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Trihydroxycoprostanic acid in the duodenal fluid of two children with intrahepatic bile duct anomalies
212
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 26864
T R I H Y D R O X Y C O P R O S T A N I C ACID IN T H E D U O D E N A L F L U I D OF TWO C H I L D R E N W I T H I N T R A H E P A T I C B I L E DUCT ANOMALIES
H. E Y S S E N , G. P A R M E N T 1 E R ,
F. C O M P E R N O L L E , J. B O O N * AND E. E G G E R M O N T * *
The Rega Institute, University of Leuven, 3000 Leuven (Belgium) (Received J a n u a r y I7th, 1972)
SUMMARY
Using thin-layer chromatography, gas-liquid chromatography and mass spectrometry, trihydroxycoprostanic acid (3~,7~,I2~-trihydroxy-5fl-cholestan-26-oic acid) was identified in the duodenal fluid of 2 subjects with anomalies of the intrahepatic bile ducts. Subject I was a case of intrahepatic cholestasis due to atresia of the interlobular bile ducts with familial incidence. The bile acid spectrum in the duodenal fluid of this patient was: 23 % chenodeoxycholic acid, 58% cholic acid and 190/0 trihydroxycoprostanic acid. Subject 2 had a Zellwegerdike syndrome with cholestasis and with scarcely developed intrahepatic bile ductuli. The bile acid spectrum in the duodenal fluid of this patient was: 18% chenodeoxycholic acid, 37% cholic acid and 45% trihydroxycoprostanic acid. No trihydroxycoprostanic acid was found in seven healthy subjects, in three cases of cholestasis of infancy, or in ten cases of various disorders of the small intestine. Obviously, the excretion of trihydroxycoprostanic acid with the bile of Patients I and 2 was due to a reduced capacity of the hepatocvtes to split off the 3 terminal carbon atoms of the side chain of trihydroxycoprostanic acid. The cause of the impaired function of the hepatocytes remains to be established.
INTRODUCTION
Bile acids are synthesized in the liver by hydrogenation and hydroxylation of the steroid nucleus of cholesterol, followed by oxidation of the side chain 1. In certain lower vertebrates, the side chain of cholesterol is oxidized but not degraded, thus giving rise to the primitive C27 bile alcohols or C2~ bile acids ~. Dihydroxycoprostanic and trihydroxycoprostanic acid, for instance, are the major bile acids in the alligator3, 4. In more evolved animals, the side chain is oxidized with removal of the terminal * D e p a r t m e n t of Pediatrics, S a i n t R a d b o u d Hospital, U n i v e r s i t y of Nijmegen, T h e N e t h e r l a n d s . ** D e p a r t m e n t of Pediatrics, S a i n t Rapha6I Hospital, U n i v e r s i t y of L e u v e n , Belgium. * C o m m o n a n d s y s t e m a t i c n a m e s of c o m p o u n d s m e n t i o n e d in t h e t e x t are as follows : t r i h y d r o x y c o p r o s t a n i c acid: 3~,7~,I2~-trihydroxy-5fl-cholestan-26-oic acid; d i h y d r o x y c o p r o s t a n i c acid: 3~,7~,-dihydroxy-5~-cholestan-26-oic acid; lithocholic acid: 3~-hydroxy-5fl-cholanoic acid; chenodeoxycholic acid : 3~,7~-dihydroxy-5fl-cholanoic acid ; deoxycholic acid : 3~, 12~-dihydroxy5fl-cholanoic acid; cholic acid; 3 a , 7 ~ , I 2 ~ - t r i h y d r o x y - 5 f l - c h o l a n o i c acid.
Biochim. Biophys. Aeta, 273 (1972) 212 221
TRIHYDROXYCOPROSTANIC
ACID IN DUODENAL FLUID
213
isopropyl fragment to yield the "modern" Cu bile acids such as cholic acid and chenodeoxycholic acid which are the predominant primary bile acids in human bile. Several investigations support the hypothesis that the reaction sequence leading to synthesis of cholic acid in mammals would reflect the evolutionary past, and that dihydroxy- and trihydroxycoprostanic acid are short-lived natural intermediates in the synthesis of chenodeoxy- and cholic acid, respectively. In rats, trihydroxycoprostanic acid is readily converted to cholic acidS, e. Small amounts of dihydroxy- and trihydroxycoprostanic acid have been found in human fistula bile 7-1°. However, in healthy subjects with an uninterrupted entero-hepatic cycle, the concentration of the coprostanic acids is too small to be detected by routine analysis of bile or duodenal fluid. In the present study, we identified trihydroxycoprostanic acid as a major bile acid in the duodenal fluid of two children with anomalies of the intrahepatic bile ducts. MATERIALS AND METHODS
Preparation of bile acid derivatives Duodenal fluid, obtained by aspiration, was centrifuged and o.2-ml portions of the clear supernatant were distributed into 16 m m × 16o m m test tubes. For quantitative studies, an internal standard of 23-nordeoxycholic acid was added at this stage. The sample was hydrolyzed in I ml 25~/o K O H in ethylene glycol at 215 °C for 15 rain as described by Evrard and Janssen n. After cooling, I ml water and I ml methanol were added and the neutral sterols were removed by three extractions with 5 ml light petroleum (b.p. 40-60 °C). The aqueous layer was then acidified to p H 2 with 6 M HC1 and extracted with diethyl ether (3 × 5 ml). The solvent phase, containing the crude bile acid mixture, was washed once with 5 ml 20°/0 NaC1, evaporated under reduced pressure, dissolved in IO ml methanol~liethyl ether (I : 9, by vol.) and methylated with diazomethane for IO min at room temperature. The methyl ester acetates were prepared by acetylation of the methyl esters with i ml of a solution of 5 ml acetic acid, 5 ml acetic anhydride and one drop of perchloric acid. After the reaction mixture had acetylated for I8 h at room temperature, 8 ml of 20% NaC1 were added, and the methyl ester acetates were extracted with diethyl ether (3 × 5 ml). The ether layers were combined, evaporated, taken up in o.5-1 ml of acetone, and chromatographed. Ketonic esters of the bile acids were prepared according to the method of Evrard and Janssen n by mild oxidation of the methyl esters with i ml of 90% acetic acid and 0.2 ml of 20% CrOs in acetic acid. After 15 min at room temperature, I ml of lO°/0 ascorbic acid and 9 ml of 20% NaC1 were added, and the ketonic esters were extracted with diethyl ether (3 × 5 ml). The combined extracts were evaporated to dryness, and the residue was dissolved in o.5-1 ml of acetone and chromatographed. Trimethylsilyl ethers were prepared by reacting the free bile acids with hexamethyldisilazane and trimethylchlorosilane in pyridine, under dry conditions, at room temperature for 30 min 1~.
Gas-liquid chromatography Gas-liquid chromatography was carried out on a Pye series lO 4 instrument Biochim. Biophys. Acta, 2 7 3 (1972) 2 1 2 - 2 2 1
214
I~. EYSSEN et al.
(Pye-Unicam, Cambridge, U.K.), with a flame-ionisation detector and with nitrogen as the carrier gas. For identification studies, the retention times of the respective peaks were compared to those of reference compounds, using several types of stationary phases: a 7-ft column packed with I % OV-I on 8O-lOO mesh Supelcoport (Supelco, Inc., Bellefonte, Pa., U.S.A.), and a 5-ft column packed with 3% QF-I on lOO-12o mesh Gas Chrom Q (Applied Science Lab. Inc., State College, Pa., U.S.A.). The temperature of the columns was kept at 252 °C. The temperatures of flash heater and detector were 270-280 °C and 250 °C, respectively. Chromatography on I °/o J X R (Applied Science Lab., State College, Pa., U.S.A.) was carried out with a temperatureprogrammed instrument provided with an argon-ionisation detector. The temperature was raised from 18o to 260 °C in 12 rain. For quantitative determinations, the methyl ester acetates were chromatographed on a column of 3% QF-I or I % OV-I with the flame-ionisation detection system. Although in this system the ionisation responses of the dihydroxy and trihydroxy bile salts are directly proportional to the actual weight of the unsubstituted parent compounds, the concentration of each bile salt was calculated from an individual standard curve. The curves were computed from the ionisation responses of a set of references (5 concentrations for each compound), run daily before and after chromatography of the samples to be quantitated.
