Europ.J.clin.1nvest. 6, 67-74 (1976)

Bile Salt Glucuronides: Identification and Quantitative Analysis in the Urine of Patients with Cholestasis W. Frohling and A . Stiehl Department of Medicine, Medizinische Universitatsklinik, Gastroenterology Unit, University of Heidelberg, Germany Received: February 20, 11975, and in revised form: July 18, 1975

Abstract. Glucuronides of lithocholate, chenodeoxycholate and cholate were synthesized enzymatically and characterized by thin layer chromatography, column chromatography and specific enzymatic hydrolysis. Bile salt glucuronides were quantitatively analysed in the urine of patients with intra- and extrahepatic cholestasis and were found to be present in 19 out of 20 patients studied. Our patients with intrahepatic cholestasis excreted 8.9 mg non-sulphated and non-glucuronidated bile salts, 18.2 mg sulphated bile salts and 7.2 mg glucuronidated bile salts. The patients with extrahepatic cholestasis excreted 14.7 mg non-sulphated and non-glucuronidated bile salts, 20.7 mg sulphated bile salts and 4.7 mg glucuronidated bile salts. These findings indicate that glucuronidation of bile salts occurs in man and represents a metabolic pathway in patients with cholestasis. Key words: Bile salts, glucuronides, sulphate esters, cholestasis, renal excretion.

Under normal conditions almost all the bile acid pool is found within the enterohepatic circulation. In cholestasis the excretion of bile salts into the bile is diminished, while elevated concentrations of bile acids are found in serum and tissues other than liver and intestine. In 1967 Palmer found sulphate esters of lithocholate in bile ( I ) . Subsequently sulphate esters of primary and secondary bile acids in serum, bile and urine were identified (2, 3 ) . Since many steroids (e.g. hormones and cholesterol) are glucuronidated, it seemed likely that bile acids can also be glucuronidated. The steroid nucleus with one, two or three reactive OH-groups and the side chain with a possibly reactive carboxyl-group make bile acids probable substrates of UDP-glucuronyltransferase. Therefore enzymatic synthesis of 14C-labelled bile salt glucuronides was used to investigate the characteristics of these substances. The synthetic glucuronides were used to identify bile aalt glucuronides excreted in the urine of patients with cholestasis. Quantitative analysis of bile salt glucuronides excreted in the urine was performed to investigate the importance of this new pathway in man.

Methods Mat er ia1s 24-14C-lithocholic acid ( I . 15 mCi/mol) was obtained from California Bionuclear Corporation,

Sun Val ley , California ; 24- I4C-chenodeoxycho1ic acid (57 mCi/mmol) from ICN, Irvine, California; 24-14C-cholic acid (52 mCi/mmol) and 14C-uridine.diphosphate-glucuronic acid ( 2 9 0 mCi/mmol) from Amersham R?diochemical Centre, Braunschweig; bovine liver 8-glucuronidase from Serva, Heidelberg; saccharolactone (glucaric acid-1, 4-lactone) from Sigma Chemical Company, St. Louis, Missouri and cholyl-glycine-hydrolase from Schwarz/Mann, Orangeburg, New York. Silica gel plates (Merck, Darmstadt) were used for thin layer chromatography and Sephadex LH-20 (Pharmacia, Frankfurt) for column chromatography. Other chemicals were analytical grade and were obtained from Merck, Darmstadt, Germany. Male Sprague-Dawley rats (150 g body weight) from Ivanovas, KiBlegg, were used for the preparation of microsomes. Patients Twenty patients with severe cholestasis and 5 healthy students were included in the sudy. Ten patients had intrahepatic cholestasb (8 alcoholic cirrhosis, 2 primary biliary cirrhosis) and ten had extrahepatic obstruction (7 had tumors and 3 choledochus stones). Serum bilirubin values ranged from 6.0 mg X to 21.5 mg %. Serum alkaline phosphatase ranged from 600 to 2100 mU/ml (upper limit of normal ZOO mU/ml). Serum transaminase va,lues ranged from 25 mU/ml to -280 mU/ml (serum glutamic oxaloacetic transaminase) and 30 mU/ml to 310 rnU/ml (serum glutamic

