in serum and urine and the urinary pattern indicated enhanced hydroxylations and sulfation to eliminate the excess of circulating bile acids. Judging from the urinary bile acid profiles, glucuronidation was of lesser importance in ICI? In contrast to the conjugated bile acids, the unconjugated bile acids were not significantly changed by ICP, suggesting that a normal uptake of bile acids from the portal blood at the basolateral membrane is maintained in the disease.

Since some bile acids and bile acid conjugates can induce cholestasis (for a review, see (11)) we searched for the presence of potentially cholestatic bile acids in the urine of the ICP patients. Three types of bile acids were found which have not previously been reported to occur in the urine of pregnant women or patients with ICI? The 4/I-hydroxylated bile acids were first identified in fetal bile (12) and they are present in amniotic fluid (13) and infant urine together with other hydroxylated derivatives of the common bile acids (14). In contrast to the l/3-, 2/?, and 6cr-hydroxylated bile acids, they were not increased in the patients with ICP, and they

TABLE 8

Ratios between concentrations of steroid sulfates in serum and urine from six patients with ICP at 31-34 weeks of gestation and three healthy pregnant women at 36-38 weeks of gestation

Ratio Steroid group Sample Patients (6) Healthy (3) Significanceb Mean (range)” Mean (range)”

5a/5P Monosulfates serum 5.6 (2. I-8.9) 8.7 (8.0-9.3) n.s. Disulfates serum 4.1 (1.5-7.0) 7.2 (5.3-8.7) n.s. Disulfates urine 7.6 (3.8-l 1)“ 11 (7.5-14)d n.s. Monosulfated N-acetylglucosaminides urine 4.2 (1.1-8.3) 11 (4.616) ns.

3al3/I Monosulfates serum 8.6 (5.3313) 2.3 (2.1-2.6)c * Disulfates serum 1.8 (0.883.4) 0.5 (0.3-0.7)c * Disulfates urine 2.1 (l.l-3.0)d 0.9 (0.8-l. 1)d * Monosulfated N-acetylglucosaminides urine 20 (9.643) 2.0 (1.9-2.1)’ *

a The calculations are based on the results for each subject. The number of subjects is given in parenthesis. b Wilcoxon’s rank sum test; n.s.=not significant. *=significant. ’ All steroid sulfates were included in the calculations. d The sulfates of the following steroids were included in the calculations: So(B)-pregnane-3a(P),20a-diols, 5a-pregnane-3a(ll),2l-diol-2O-one, and

5a-pregnane-3a,20a,2l-triol. e All sulfated GlcNAc conjugates were included in the calculation.

354

Steroids und bile aids in ICP

are likely to be formed in the fetal compartment and not to be related to the cholestasis.

Three isomeric short-chain bile acids were found in urine from the patients with ICP but not in urine from the healthy pregnancies. The major isomer was 3a-hydroxy-5a-androstane-17/Gcarboxylic acid. This acid has previously been found in human meconium (15). In vitro studies with rat and human liver microsomes and purified enzymes have shown that the short-chain bile acids are conjugated with glucuronic acid at C-3 or at the carboxyl group (1619). In agreement with these findings, the short-chain acids in urine from patients with ICP were found in the glucuronide fraction. The effects of 3a-hydroxy-5a-androstane-17/Lcarb- oxylic acid or its glucuronides on bile secretion are not known. However, the S/$isomer is choleretic in rats

(20). The 3a-hydroxy-5a-androstane- 17a-carboxylic acid

has the same configuration at C-3 and C-5 as the major sulfated progesterone metabolites (see below). In addition, 5a-pregnane-3a,20a, 21-trio1 is a major metabolite which is increased in serum and urine of patients with ICP (5, this study). This compound is a potential precursor of the 17/j’-carboxylic acid. However, it should be noted that the trio1 is excreted as a sulfate whereas the carboxylic acid is a glucuronide.

The finding of 7a, 12a-dihydroxy-3-oxo-4-cholenoic acid as a major bile acid in both unconjugated and aminoacyl amidated forms is of particular interest. Ex- cretion of 3-0x0-A4 bile acids has been observed in con- nection with cholestasis due to inborn deficiency of 3- oxo-A4-steroid 5a-reductase (21) or a relative deficiency of the enzyme caused by liver disease (22,23). Infants who are believed to have a “physiological cholestasis” in the neonatal period, also excrete 7a,l2a-dihydroxy- 3-oxo-4-cholenoic acid in urine (24). It has been shown that 7a-hydroxy-3-oxo-4-cholenoyltaurine strongly in- hibits ATP-dependent canalicular bile acid transport (25). It is not known whether this is also the case with the 12a-hydroxylated analogue now found in urine during pregnancy. It may be speculated that the large load of 3-0x0-A4 steroids to be reduced in the liver in pregnancy (about 250-300 mg of progesterone is me- tabolized in late pregnancy) could result in incomplete reduction of precursors in bile acid biosynthesis. The 7a, 12a-dihydroxy-3-oxo-4-cholenoic acid was excreted in urine both in the healthy pregnant women and the patients with ICI? Although the excretion of the aminoacyl amidated form was increased in ICP, the excretion relative to that of cholic and chenodeoxycholic acids was not increased, indicating that the formation of the 3-0x0-A4 bile acid was not selectively increased in ICI?