Mass spectrometry Mass spectra of reference compounds were recorded by using the direct insertion system of an AEI-MSI2 mass spectrometer (AEI, Manchester, U.K.) at an ionsource temperature of 12o 14o °C. Mass spectra of the bile acids in the duodenal fluid of patients were run via a gas-liquid chromatography-mass spectrometry combination technique. The temperature of membrane separator (V-562o Molecular Separator, Varian, Palo Alto, Calif., U.S.A.) and ion source was maintained at 220 25o °C. Gas liquid chromatography was carried out on a Pye series lO4 apparatus (Pye-Unicam, Cambridge, Great Britain) with helium as the carrier gas.
Preparatios of trihydroxycoprostanic acid from alligator bile IO ml bile from Alligator missisipiensis were diluted with 5 vol. of ethanol. The precipitate was filtered, the filtrate was evaporated to dryness, and the residue was hydrolyzed in IOO ml of 25% K O H in ethylene glycol for 25 rain at 220 °C. An equal volume of 2o°/~ NaC1 and methanol was added, and the neutral sterols were extracted with light petroleum (3 × 300 ml). The water layer was acidified to pH 1 with conc. HC1, and the crude bile acids were extracted with diethyl ether (4 × IOO nil). The ether layers were combined, washed with water, dried over Na~S04 and evaporated to dryness. The residue was methylated with diazomethane for 15 rain at room temperature in methanol-diethyl ether (i : 9, by vol.). On thin-layer chromatography in chloroform-methanol-acetone (14:1:5, by vol.), the methyl ester fraction showed 6 spots with phosphomolybdic acid reagent. The mixture of the methyl esters was separated by column chromatography on 58 g silica gel and stepwise elution with chloroform, 500 ml; chloroform-acetone (9 :i, 8:2 and 7:3, by vol.), 500 ml of each; chloroform acetone methanol (7° :25:5, by vol.), 500 ml. Fractions of 25 ml were collected, and each fraction was examined by thin-layer chromatography.
Biochim. Biophys. Acta, 273 (1972) 212 221
TRIHYDROXYCOPROSTANIC
ACID IN DUODENAL FLUID
215
Two major compounds were isolated and characterized by gas-liquid chromatography, thin-layer chromatography and mass spectrometry. Compound I was identified as the methyl ester of 3a,7x, I2a-trihydroxy-5fl-cholestan-26-oic acid (trihydroxycoprostanic acid). On recrystallization from diethyl ether-light petroleum (b.p. 60-80 °C) this compound yielded 19o mg of white needles with a melting point of 155156 °C, which corresponds to the figures (m.p. 153-155 °C) given by Haslewood 3. Compound n was identified as the methyl ester of 3a,7a-dihydroxy-5fl-cholestan26-oic acid (dihydroxycoprostanic acid). The fractions containing the major amounts of dihydroxycoprostanic acid were combined and yielded 75 mg of an amorphous powder which was contaminated with trace amounts (less than I °/o) of an unidentified substance. Case histories Case I. This male infant was born at term after a normal pregnancy. Two
other sisters were normal but one older brother died at the age of 4 months from obstructive jaundice due to cholestasis and atresia of the intrahepatic bile ducts. The patients had no malformations but a number of congenital stigmata: simian crease on the left hand, bilateral inner epicantal fold, frontal bossing and nystagmus. He had obstructive jaundice from the age of 2.5 months up Lill 4 months. Histological examination of a liver biopsy specimen suggested that the obstructive jaundice was due to cholestasis and partial atresia of the interlobular bile ducts. The patient died at the age of 6 months from pneumonia. The clinical and pathological data on this patient and his brother will be described in more detail elsewhere*. Case 2. This female infant was born at term by cesarean section. The pregnancy was complicated b y vaginal bleeding during the first trimester. The patient was the first child of unrelated parents. She was first seen at the age of 3 months for diarrhoea and jaundice. She measured 58 cm (p25), and weighed 3820 g ( < P 3 ) and had a number of congenital anomalies: clump feet, frontal bossing, broad metopic suture, third fontanel, bilateral epicanthal fold as well as simian creases. The histological examination of a liver biopsy specimen suggested that the jaundice was due to cholestasis. Ductular structures were rather scarse. The icterus disappeared at the age of 5 months. However, the child failed to thrive and was mentally retarded. She died at the age of 8.5 months. At that time she weighed only 405 ° g (
Bile acids in the duodenal fluid
The results of the bile salt determinations are shown in Table I. Variability of total bile acid concentration was high, as could be expected from determinations on duodenal fluid. However, in six out of seven healthy subjects, all values ranged * J. Boon, J. Bakkeren, J. Miser6, E. Schretlen, G. Parmentier and H. Eyssen, submitted for publication. ** E. Eggermont, J. P. Frijns, B. Van Damme and H. Eyssen, unpublished. Biochim. Biophys. Acta, 273 (1972) 212-22i
1,)
~o
a
3.5months
2
3months 4months 7months 7 months 15 m o n t h s 3.5 y e a r s 5 years 2 months 3months 3.5 m o n t h s 3months i2months 6months 8months
7 months
16 m o n t h s
2 years 2 years 6 years 3.5 y e a r s
3 4 5 6 7 8 9 IO ii 12 13 14 15 16
I7
18
19 20 21 22
Controls
4.5 m o n t h s
Age
I
Patients
Subjects
F F F M
F
M
M IV[ M F M M M F M F M F M M
F
M
Sex
Normal Normal Normal Normal Normal Normal Normal Cholestatic icterus Cholestatic ieterus Cholestatic icterus Cow milk protein intolerance Cow milk protein intolerance Tropical sprue Malabsorption due to chronic intestinal infection Contaminated small bowel syndrome Transitory pancreatic insufficiency Congenital lymphedema Coeliac disease Coeliac disease Hirschsprung's disease
A t r e s i a of t h e i n t r a h e p a t i c bile ducts Zellweger-like syndrome
Diagnosis
t 15 ~25 899 975
166
152
251 424 193 54 131 240 158 19 ii 126 364 lO6 798 373
61
26
o o o Tr
o
o
o o o o o Tr Tr o o o o o o o
o
o
28 61 53 27
28
56
51 39 27 34 59 28 39 51 20 32 33 29 22 12
18
23
Bile salts in the duodenal fluid Total amount Relative concentration ( % ) (mg/Ioo ml) Lithocholic Chenodeoxyacid cholic acid
21 o 14 15
14
o
o Tr 6 o Tr 22 14 o o o o 5 o o
o
o
Deoxycholic acid
51 39 33 58
58
44
49 61 67 66 4I 50 46 49 8o 68 69 66 78 88
37
58
Cholic acid
o o o o
o
o
o o o o o o o o u o o o o o
45
19
Trihydroxycoprostanic acid
T h e b i l e s a l t s in t h e d u o d e n a l f l u i d w e r e d e t e r m i n e d b y g a s - l i q u i d c h r o m a t o g r a p h y o f t h e m e t h y l e s t e r a c e t a t e s o n a c o l u m n o f 3 % Q F - I , a t a t e m p e r a t u r e of 252°C, u s i n g 2 3 - n o r d e o x y c h o l i c a c i d m e t h y l e s t e r a c e t a t e a s a n i n t e r n a l s t a n d a r d . W h e n a s a m p l e w a s a n a l y s e d i n i o - f o l d , t h e s t a n d a r d d e v i a t i o n f o r t o t a l b i l e s a l t s w a s ± 3 . 5 1 % ; t h e s t a n d a r d d e v i a t i o n f o r t h e i n d i v i d u a l b i l e s a l t s r a n g e d f r o m ~: 2 . 5 % f o r c h e n o d e o x y c h o l i c a c i d t o :[_ 4.2 % f o r c h o l i c a c i d . T r m e a n s t r a c e a m o u n t s , i.e. less t h a n 2 % of t o t a l b i l e a c i d s .