68

pyruvic transaminase). The duration of cholestasis ranged from 1 to 6 weeks. Three of the ten patients with cirrhosis had ascites. The diagnosis was based on physical signs and biochemical tests and was confirmed by laparoscopy or liver biopsy ox at autopsy. fieparstion of Microsones: Rat liver microSomes were prepared as described earlier (8) and were finally suspended in 50 mM Tris-HCI pH 6 . 5 . Microsoma1 protein was determined by the method of Lowry et aZ. ( 4 ) . EnzSmatic Synthesis of Bile Salt Ghcuronides: Ra: liver microsomes (5 mg microsomal protein) were incubated with I4C-labelled bile salts (lithocholate, chenodeoxycholate and cholate, 0.1 pCi/pmol, final concentration 1 m M ) , 3 mM uridine-diphospho-glucuronic acid (UDPGA), 5 mM IigC12 and 50 mM Tris-HC1-buffer pH 6 . 5 . Potassium salts (lithocholate) or sodium salts (chenodeoxycholate, cholate) of the free bile acids were used. Controls were incubated without UDPGA. Incubation was performed for 30 minutes in a shaking water bath. The incubation was stopped by adding 9 volumes of ethanol, the precipitated protein was washed 3 times with ethanol and the combined supernatants were dried in vacuo. Ether Eztraction: Protein was precipitated with trichloracetic acid, the protein-free supernatant was acidified to pH 1 with HC1 and extracted 3 times with 5 volumes of diethylether. The bile salt radioactivity remaining in the water phase was determined. Thin Layer Chromatography was performed on silica gel G plates with butanol: acetic acid: water (50:5:10) as solvent. The plates were dried and the silica gel scraped off and eluted with methanol. The radioactivity in the methanol eluates was determined. C o ~ Chromatography: m Bile salt glucuronides were prepared as described above and were chromatographed on 3 g Sephadex LH-20-col~~~ns equilibrated with chlorofom/methanol ( 1 :1 ) containing 0.01 M NaC1. Elution was performed with the same solvent system and the radioactivity in every 5 ml portion was determined. Petemination of Radioactiuity:The samples were counted in a Packard Tri Carb liquid scintillation counter using Bray's solution (5) as scintillation fluid. Quench correction was performed with an internal 4C-toluene standard. The recovery was 72 %. Identification of ?adioactive Bile Salt ;~uczcrQiidesir. MGX: Urine was collected in 24 hour specimens for 5 days after the intravenous administration of 15 UCi 14C-chenodeoxycholate to 2 patients with alcoholic cirrhosis. 5 ml portions of urine were chromatographed on Sephadex LH-20: l)untreated, 2) after hydrolysis o f glucuronides, 3) after treatment with Bglucuronidase, inhibited by the addition of saccharolactone and 4) after solvolysis of the suluhates. A n a l y s i s of Urine of Patients with Cholestasis: 5 ml of urine were chromatographed on 3 g Sephadex LH-20-columns. Free, taurine- and glycine-con-

W. Frohling and A . Stiehl: Bile Salt Glucuronides jugated bile salts were eluted with 40 ml chlorofodmethanol ( I : ] ) 'containing 0.01 M NaC1. The second fraction, containing glucuronides and sulphates, was eluted with 200 ml methanol. Enzymatic HydroZysis of Taurine- and GlycineConjugated Bile Salts: Cholyl-glycine-hydrolase was used according to Nair e t aZ. (6). The incubation was extended to 12 hours. Hydrolysis of GZucwonides:' Sulphate and glucuronide containing fractions were eluted from the Sephadex column and dried in vacuo. The fractions were then dissolved in 50 mM acetate buffer pH 4.5 and incubated with B-glucuronidase (1000 I.U. per ml initial urine volume) for 24 hours at 37OC in a shaking water bath. Two controls were performed. The first contained 5 mM saccharolactone, a specific 8-glucuronidaseinhibitor. The second control contained only 8-glucuronidase in acetate-buffer and enabled a correction for bile salt contamination (espe-