Progesterone metabolites In agreement with the early studies (5,6), the total levels of sulfated steroids in plasma and their excretion in urine were increased in patients with ICP This finding argues against the recent suggestion that impairment of sulfation may contribute to the pathogenesis of ICP (26). However, it is possible that the sulfation of large amounts of progesterone metabolites may decrease the capacity for sulfation of other compounds, both in healthy pregnancies and particularly in ICP.

In contrast to the sulfates, the glucuronidated metabolites were not increased in the serum or in the urine of patients with ICl? The major glucuronidated metabolites have a 3a-hydroxy-5P(H) configuration. The mono- and disulfate of 5/Gpregnane-3a,20a-diol increased markedly in serum and urine of patients with ICP while the glucuronide was unchanged or decreased. This indicates that the relative rates of sulfation and glucuronidation are changed in patients with ICI?

A most characteristic change in ICP is the relative and absolute increase in sulfated metabolites having a 3a-hydroxy-Sar(H) structure and a relative decrease in the 3B-epimers. Theoretically, 3a-hydroxy-/3ghydroxy- steroids are interconvertible, e.g. in the liver or during an enterohepatic circulation with participation of enzymes in the intestinal microflora. However, studies in normal pregnancies have shown that these epimers are not interconverted (27,28). Participation of the intestinal microflora would be even less likely in patients with ICP who have impaired enterohepatic circulation and decreased formation of deoxycholic acid. It can there- fore be concluded that the 3a- and 3/Ghydroxy epimers are primary metabolites of progesterone. This is also supported by a study of progesterone metabolism in a bile fistula patient (29).

Pregnanediols and pregnanetriols conjugated with N-acetylglucosamine and either sulfuric acid or glucuronic acid were recently found as steroid metabolites in the urine of pregnant women (9). The profiles of these conjugates were also changed in patients with ICI! The urinary excretion of the sulfated forms was increased and that of the glucuronidated forms decreased in our patients with ICI? The ratio of 3a-hydroxy- to 3/3- hydroxysteroids was markedly increased for the sulfated N-acetylglucosaminides (Table 8) further sup- porting an increased formation of 3a-hydroxy-Sa(H) metabolites of progesterone in patients with ICI?

The combined results of the previous (l-7) and present studies suggest that patients with ICP have a changed metabolism of progesterone with an increased formation of 3a-hydroxy-Sa(H) metabolites and sulfates. It is not possible with certainty to distinguish

35.5

L. J. Meng et al.

between primary changes in progesterone metabolism and secondary changes caused by the cholestasis. Sul- fated and glucuronidated progesterone metabolites are secreted in bile (30) but the canalicular transporters involved in their secretion have not been defined. About 30% of the biliary steroids are disulfates (30) which are likely to be transported by the multispecific organic anion transporter (31). The serum levels and urinary excretion of the disulfated progesterone metabolites are increased in ICI? This may reflect an impaired biliary secretion. On the other hand, pregnancy bile also contains a high percentage of glucuronides (30) and these do not increase in serum or urine of patients with ICI? These results indicate selective de- fects in the biliary secretion of sulfated steroids in ICI? However, the increase of the ratio of 3a-hydroxy to 3p- hydroxysteroid sulfates in ICP is unlikely to be secondary to such a defect. If this change were due to a selective decrease in the biliary secretion of 3a-hydroxysteroid sulfates, the 3a-hydroxy-/3&hydroxysteroid ratio would be expected to decrease in bile. This was not the case in the two subjects studied previously (6). Thus, it is likely that patients with ICP have a primary change in the metabolism of progesterone, resulting in the formation of an increased proportion of metabolites with a 3a-hydroxy-Sa(H) configuration. This change was observed in two patients before the onset of pruritus and abnormal liver function tests indicative of ICP (5). This metabolic change appears to be a most important finding for the early diagnosis of ICI? The possible role of progesterone or its metabolites in the pathogenesis of ICP has recently been brought to attention by the finding that 10 of the 13 ICP patients studied by Bacq et al. (3) had been given daily doses of 0.2-lg of progesterone (Utrogestan@) shortly before the development of ICI? This would greatly increase the substrate load on enzymes involved in the reductive metabolism of progesterone and the sulfation of the Sa-reduced metabolites (29). The possible mechanisms by which some metabolites of progesterone or their sulfates may induce cholestasis remain to be established.

Acknowledgements This work was supported by grants from the Swedish Medical Research Council (no. 03x-219), Karolinska Instututet and Fondecyt (Chile) (no. 1940420).

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