FLUID
~"
I
BILE SALTS IN THE DUODENAL
TABLE
~'
o~
tO
217
TRIHYDROXYCOPROSTANIC ACID IN DUODENAL FLUID OAc
® AcO..~
cOOCH~
~
AcO
laJ U3 Z
AC.~.OA¢
OOCH3 COOCH3
AcO*"
AcO
O
COOCH3 CH3
¢H3
n t,.3 LLI n-
AcO'" 5
\ o
2J0
1~o
3~0
M IN
410
50
@
I
2
5
O (9 LO LU O
lO
2i0
30
MIN
Fig. I. Gas-liquid chromatography of the methyl ester acetates of reference bile acids (a) and of the bile acids in the duodenal fluid from patient No. 2 (b). Stationary phase: 3% QF-I at 252 °C. I, internal standard (23-nordeoxycholic acid); 2, chenodeoxycholic acid; 3, dihydroxycoprostanic acid; 4, cholic acid; 5, trihydroxycoprostanic acid.
Biochim. Biophys. Acta, 273 (1972) 212-221
218
I~. EYSSEN et a/~.
between 131 and 424 mg/Ioo ml, the average being 233 mg/Ioo ml. On the other hand, Case I and Case 2 had subnormal amounts of bile acids in the duodenal fluid (26 and 61 mg/ioo ml, respectively). It should be mentioned that these samples were obtained during the remission period following the obstructive jaundice. Low concentrations of bile acids were also found in three patients with cholestasis (ii, 19 and 126 mg/Ioo ml, respectively). In ten patients with various disorders of the small intestine, the bile acid concentration in the duodenum was within the normal limits. As shown in Table I, cholic acid and chenodeoxycholie acid were the major bile acids in infants and children. No, or only in trace amounts, lithocholic acid was found. Deoxycholic acid was not present in the younger infants, but reached relative concentrations of 15-2o% in most of the children above the age of 15 months. The most striking difference in the bile acid pattern was the presence in Patients I and 2 of a peak which was not found in any of the healthy controls or in the patients with cholestasis or various disorders of the small intestine (Peak 5, Fig. I). This peak accounted for 19 and 45% of the total bile acids in Cases I and 2, respectively. This bile acid eluted from the columns well after cholic acid and was different from cholic acid, chenodeoxyeholic acid and the secondary bile salts deoxycholic acid and lithocholic acid. In addition to the three major peaks, trace amounts of at least four other compounds were detected. These peaks were too small to be identified. However, on both the QF-I and the OV-I stationary phases, one of them (Peak 3, Fig. I) had the same relative retention time as dihydroxycoprostanic acid prepared from alligator bile. The peak was not detected in any of the control subjects.
Identification of trihydroxycoprosta~ic acid On gas-liquid and thin-layer chromatography, the relative retention times and the RF values of the methyl ester acetate and the methyl ester ketone of Peak 5 corresponded to those of authentic trihydroxycoprostanic acid (Table II and Fig. I). Peaks 2 and 4 corresponded to chenodeoxycholic and eholie acid, respectively. The identity of these peaks was further confirmed by mass spectrometry. A detailed study of the mass spectra of bile acids has been published by Egger 14. By comparison with reference compounds, Peaks 2 and 4 could be easily identified as ehenodeoxycholic and eholic acid, respectively. When the mass spectrum of Peak 5 was recorded as the methyl ester acetate, a peak of the molecular ion (role 59 o) was not found, because acetic acid was too easily eliminated from the molecular ion. However, other characteristic fragment ions were recorded at rn/e 530 (M--HOAc), role 499 (M--HOAc-OCH3), role 488 (M--HOAc CH2=CO), role 470 (M--2HOAc), role 41o (M--3HOAc), role 313 ( M - s i d e chain-2HOAc), role 253 (M--side chain-3HOAc). When the methyl ester ketones were subjected to mass spectrometry, the spectrum showed a peak of the molecular ion at role 458 (Fig. 2). Other characteristic ion fragments were found at role 440 (M--H~O), m/e 427 (M--OCH3), role 371 (M--CH3-'CHCOOCH3), role 3oi (M--side chain), role 283 (M--side chain-H~O), role 261 (fragmentation of the D-ring). Compared to cholic acid, the compound giving rise to Peak 5 had an identical substitution pattern for the steroid nucleus, but a side chain differing by presence of 3 additional carbon atoms. This conclusion was based on the observation that, for both the ketones and the. acetates, the fragment ions corresponding to loss of the side chain and to ring I~ fragmentation of the unknown compound in Biochim. Biophys. Acta, 273 (I972) 212-221
219
T R I H Y D R O X Y C O P R O S T A N I C ACID IN D U O D E N A L F L U I D TABLE
II
GAS--LIQUID OF THE
CHROMATOGRAPHY
BILE ACIDS IN THE
OF THE
DUODENAL
METHYL
FLUID
ESTER
ACETATES
OF PATIENT
NO.
AND
METHYL
ESTER
KETONES
2
R e s u l t s a r e g i v e n as a v e r a g e of i o d e t e r m i n a t i o n s 4- s t a n d a r d e r r o r of m e a n .
Relative retention times compared to 23-nordeoxycholic acid Methyl ester ketones on Methyl ester acetates on 3% QF-z x% JXR z % OV-z z% JXR z % OV-z 2 3-Nordeoxycholic acid Peak 2 Chenodeoxycholic acid Peak 3 Dihydroxycoprostanic acid Peak 4 Cholic acid Peak 5 Trihydroxycoprostanic acid
i.oo 1.51 4- 0.02 1.51 4- o . o i 2.13 4- o . o i
I.OO 1.23 4- O.Ol 1.23 4- o . o i --
I.OO 1.46 4- o . o i 1.45 4- o . o i (2.40 4- 0.03)*
I.OO 1.13 4- o . o i 1.13 ± o . o i --
I.OO 1.24 4- o . o i 1.23 4- o . o i --
2.13 2.75 2.76 4.00
0.02 o.04 o.o 3 o.04
-1.28 4- o . o i 1.28 4- o . o i 1.6o 4- o . o i
2.39 1.59 1.59 2.6o
o.oi o.oi o.oi o.o 3
-1.31 4- o . o i 1.31 4- o . o i 1.69 4- o . o i
-1.65 4- o . o i 1.66 4- o . o i 2.88 4- o . o i
4.Ol 4- o.04
1.61 + o . o i
2.60 ± 0.02
1.69 ± o . o i
2.87 ± o.o2
444i
44:¢4-
* I t r e m a i n s t o b e e s t a b l i s h e d w h e t h e r t h i s p e a k c o r r e s p o n d s t o p e a k 3 of t h e m e t h y l e s t e r a c e t a t e s on 3% QF-I.
~I>"'7',
~
26~i
euc >
371
OOCH3 H3
301
: 2sl
1
,~L~,I..... ~0
,.
150
200
250
m/e
~,,j . .
. .
i, ..........
J......
300
i,,
261
100
75c
-
50-
I
307
;
7"2s.
) ~
tllJL [ 1
o,l,hthL, 150
200
2~o
m/e
360
3go
"
~6o
~so
s00
F i g . 2. M a s s s p e c t r u m of t h e m e t h y l e s t e r k e t o n e of a s a m p l e of c r y s t a l l i n e t r i h y d r o x y c o p r o s t a n i c a c i d p r e p a r e d f r o m t h e b i l e of Alligator missisipiensis (a). T h e m a s s s p e c t r u m w a s o b t a i n e d b y u s i n g t h e d i r e c t i n s e r t i o n s y s t e m of t h e m a s s s p e c t r o m e t e r . (b) g i v e s t h e m a s s s p e c t r u m of t h e m e t h y l e s t e r k e t o n e of P e a k 5 f r o m t h e d u o d e n a l f l u i d of p a t i e n t No. 2. T h i s s p e c t r u m w a s r e c o r d e d via a g a s - l i q u i d 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 c o m b i n a t i o n t e c h n i q u e . P e a k s a t role 3 5 5 a n d 2 8 i a r e d u e t o t h e b a c k g r o u n d of t h e g a s c h r o m a t o g r a p h i c s y s t e m .