I

Lithocholate

3

rng

'*lI

6

9

,I2

microsomal protein

Chenodeoxycholate

lo

minutes

Fig. I . Enzymatic synthesis of bile salt glucuronides. 24-14C-lithocholate and 24-1 4C-chenodeoxycholate were incubated with UDPGA and microsomes. The amount of radioactivity which is not extractable into ether at pH I is dependent on the time of incubation and the amount of microsoma1 protein. No increase of non-extractable radioactivity was observed in controls without UDPGA

W. Fr6hling and A . Stiehl: Bile Salt Glucuronides cially cholate) in commercially available preparations of bovine liver 8-glucuronidase to be made. Hydrolysis of Sulphates: The solvolysis procedure described by Palmer e t aZ. (7) was used. Following hydrolysis of glucuronides, sulphates, taurine- and glycine-conjugates the free bile acids were extracted into ether at pH 1 and prepared for gas liquid chromatography.

69

Gas Liquid Chromatography: Trifluoracetate substituted methyl cholanates were prepared according to the method of Nair e t a l . (6). Gas liquid chromatography was performed using 3 % qF1 columns and 0.5 X HI Eff-8BP-columns in a HI? 1610 A instrument, which was equipped with a flame ionization detector. The recovery was determined by adding 24-14C-taurocholate and 24- C-t aurocho 1 ate sulphate as interna1 standards.

Fig. 2. Thin layer chromatography of 14C-chenodeoxycholate glucuronide. M

c --.

2

SEM is indicated

- CDC (n=10)

^ 7 +~C D C ( n = l O )

\ \

Fig. 3 . Thin layer chromatography of chenodeoxych01ate-~4C-glucuronide. UDPGA was 14C-labelled and chenodeoxycholate was unlabelled. M C SEM is indicated

W. Frahling and A. Stiehl: Bile Salt Glucuronides

70

can be found (Fig. 3) when 14C-UDP-glucuronic acid was used for labelling the glucuronide molecule, instead of 24-14C-chenodeoxycholate. Free 14C-UDPGA does not move in this solvent system and is found at the origin. No glucuronides are found in controls incubated without chenodeoxycholate. For further identification enzymatically synthesized lithocholate glucuronide was incubated with 8-glucuronidase for 24 hours (Table 1 ) . In control experiments a-glucuronidase was inhibited by the addition of 5 mM saccharolactone. Lithocholate glucuronide was chromatographed on thin layer plates and the radioactivity was measured. The glucuronide was hydrolysed by glucuronidase and the hydrolysis was inhibited by the specific R-glucuronidase-inhibitor saccharolactone (Table 1 ) . Column chromatography of lithocholate glucuronide on Sephadex

t?esuZts

After incubation of bile salts with microsomes and UDPGA an accumulation of polar bile salt metabolites was observed (Fig. 1 ) . An increasing amount of bile salt radioactivity, which is not extractable into ether at pH 1 , is found on increasing the time of incubation and the amount of microsomal protein. The synthesis of this metabolite is dependent on the presence of UDPGA and is not found in controls incubated without UDPGA. Thin layer chromatography of chenodeoxycholate glucuronide is shown in Fig. 2. In controls without UDPGA the whole bile salt radioactivity is found at the solvent front. In incubations with UDPGA the bile salt glucuronide forms a second peak with an RF of about 0.27. The same peak of chenodeoxycholate glucuroni.de

Table 1 . 8-Glucuronidase-treatment of lithocholate glucuronide procedure Procedure enzymatic synthesis + 8-glucuronidase

enzymatic synthesis

enzymatic synthesis + R-glucuronidase + saccharolactone

1ithocholat e glucuronide (n mol)

I 06a

84

10

i 10

avalues represent Mean

?