Peak 5 had identical m/e values to those derived from cholic acid. On the other hand, the molecular ions and fragment ions derived from them by loss of water (ketones) or acetic acid (acetates) indicated a difference of 42 mass units (C3H6) between cholic Biochim. Biophys. Acta, 273 (1972) 2 1 2 - 2 2 1
220
H. EYSSEN et al.
acid and the substance in Peak 5. In particular, the spectrum of the triketone methyl ester from cholic acid showed a loss of a --CH2--COOCH3 radical (73 mass units) from the side chain. A corresponding loss of 87 mass units in the spectrum obtained from Peak 5 showed that a carboxyl group was present in the C-26 or C-27 position, and indicated the presence of a methyl group in the a-position of the carboxyl group. These data were compatible with a C27 bile acid such as trihydroxycoprostanic acid. Further evidence for this was provided by the observation that the mass spectrum of the triketone methyl ester derived from authentic trihydroxycoprostanic acid was identical to that obtained from Peak 5 (Fig. 2). DISCUSSION
Several studies indicate that trihydroxycoprostanic acid is a natural intermediate in the synthesis of cholic acid in man. Small amounts of trihydroxycopros tanic acid have been crystallized from human fistula bile 8. Moreover, injection of C14-1abeled cholesterol gives rise to a radioactive fraction in bile, that crystallizes to constant specific activity with added trihydroxycoprostanic acid 7,9, and that upon reinjection is easily converted into cholic acid 9. The present investigation demonstrates that trihydroxycoprostanic acid is a major bile acid in the duodenal fluid of certain patients with anomalies of the intrahepatic bile ducts. In two patients, trihydroxycoprostanic acid accounted for 19 and 45%, respectively, of the total bile acids in the duodenal fluid. The relative amounts of chenodeoxycholic acid were 23% and 18%, and those of cholic acid were 58o/0 and 37%, in Patients I and 2, respectively. Bladder bile was not available for analysis, but there is no obvious reason to suspect that the pattern of the bile acids in the bladder bile would be significantly different. In addition to chenodeoxycholic acid, cholic acid and trihydroxycoprostanic acid, trace amounts of several other compounds were detected by gas liquid chromatography. Although final proof is lacking, one of these smaller peaks could have been dihydroxycoprostanic acid. It has been established that the steroid nucleus of cholesterol is first hydroxylated before the side chain is oxidized to yield trihydroxycoprostanic acid 15,16. Finally, the terminal isopropyl fragment of the side chain is split off as propionyl-CoA, thus yielding the C24 bile acids 5. The latter reaction is assumed to be a function of the mitochondria6,17. Obviously, the excretion of trihydroxycoprostanic acid with the bile of Patients i and 2 resulted from a reduced rate of degradation of the side chain. The cause of the impaired function of the hepatocytes remains to be established. Patient I was a case of partial atresia of the interlobular bile ducts, whereas Patient 2 had a Zellweger-like syndrome. Although the clinical features were not identical, cholestasis and congenital stigmata were common to both cases. As no trihydroxycoprostanic acid was found in three other cases of cholestasis, it seems unlikely that cholestasis by itself was the primary cause of the inhibition of the side chain oxidase system. The metabolic block could result from a gene defect, from a delayed maturation process, from specific inhibition of the side chain oxidase system, or from a more generalized damage to the mitochondria, the natural site of the conversion of trihydroxycoprostanic acid into cholic acid. A primary alteration of the cell membrane, which would result in premature leakage of trihydroxyeoprostanic acid out of the cytoplasm, has to be considered as an alternative hypothesis. Biochim. Biophys. Acta, 273 (I972) 2 1 2 - 2 2 i
TRIHYDROXYCOPROSTANIC ACID IN DUODENAL FLUID
22r
The present observation still raises several unanswered questions. The fact that the "normal" bile acids, cholic acid and chenodeoxycholic acid, were still formed would suggest that either an alternative pathway was operating, or that the metabolic block was incomplete. I t has been demonstrated that bishomocholic acid and scymnol, for instance, could be precursors of cholic acid18, TM. Although this pathway is assumed to be quantitatively unimportant in healthy subjects, it cannot be excluded that a similar mechanism was operating in Patients I and 2. It also remains to be established whether the occurrence of trihydroxycoprostanic acid could have contributed to the cholestasis and the atresia of thc intrahepatic bile ducts. Monohydroxy bile salts, such as lithocholic acid, are known to alter the hepatic structure and function2°, ~1. Administration of taurolithocholate, for instance, produces the electron microscopic and functional picture of cholestasis 2~. So far, no information is available on possible hepatoxic effects of the C27 bile acids in man. The present study demonstrates that formation of excessive amounts of trihydroxycoprostanic acid can be a feature of some cases of cholestasis. However, more cases will have to be studied to elucidate the mechanism of the incomplete conversion of trihydroxycoprostanic acid to cholic acid, and the possible role of the C~7 bile acids in the etiology of cholestasis and atresia of the intrahepatic bile ducts. ACKNOWLEDGMENTS
The authors wish to thank Miss R. Massonet and Mr J. Mertens for skilful technical assistance. Bile from Alligator missisipiensis was provided by Professor J. Mortelmans, Institute for Tropical Medicine, Antwerp. This work was supported by a grant from the "Fonds voor Collectief Fundamenteel Onderzoek", Contract No. 43 of the "Onderling 0verlegde Acties". REFERENCES I S. B e r g s t r 6 m , H. Danielsson a n d 13. S a m u e l s o n , in K. Bloch, Lipid Metabolism, Formation and Metabolism of Bile Acids, Wiley, N e w York, 196o, p. 291. 2 G. A. D. Haslewood, Physiol. Rev., 35 (1955) 178. 3 G. A. D. Haslewood, Biochem. J., 52 (1952) 583 . 4 Th. Briggs, M. W. W h i t e h o u s e a n d E. Staple, Arch. Biochem., 85 (1959) 275. 5 R. J. B r i d g e w a t e r a n d S. L i n d s t e d t , Acta Chem. Scand., i i (1957) 4o9. 6 Th. Briggs, M. W. W h i t e h o u s e a n d E. Staple, J. Biol. Chem., 236 (1961) 688. 7 E. Staple a n d J. L. R a b i n o w i t z , Biochim. Biophys. Acta, 59 (1962) 735. 8 J. 13. Carey, J r a n d G. A. D. Haslewood, J. Biol. Chem., 238 (1963) 855. 9 J. B. Carey, Jr., J. Clin. Invest., 43 (1964) 1443. IO R. F. H a n s o n a n d G. Williams, Biochem. J., 121 (1971) 863. 11 E. E v r a r d a n d G. J a n s s e n , J. Lipid Res., 9 (1968) 226. 12 U. M a k i t a a n d W. W. Wels, Anal. Biochem., 5 (1963) 523. 13 J. M. Opitz, G . M . zu Rhein, L. Vitale, N. T. Shahidi, J. J. Howe, S. M. Ghou, D. R. Shanklin, H. D. Sybers, A. R. Dood a n d T. Gerritsen, Birth Defects. Original Article Series, 5 (1969) 144. 14 H. Egger, Monatsch. Chem., 99 (1968) 1163. 15 S. 13ergstr6m, I n G. E. W. W o l s t e n h o l m e a n d M. O'Connor, CibaFound. Syrup. Synthesis of Terpenes and Sterols, Bile Acids, Formation and Metabolism, Little Brown, Boston, Mass., 1959, p. 185. 16 H. Danielsson, Advan. Lipid Res., i (1963) 335. 17 H. M. Suld, E. Staple a n d S. Gurin, J. Biol. Chem., 237 (1962) 338. 18 S. L i n d s t e d t a n d N. T r y d i n g , Arhiv Kemi, i i (1957) 137. 19 H. Danielsson, A. Kallner, R. J. Bridgewater, Th. Briggs a n d G. A. D. Haslewood, Acta Chem. Scand., 16 (1962) 1765. 20 G. A. Leveille, H. E. Sauberlich a n d R. D. H u n t , Poultry Sci., 41 (1962) 199121 H. E y s s e n , M. V a n d e p u t t e a n d E. E v r a r d , Arch. Int. Pharmacodyn., 158 (1965) 292. 22 F. Schaffner a n d N. 13. J a v i t t , Lab. Invest., 15 (1966) 1783.
Biochim. Biophys. Acta, 273 (1972) 212-221
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 26864
T R I H Y D R O X Y C O P R O S T A N I C ACID IN T H E D U O D E N A L F L U I D OF TWO C H I L D R E N W I T H I N T R A H E P A T I C B I L E DUCT ANOMALIES
H. E Y S S E N , G. P A R M E N T 1 E R ,
F. C O M P E R N O L L E , J. B O O N * AND E. E G G E R M O N T * *
The Rega Institute, University of Leuven, 3000 Leuven (Belgium) (Received J a n u a r y I7th, 1972)
SUMMARY
Using thin-layer chromatography, gas-liquid chromatography and mass spectrometry, trihydroxycoprostanic acid (3~,7~,I2~-trihydroxy-5fl-cholestan-26-oic acid) was identified in the duodenal fluid of 2 subjects with anomalies of the intrahepatic bile ducts. Subject I was a case of intrahepatic cholestasis due to atresia of the interlobular bile ducts with familial incidence. The bile acid spectrum in the duodenal fluid of this patient was: 23 % chenodeoxycholic acid, 58% cholic acid and 190/0 trihydroxycoprostanic acid. Subject 2 had a Zellwegerdike syndrome with cholestasis and with scarcely developed intrahepatic bile ductuli. The bile acid spectrum in the duodenal fluid of this patient was: 18% chenodeoxycholic acid, 37% cholic acid and 45% trihydroxycoprostanic acid. No trihydroxycoprostanic acid was found in seven healthy subjects, in three cases of cholestasis of infancy, or in ten cases of various disorders of the small intestine. Obviously, the excretion of trihydroxycoprostanic acid with the bile of Patients I and 2 was due to a reduced capacity of the hepatocvtes to split off the 3 terminal carbon atoms of the side chain of trihydroxycoprostanic acid. The cause of the impaired function of the hepatocytes remains to be established.
INTRODUCTION
Bile acids are synthesized in the liver by hydrogenation and hydroxylation of the steroid nucleus of cholesterol, followed by oxidation of the side chain 1. In certain lower vertebrates, the side chain of cholesterol is oxidized but not degraded, thus giving rise to the primitive C27 bile alcohols or C2~ bile acids ~. Dihydroxycoprostanic and trihydroxycoprostanic acid, for instance, are the major bile acids in the alligator3, 4. In more evolved animals, the side chain is oxidized with removal of the terminal * D e p a r t m e n t of Pediatrics, S a i n t R a d b o u d Hospital, U n i v e r s i t y of Nijmegen, T h e N e t h e r l a n d s . ** D e p a r t m e n t of Pediatrics, S a i n t Rapha6I Hospital, U n i v e r s i t y of L e u v e n , Belgium. * C o m m o n a n d s y s t e m a t i c n a m e s of c o m p o u n d s m e n t i o n e d in t h e t e x t are as follows : t r i h y d r o x y c o p r o s t a n i c acid: 3~,7~,I2~-trihydroxy-5fl-cholestan-26-oic acid; d i h y d r o x y c o p r o s t a n i c acid: 3~,7~,-dihydroxy-5~-cholestan-26-oic acid; lithocholic acid: 3~-hydroxy-5fl-cholanoic acid; chenodeoxycholic acid : 3~,7~-dihydroxy-5fl-cholanoic acid ; deoxycholic acid : 3~, 12~-dihydroxy5fl-cholanoic acid; cholic acid; 3 a , 7 ~ , I 2 ~ - t r i h y d r o x y - 5 f l - c h o l a n o i c acid.
Biochim. Biophys. Aeta, 273 (1972) 212 221
TRIHYDROXYCOPROSTANIC
ACID IN DUODENAL FLUID
213
isopropyl fragment to yield the "modern" Cu bile acids such as cholic acid and chenodeoxycholic acid which are the predominant primary bile acids in human bile. Several investigations support the hypothesis that the reaction sequence leading to synthesis of cholic acid in mammals would reflect the evolutionary past, and that dihydroxy- and trihydroxycoprostanic acid are short-lived natural intermediates in the synthesis of chenodeoxy- and cholic acid, respectively. In rats, trihydroxycoprostanic acid is readily converted to cholic acidS, e. Small amounts of dihydroxy- and trihydroxycoprostanic acid have been found in human fistula bile 7-1°. However, in healthy subjects with an uninterrupted entero-hepatic cycle, the concentration of the coprostanic acids is too small to be detected by routine analysis of bile or duodenal fluid. In the present study, we identified trihydroxycoprostanic acid as a major bile acid in the duodenal fluid of two children with anomalies of the intrahepatic bile ducts. MATERIALS AND METHODS
Preparation of bile acid derivatives Duodenal fluid, obtained by aspiration, was centrifuged and o.2-ml portions of the clear supernatant were distributed into 16 m m × 16o m m test tubes. For quantitative studies, an internal standard of 23-nordeoxycholic acid was added at this stage. The sample was hydrolyzed in I ml 25~/o K O H in ethylene glycol at 215 °C for 15 rain as described by Evrard and Janssen n. After cooling, I ml water and I ml methanol were added and the neutral sterols were removed by three extractions with 5 ml light petroleum (b.p. 40-60 °C). The aqueous layer was then acidified to p H 2 with 6 M HC1 and extracted with diethyl ether (3 × 5 ml). The solvent phase, containing the crude bile acid mixture, was washed once with 5 ml 20°/0 NaC1, evaporated under reduced pressure, dissolved in IO ml methanol~liethyl ether (I : 9, by vol.) and methylated with diazomethane for IO min at room temperature. The methyl ester acetates were prepared by acetylation of the methyl esters with i ml of a solution of 5 ml acetic acid, 5 ml acetic anhydride and one drop of perchloric acid. After the reaction mixture had acetylated for I8 h at room temperature, 8 ml of 20% NaC1 were added, and the methyl ester acetates were extracted with diethyl ether (3 × 5 ml). The ether layers were combined, evaporated, taken up in o.5-1 ml of acetone, and chromatographed. Ketonic esters of the bile acids were prepared according to the method of Evrard and Janssen n by mild oxidation of the methyl esters with i ml of 90% acetic acid and 0.2 ml of 20% CrOs in acetic acid. After 15 min at room temperature, I ml of lO°/0 ascorbic acid and 9 ml of 20% NaC1 were added, and the ketonic esters were extracted with diethyl ether (3 × 5 ml). The combined extracts were evaporated to dryness, and the residue was dissolved in o.5-1 ml of acetone and chromatographed. Trimethylsilyl ethers were prepared by reacting the free bile acids with hexamethyldisilazane and trimethylchlorosilane in pyridine, under dry conditions, at room temperature for 30 min 1~.
Gas-liquid chromatography Gas-liquid chromatography was carried out on a Pye series lO 4 instrument Biochim. Biophys. Acta, 2 7 3 (1972) 2 1 2 - 2 2 1
214
I~. EYSSEN et al.
(Pye-Unicam, Cambridge, U.K.), with a flame-ionisation detector and with nitrogen as the carrier gas. For identification studies, the retention times of the respective peaks were compared to those of reference compounds, using several types of stationary phases: a 7-ft column packed with I % OV-I on 8O-lOO mesh Supelcoport (Supelco, Inc., Bellefonte, Pa., U.S.A.), and a 5-ft column packed with 3% QF-I on lOO-12o mesh Gas Chrom Q (Applied Science Lab. Inc., State College, Pa., U.S.A.). The temperature of the columns was kept at 252 °C. The temperatures of flash heater and detector were 270-280 °C and 250 °C, respectively. Chromatography on I °/o J X R (Applied Science Lab., State College, Pa., U.S.A.) was carried out with a temperatureprogrammed instrument provided with an argon-ionisation detector. The temperature was raised from 18o to 260 °C in 12 rain. For quantitative determinations, the methyl ester acetates were chromatographed on a column of 3% QF-I or I % OV-I with the flame-ionisation detection system. Although in this system the ionisation responses of the dihydroxy and trihydroxy bile salts are directly proportional to the actual weight of the unsubstituted parent compounds, the concentration of each bile salt was calculated from an individual standard curve. The curves were computed from the ionisation responses of a set of references (5 concentrations for each compound), run daily before and after chromatography of the samples to be quantitated.