SEM

A

E l u t i o n volume ( m l ,

Fig. 4 . Column chromatography of lithocholate glucuronide on Sephadex LH-20. Free bile salts were eluted with 5 - 40 ml chloroform/methanol. Glucuronidated bile salts were eluted with a second peak between 40 and 85 ml chloroform/methanol. M ? SEM is indicated

W. FrBhling and A . Stiehl: Bile Salt Glucuronides is shown in Fig. 4 . In controls without UDPGA only the free bile salt is eluted between 5 and 40 ml. In incubations with UDPGA lithocholate glucuronide is eluted with a second peak between 40 and 85 ml. In two patients glucuronides were identified after intravenous administration of 14C-chenodeoxycholate. Following hydrolysis of taurineand glycine-conjugates the metabolites extracted from the urine had identical chromatographic properties as the synthesized bile salt glucuronides on thin layer and column chromatography. Enzymatic hydrolysis by 8-glucuronidase resulted in a substance with the chromatographic properties of chenodeoxycholate on thin layer and gas liquid chromatography. The hydrolysis by 8-glucuronidase was significantly inhibited by saccharolactone. Thus the metabolite extracted from the urine of these patients was identical with the chenodeoxycholate glucuronide synthesized enzymatically (Fig. 5 ) . Quantitative analysis of bile salts was performed in the urine of 20 patients with intraand extrahepatic cholestasis (Table 2 and 3 ) . For quantitative analysis the glucuronide containing fraction was hydrolysed by 8-glucuronidase and analysed by gas liquid chromatography. In control experiments performed with every single sample this reaction could be in-

71 hibited by saccharolactone, a specific 13-glucuronidase-inhibitor. A wide individual variation of glucuronidation as well as sulphation of bile salts was observed. Glucuronides were found in all but one patient (Patient No. 2, Table 2, intrahepatic cholestasis due to alcoholic cirrhosis), who excreted most of the bile salts as sulphate esters. In total, 16 % of the bile salts excreted in the urine were glucuronidated, 52 iZ were sulphated and 32 iZ were neither glucuronidated nor sulphated. Chenodeoxycholate and deoxycholate were excreted predominantly as sulphates and cholate predominantly in neither the glucuronidated nor the sulphated form. The amount of sulphates was correlated with the total amount of bile salts excreted, whereas no correlation could be found between glucuronides and total bile salts. Only trace amounts of bile salt glucuronides were found in the urine of healthy control persons.

Discussion Bile salt glucuronides were synthesized enzymatically. Rat liver microsomes were used to prepare UDP-glucuronyltransferase and 14Clabelled bile salts and UDPGA were used as sub-

.-

-

untreated hydrolysis o f g l u c u r o n i d e r ~ a l v a l y s i sof s u l f o t e r

h y d r o l y s i s of glucuronides r s o l r o l y s i s of sulfates

Elution volume ( m i )

Fig. 5 . Column chromatography of chenodeoxycholate glucuronide in hcman urine. Two patients with intrahepatic cholestasis due to alcoholic cirrhosis were treated with 15 pCi 14C-chenodeoxycholate intravenously. Urine was chromatographed on a Sephadex LH-20-column. Free, taurine- and glycineconjugated bile salts are eluted between 5 and 40 ml. The second peak between 50 and 110 ml consists of chenodeoxycholate-glucuronide and -sulphate, as can be shown after hydrelysis by B-glucuronidase and solvolysis. Hydrolysis by 8-glucuronidase was signifkcantly inhibited by the addition of saccharolactone

W. Frijhling and A . Stiehl: Bile Salt Glucuronides

72

Table 2. Urinary excretion o f bile salts, bile salt sulphates and bile salt glucuronides in patients with extrahepatic cholestasis (mg/24 h) Pat. non-sulph. non-glucura N o . LCb DCC CDCd Ce

0.6