Mass spectrometry Mass spectra of reference compounds were recorded by using the direct insertion system of an AEI-MSI2 mass spectrometer (AEI, Manchester, U.K.) at an ionsource temperature of 12o 14o °C. Mass spectra of the bile acids in the duodenal fluid of patients were run via a gas-liquid chromatography-mass spectrometry combination technique. The temperature of membrane separator (V-562o Molecular Separator, Varian, Palo Alto, Calif., U.S.A.) and ion source was maintained at 220 25o °C. Gas liquid chromatography was carried out on a Pye series lO4 apparatus (Pye-Unicam, Cambridge, Great Britain) with helium as the carrier gas.
Preparatios of trihydroxycoprostanic acid from alligator bile IO ml bile from Alligator missisipiensis were diluted with 5 vol. of ethanol. The precipitate was filtered, the filtrate was evaporated to dryness, and the residue was hydrolyzed in IOO ml of 25% K O H in ethylene glycol for 25 rain at 220 °C. An equal volume of 2o°/~ NaC1 and methanol was added, and the neutral sterols were extracted with light petroleum (3 × 300 ml). The water layer was acidified to pH 1 with conc. HC1, and the crude bile acids were extracted with diethyl ether (4 × IOO nil). The ether layers were combined, washed with water, dried over Na~S04 and evaporated to dryness. The residue was methylated with diazomethane for 15 rain at room temperature in methanol-diethyl ether (i : 9, by vol.). On thin-layer chromatography in chloroform-methanol-acetone (14:1:5, by vol.), the methyl ester fraction showed 6 spots with phosphomolybdic acid reagent. The mixture of the methyl esters was separated by column chromatography on 58 g silica gel and stepwise elution with chloroform, 500 ml; chloroform-acetone (9 :i, 8:2 and 7:3, by vol.), 500 ml of each; chloroform acetone methanol (7° :25:5, by vol.), 500 ml. Fractions of 25 ml were collected, and each fraction was examined by thin-layer chromatography.
Biochim. Biophys. Acta, 273 (1972) 212 221
TRIHYDROXYCOPROSTANIC
ACID IN DUODENAL FLUID
215
Two major compounds were isolated and characterized by gas-liquid chromatography, thin-layer chromatography and mass spectrometry. Compound I was identified as the methyl ester of 3a,7x, I2a-trihydroxy-5fl-cholestan-26-oic acid (trihydroxycoprostanic acid). On recrystallization from diethyl ether-light petroleum (b.p. 60-80 °C) this compound yielded 19o mg of white needles with a melting point of 155156 °C, which corresponds to the figures (m.p. 153-155 °C) given by Haslewood 3. Compound n was identified as the methyl ester of 3a,7a-dihydroxy-5fl-cholestan26-oic acid (dihydroxycoprostanic acid). The fractions containing the major amounts of dihydroxycoprostanic acid were combined and yielded 75 mg of an amorphous powder which was contaminated with trace amounts (less than I °/o) of an unidentified substance. Case histories Case I. This male infant was born at term after a normal pregnancy. Two
other sisters were normal but one older brother died at the age of 4 months from obstructive jaundice due to cholestasis and atresia of the intrahepatic bile ducts. The patients had no malformations but a number of congenital stigmata: simian crease on the left hand, bilateral inner epicantal fold, frontal bossing and nystagmus. He had obstructive jaundice from the age of 2.5 months up Lill 4 months. Histological examination of a liver biopsy specimen suggested that the obstructive jaundice was due to cholestasis and partial atresia of the interlobular bile ducts. The patient died at the age of 6 months from pneumonia. The clinical and pathological data on this patient and his brother will be described in more detail elsewhere*. Case 2. This female infant was born at term by cesarean section. The pregnancy was complicated b y vaginal bleeding during the first trimester. The patient was the first child of unrelated parents. She was first seen at the age of 3 months for diarrhoea and jaundice. She measured 58 cm (p25), and weighed 3820 g ( < P 3 ) and had a number of congenital anomalies: clump feet, frontal bossing, broad metopic suture, third fontanel, bilateral epicanthal fold as well as simian creases. The histological examination of a liver biopsy specimen suggested that the jaundice was due to cholestasis. Ductular structures were rather scarse. The icterus disappeared at the age of 5 months. However, the child failed to thrive and was mentally retarded. She died at the age of 8.5 months. At that time she weighed only 405 ° g (
Bile acids in the duodenal fluid
The results of the bile salt determinations are shown in Table I. Variability of total bile acid concentration was high, as could be expected from determinations on duodenal fluid. However, in six out of seven healthy subjects, all values ranged * J. Boon, J. Bakkeren, J. Miser6, E. Schretlen, G. Parmentier and H. Eyssen, submitted for publication. ** E. Eggermont, J. P. Frijns, B. Van Damme and H. Eyssen, unpublished. Biochim. Biophys. Acta, 273 (1972) 212-22i
1,)
~o
a
3.5months
2
3months 4months 7months 7 months 15 m o n t h s 3.5 y e a r s 5 years 2 months 3months 3.5 m o n t h s 3months i2months 6months 8months
7 months
16 m o n t h s
2 years 2 years 6 years 3.5 y e a r s
3 4 5 6 7 8 9 IO ii 12 13 14 15 16
I7
18
19 20 21 22
Controls
4.5 m o n t h s
Age
I
Patients
Subjects
F F F M
F
M
M IV[ M F M M M F M F M F M M
F
M
Sex
Normal Normal Normal Normal Normal Normal Normal Cholestatic icterus Cholestatic ieterus Cholestatic icterus Cow milk protein intolerance Cow milk protein intolerance Tropical sprue Malabsorption due to chronic intestinal infection Contaminated small bowel syndrome Transitory pancreatic insufficiency Congenital lymphedema Coeliac disease Coeliac disease Hirschsprung's disease
A t r e s i a of t h e i n t r a h e p a t i c bile ducts Zellweger-like syndrome
Diagnosis
t 15 ~25 899 975
166
152
251 424 193 54 131 240 158 19 ii 126 364 lO6 798 373
61
26
o o o Tr
o
o
o o o o o Tr Tr o o o o o o o
o
o
28 61 53 27
28
56
51 39 27 34 59 28 39 51 20 32 33 29 22 12
18
23
Bile salts in the duodenal fluid Total amount Relative concentration ( % ) (mg/Ioo ml) Lithocholic Chenodeoxyacid cholic acid
21 o 14 15
14
o
o Tr 6 o Tr 22 14 o o o o 5 o o
o
o
Deoxycholic acid
51 39 33 58
58
44
49 61 67 66 4I 50 46 49 8o 68 69 66 78 88
37
58
Cholic acid
o o o o
o
o
o o o o o o o o u o o o o o
45
19
Trihydroxycoprostanic acid
T h e b i l e s a l t s in t h e d u o d e n a l f l u i d w e r e d e t e r m i n e d b y g a s - l i q u i d c h r o m a t o g r a p h y o f t h e m e t h y l e s t e r a c e t a t e s o n a c o l u m n o f 3 % Q F - I , a t a t e m p e r a t u r e of 252°C, u s i n g 2 3 - n o r d e o x y c h o l i c a c i d m e t h y l e s t e r a c e t a t e a s a n i n t e r n a l s t a n d a r d . W h e n a s a m p l e w a s a n a l y s e d i n i o - f o l d , t h e s t a n d a r d d e v i a t i o n f o r t o t a l b i l e s a l t s w a s ± 3 . 5 1 % ; t h e s t a n d a r d d e v i a t i o n f o r t h e i n d i v i d u a l b i l e s a l t s r a n g e d f r o m ~: 2 . 5 % f o r c h e n o d e o x y c h o l i c a c i d t o :[_ 4.2 % f o r c h o l i c a c i d . T r m e a n s t r a c e a m o u n t s , i.e. less t h a n 2 % of t o t a l b i l e a c i d s .
FLUID
~"
I
BILE SALTS IN THE DUODENAL
TABLE
~'
o~
tO
217
TRIHYDROXYCOPROSTANIC ACID IN DUODENAL FLUID OAc
® AcO..~
cOOCH~
~
AcO
laJ U3 Z
AC.~.OA¢
OOCH3 COOCH3
AcO*"
AcO
O
COOCH3 CH3
¢H3
n t,.3 LLI n-
AcO'" 5
\ o
2J0
1~o
3~0
M IN
410
50
@
I
2
5
O (9 LO LU O
lO
2i0
30
MIN
Fig. I. Gas-liquid chromatography of the methyl ester acetates of reference bile acids (a) and of the bile acids in the duodenal fluid from patient No. 2 (b). Stationary phase: 3% QF-I at 252 °C. I, internal standard (23-nordeoxycholic acid); 2, chenodeoxycholic acid; 3, dihydroxycoprostanic acid; 4, cholic acid; 5, trihydroxycoprostanic acid.
Biochim. Biophys. Acta, 273 (1972) 212-221
218
I~. EYSSEN et a/~.
between 131 and 424 mg/Ioo ml, the average being 233 mg/Ioo ml. On the other hand, Case I and Case 2 had subnormal amounts of bile acids in the duodenal fluid (26 and 61 mg/ioo ml, respectively). It should be mentioned that these samples were obtained during the remission period following the obstructive jaundice. Low concentrations of bile acids were also found in three patients with cholestasis (ii, 19 and 126 mg/Ioo ml, respectively). In ten patients with various disorders of the small intestine, the bile acid concentration in the duodenum was within the normal limits. As shown in Table I, cholic acid and chenodeoxycholie acid were the major bile acids in infants and children. No, or only in trace amounts, lithocholic acid was found. Deoxycholic acid was not present in the younger infants, but reached relative concentrations of 15-2o% in most of the children above the age of 15 months. The most striking difference in the bile acid pattern was the presence in Patients I and 2 of a peak which was not found in any of the healthy controls or in the patients with cholestasis or various disorders of the small intestine (Peak 5, Fig. I). This peak accounted for 19 and 45% of the total bile acids in Cases I and 2, respectively. This bile acid eluted from the columns well after cholic acid and was different from cholic acid, chenodeoxyeholic acid and the secondary bile salts deoxycholic acid and lithocholic acid. In addition to the three major peaks, trace amounts of at least four other compounds were detected. These peaks were too small to be identified. However, on both the QF-I and the OV-I stationary phases, one of them (Peak 3, Fig. I) had the same relative retention time as dihydroxycoprostanic acid prepared from alligator bile. The peak was not detected in any of the control subjects.
Identification of trihydroxycoprosta~ic acid On gas-liquid and thin-layer chromatography, the relative retention times and the RF values of the methyl ester acetate and the methyl ester ketone of Peak 5 corresponded to those of authentic trihydroxycoprostanic acid (Table II and Fig. I). Peaks 2 and 4 corresponded to chenodeoxycholic and eholie acid, respectively. The identity of these peaks was further confirmed by mass spectrometry. A detailed study of the mass spectra of bile acids has been published by Egger 14. By comparison with reference compounds, Peaks 2 and 4 could be easily identified as ehenodeoxycholic and eholic acid, respectively. When the mass spectrum of Peak 5 was recorded as the methyl ester acetate, a peak of the molecular ion (role 59 o) was not found, because acetic acid was too easily eliminated from the molecular ion. However, other characteristic fragment ions were recorded at rn/e 530 (M--HOAc), role 499 (M--HOAc-OCH3), role 488 (M--HOAc CH2=CO), role 470 (M--2HOAc), role 41o (M--3HOAc), role 313 ( M - s i d e chain-2HOAc), role 253 (M--side chain-3HOAc). When the methyl ester ketones were subjected to mass spectrometry, the spectrum showed a peak of the molecular ion at role 458 (Fig. 2). Other characteristic ion fragments were found at role 440 (M--H~O), m/e 427 (M--OCH3), role 371 (M--CH3-'CHCOOCH3), role 3oi (M--side chain), role 283 (M--side chain-H~O), role 261 (fragmentation of the D-ring). Compared to cholic acid, the compound giving rise to Peak 5 had an identical substitution pattern for the steroid nucleus, but a side chain differing by presence of 3 additional carbon atoms. This conclusion was based on the observation that, for both the ketones and the. acetates, the fragment ions corresponding to loss of the side chain and to ring I~ fragmentation of the unknown compound in Biochim. Biophys. Acta, 273 (I972) 212-221
219
T R I H Y D R O X Y C O P R O S T A N I C ACID IN D U O D E N A L F L U I D TABLE
II
GAS--LIQUID OF THE
CHROMATOGRAPHY
BILE ACIDS IN THE
OF THE
DUODENAL
METHYL
FLUID
ESTER
ACETATES
OF PATIENT
NO.
AND
METHYL
ESTER
KETONES
2
R e s u l t s a r e g i v e n as a v e r a g e of i o d e t e r m i n a t i o n s 4- s t a n d a r d e r r o r of m e a n .
Relative retention times compared to 23-nordeoxycholic acid Methyl ester ketones on Methyl ester acetates on 3% QF-z x% JXR z % OV-z z% JXR z % OV-z 2 3-Nordeoxycholic acid Peak 2 Chenodeoxycholic acid Peak 3 Dihydroxycoprostanic acid Peak 4 Cholic acid Peak 5 Trihydroxycoprostanic acid
i.oo 1.51 4- 0.02 1.51 4- o . o i 2.13 4- o . o i
I.OO 1.23 4- O.Ol 1.23 4- o . o i --
I.OO 1.46 4- o . o i 1.45 4- o . o i (2.40 4- 0.03)*
I.OO 1.13 4- o . o i 1.13 ± o . o i --
I.OO 1.24 4- o . o i 1.23 4- o . o i --
2.13 2.75 2.76 4.00
0.02 o.04 o.o 3 o.04
-1.28 4- o . o i 1.28 4- o . o i 1.6o 4- o . o i
2.39 1.59 1.59 2.6o
o.oi o.oi o.oi o.o 3
-1.31 4- o . o i 1.31 4- o . o i 1.69 4- o . o i
-1.65 4- o . o i 1.66 4- o . o i 2.88 4- o . o i
4.Ol 4- o.04
1.61 + o . o i
2.60 ± 0.02
1.69 ± o . o i
2.87 ± o.o2
444i
44:¢4-
* I t r e m a i n s t o b e e s t a b l i s h e d w h e t h e r t h i s p e a k c o r r e s p o n d s t o p e a k 3 of t h e m e t h y l e s t e r a c e t a t e s on 3% QF-I.
~I>"'7',
~
26~i
euc >
371
OOCH3 H3
301
: 2sl
1
,~L~,I..... ~0
,.
150
200
250
m/e
~,,j . .
. .
i, ..........
J......
300
i,,
261
100
75c
-
50-
I
307
;
7"2s.
) ~
tllJL [ 1
o,l,hthL, 150
200
2~o
m/e
360
3go
"
~6o
~so
s00
F i g . 2. M a s s s p e c t r u m of t h e m e t h y l e s t e r k e t o n e of a s a m p l e of c r y s t a l l i n e t r i h y d r o x y c o p r o s t a n i c a c i d p r e p a r e d f r o m t h e b i l e of Alligator missisipiensis (a). T h e m a s s s p e c t r u m w a s o b t a i n e d b y u s i n g t h e d i r e c t i n s e r t i o n s y s t e m of t h e m a s s s p e c t r o m e t e r . (b) g i v e s t h e m a s s s p e c t r u m of t h e m e t h y l e s t e r k e t o n e of P e a k 5 f r o m t h e d u o d e n a l f l u i d of p a t i e n t No. 2. T h i s s p e c t r u m w a s r e c o r d e d via a g a s - l i q u i d 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 c o m b i n a t i o n t e c h n i q u e . P e a k s a t role 3 5 5 a n d 2 8 i a r e d u e t o t h e b a c k g r o u n d of t h e g a s c h r o m a t o g r a p h i c s y s t e m .
Peak 5 had identical m/e values to those derived from cholic acid. On the other hand, the molecular ions and fragment ions derived from them by loss of water (ketones) or acetic acid (acetates) indicated a difference of 42 mass units (C3H6) between cholic Biochim. Biophys. Acta, 273 (1972) 2 1 2 - 2 2 1
220
H. EYSSEN et al.
acid and the substance in Peak 5. In particular, the spectrum of the triketone methyl ester from cholic acid showed a loss of a --CH2--COOCH3 radical (73 mass units) from the side chain. A corresponding loss of 87 mass units in the spectrum obtained from Peak 5 showed that a carboxyl group was present in the C-26 or C-27 position, and indicated the presence of a methyl group in the a-position of the carboxyl group. These data were compatible with a C27 bile acid such as trihydroxycoprostanic acid. Further evidence for this was provided by the observation that the mass spectrum of the triketone methyl ester derived from authentic trihydroxycoprostanic acid was identical to that obtained from Peak 5 (Fig. 2). DISCUSSION
Several studies indicate that trihydroxycoprostanic acid is a natural intermediate in the synthesis of cholic acid in man. Small amounts of trihydroxycopros tanic acid have been crystallized from human fistula bile 8. Moreover, injection of C14-1abeled cholesterol gives rise to a radioactive fraction in bile, that crystallizes to constant specific activity with added trihydroxycoprostanic acid 7,9, and that upon reinjection is easily converted into cholic acid 9. The present investigation demonstrates that trihydroxycoprostanic acid is a major bile acid in the duodenal fluid of certain patients with anomalies of the intrahepatic bile ducts. In two patients, trihydroxycoprostanic acid accounted for 19 and 45%, respectively, of the total bile acids in the duodenal fluid. The relative amounts of chenodeoxycholic acid were 23% and 18%, and those of cholic acid were 58o/0 and 37%, in Patients I and 2, respectively. Bladder bile was not available for analysis, but there is no obvious reason to suspect that the pattern of the bile acids in the bladder bile would be significantly different. In addition to chenodeoxycholic acid, cholic acid and trihydroxycoprostanic acid, trace amounts of several other compounds were detected by gas liquid chromatography. Although final proof is lacking, one of these smaller peaks could have been dihydroxycoprostanic acid. It has been established that the steroid nucleus of cholesterol is first hydroxylated before the side chain is oxidized to yield trihydroxycoprostanic acid 15,16. Finally, the terminal isopropyl fragment of the side chain is split off as propionyl-CoA, thus yielding the C24 bile acids 5. The latter reaction is assumed to be a function of the mitochondria6,17. Obviously, the excretion of trihydroxycoprostanic acid with the bile of Patients i and 2 resulted from a reduced rate of degradation of the side chain. The cause of the impaired function of the hepatocytes remains to be established. Patient I was a case of partial atresia of the interlobular bile ducts, whereas Patient 2 had a Zellweger-like syndrome. Although the clinical features were not identical, cholestasis and congenital stigmata were common to both cases. As no trihydroxycoprostanic acid was found in three other cases of cholestasis, it seems unlikely that cholestasis by itself was the primary cause of the inhibition of the side chain oxidase system. The metabolic block could result from a gene defect, from a delayed maturation process, from specific inhibition of the side chain oxidase system, or from a more generalized damage to the mitochondria, the natural site of the conversion of trihydroxycoprostanic acid into cholic acid. A primary alteration of the cell membrane, which would result in premature leakage of trihydroxyeoprostanic acid out of the cytoplasm, has to be considered as an alternative hypothesis. Biochim. Biophys. Acta, 273 (I972) 2 1 2 - 2 2 i
TRIHYDROXYCOPROSTANIC ACID IN DUODENAL FLUID
22r
The present observation still raises several unanswered questions. The fact that the "normal" bile acids, cholic acid and chenodeoxycholic acid, were still formed would suggest that either an alternative pathway was operating, or that the metabolic block was incomplete. I t has been demonstrated that bishomocholic acid and scymnol, for instance, could be precursors of cholic acid18, TM. Although this pathway is assumed to be quantitatively unimportant in healthy subjects, it cannot be excluded that a similar mechanism was operating in Patients I and 2. It also remains to be established whether the occurrence of trihydroxycoprostanic acid could have contributed to the cholestasis and the atresia of thc intrahepatic bile ducts. Monohydroxy bile salts, such as lithocholic acid, are known to alter the hepatic structure and function2°, ~1. Administration of taurolithocholate, for instance, produces the electron microscopic and functional picture of cholestasis 2~. So far, no information is available on possible hepatoxic effects of the C27 bile acids in man. The present study demonstrates that formation of excessive amounts of trihydroxycoprostanic acid can be a feature of some cases of cholestasis. However, more cases will have to be studied to elucidate the mechanism of the incomplete conversion of trihydroxycoprostanic acid to cholic acid, and the possible role of the C~7 bile acids in the etiology of cholestasis and atresia of the intrahepatic bile ducts. ACKNOWLEDGMENTS
The authors wish to thank Miss R. Massonet and Mr J. Mertens for skilful technical assistance. Bile from Alligator missisipiensis was provided by Professor J. Mortelmans, Institute for Tropical Medicine, Antwerp. This work was supported by a grant from the "Fonds voor Collectief Fundamenteel Onderzoek", Contract No. 43 of the "Onderling 0verlegde Acties". REFERENCES I S. B e r g s t r 6 m , H. Danielsson a n d 13. S a m u e l s o n , in K. Bloch, Lipid Metabolism, Formation and Metabolism of Bile Acids, Wiley, N e w York, 196o, p. 291. 2 G. A. D. Haslewood, Physiol. Rev., 35 (1955) 178. 3 G. A. D. Haslewood, Biochem. J., 52 (1952) 583 . 4 Th. Briggs, M. W. W h i t e h o u s e a n d E. Staple, Arch. Biochem., 85 (1959) 275. 5 R. J. B r i d g e w a t e r a n d S. L i n d s t e d t , Acta Chem. Scand., i i (1957) 4o9. 6 Th. Briggs, M. W. W h i t e h o u s e a n d E. Staple, J. Biol. Chem., 236 (1961) 688. 7 E. Staple a n d J. L. R a b i n o w i t z , Biochim. Biophys. Acta, 59 (1962) 735. 8 J. 13. Carey, J r a n d G. A. D. Haslewood, J. Biol. Chem., 238 (1963) 855. 9 J. B. Carey, Jr., J. Clin. Invest., 43 (1964) 1443. IO R. F. H a n s o n a n d G. Williams, Biochem. J., 121 (1971) 863. 11 E. E v r a r d a n d G. J a n s s e n , J. Lipid Res., 9 (1968) 226. 12 U. M a k i t a a n d W. W. Wels, Anal. Biochem., 5 (1963) 523. 13 J. M. Opitz, G . M . zu Rhein, L. Vitale, N. T. Shahidi, J. J. Howe, S. M. Ghou, D. R. Shanklin, H. D. Sybers, A. R. Dood a n d T. Gerritsen, Birth Defects. Original Article Series, 5 (1969) 144. 14 H. Egger, Monatsch. Chem., 99 (1968) 1163. 15 S. 13ergstr6m, I n G. E. W. W o l s t e n h o l m e a n d M. O'Connor, CibaFound. Syrup. Synthesis of Terpenes and Sterols, Bile Acids, Formation and Metabolism, Little Brown, Boston, Mass., 1959, p. 185. 16 H. Danielsson, Advan. Lipid Res., i (1963) 335. 17 H. M. Suld, E. Staple a n d S. Gurin, J. Biol. Chem., 237 (1962) 338. 18 S. L i n d s t e d t a n d N. T r y d i n g , Arhiv Kemi, i i (1957) 137. 19 H. Danielsson, A. Kallner, R. J. Bridgewater, Th. Briggs a n d G. A. D. Haslewood, Acta Chem. Scand., 16 (1962) 1765. 20 G. A. Leveille, H. E. Sauberlich a n d R. D. H u n t , Poultry Sci., 41 (1962) 199121 H. E y s s e n , M. V a n d e p u t t e a n d E. E v r a r d , Arch. Int. Pharmacodyn., 158 (1965) 292. 22 F. Schaffner a n d N. 13. J a v i t t , Lab. Invest., 15 (1966) 1783.
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