Upsala J Med Sci 88: 1-8, 1983 Bile Salt Sulphation in Man Liver bile salt sulphotransferase activity in patients with primary biliary cirrhosis Lars Loof and Anders Nyberg Department of Internal Medicine, University Hospital, Uppsala, Sweden ABSTRACT Bile salt sulphation in primary biliary cirrhosis was studied by measure- ments of the liver bile salt sulphotransferase levels in 16 patients. Although the enzyme activity varied among the patients it did not correlate with the severity of cholestasis. Furthermore, the mean bile salt sulphotransferase magnitude in patients with primary biliary cirrhosis did not differ signifi- cantly from corresponding enzyme activity in patients with non-cholestatic, alcohol induced liver disease. The present data indicates that chronic chole- stasis, as evidenced in patients with primary biliary cirrhosis, does not lead to increased levels of liver bile salt sulphotransferase. It is suggested that mechanisms other than enzymic induction are responsible for the increased bile salt sulphate synthesis as observed in primary biliary cirrhosis. INTRODUCTION Sulphation, identified by Palmer (14) as one pathway of the human bile salt metabolism, occurs to only a minor extent i n the healthy man (1, 12, 21, 26). Patients with cholestasis eliminate large amounts o f bile salt sulphates in the urine (1, 1 2 , 21, 23, 26). Although the pathophysiological significance of bile salt sulphation has not been determined it is regarded to be important for the elimination o f potentially toxic bile salts (22). However, few data concerning the enzymatic mechanisms for bile salt sulphate synthesis in clini- cal conditions with cholestasis are available. The enzyme, bile salt sulpho- transferase, which transfers sulphate groups form 3'-phosphoadenosine-5'-phos- phosulphate (PAPS) to bile salts, has been identified in the human liver, (5, 8), small intestine ( 9 ) and adrenals (11). In the present investigation bile salt sulphation in primary biliary cirrhosis (PBC), a liver disease characte- rised by a chronic intrahepatic cholestasis ( 1 6 ) , has been studied by measu- rements of the bile salt sulphotransferase levels in percutaneous liver biopsy specimens. 1-832859 1 MATERIAL AND METHODS Patients The study was carried out o n patients admitted for clinical investigation of liver disease to the Department of Internal Medicine, University Hospital, Uppsala, Sweden. Sixteen patients with primary biliary cirrhosis were examined (Table I). Drug treatment at the time of the study included prednisolone (pat. n o . 2, 5 and l o ) , spironolactone (pat. n o . 1 and 2), cholestyramine (pat. n o . 4 , 7, 11 and 1 4 ) , cimetidine i levothyroxin (pat. n o . 14). The criteria f o r the diagnosis were: a) blood chemistry analyses of liver function indicative of cholestasis; b) liver histology diagnostic o r highly suggestive of PBC. C) normal extrahepatic bile ducts on cholangiography. d) positive serum mitocond- rial antibody. Routine percutaneous liver biopsies were performed ad modum Menghini (13). Liver histology was analysed and classified in four stages by one pathologist. (Stage 1 florid duct lesion, stage 2 = ductular proliferation, lymphoid aggregates with preserved lobular architecture, stage 3 = fibrosis with septum formation and disturbed lobular architecture, stage 4 = cirrhosis (16). Liver function was also evaluated by the intravenous galactose tolerance test ( 2 4 ) . Eight patients with alcoholic liver disease were also included in the study (Table 11). The patients had no drug therapy. The diagnosis was based on a history of alcohol abuse together with abnormal liver function tests. The clinical investigation did not reveal any alternative etiology to the liver disease. I n these patients blood chemistry analyses of liver function were not indicative of cholestasis. Chemicals Chemicals used were of analytical grade and obtained from Kebo Grave, Stockholm, Sweden, unless otherwise specified. The sodium salt of glycolitho- cholate (Calbiochem) was stored dessicated at +4OC. For preparation of buffers and other solutions deionized water was used. Carrier-free (35S)-labelled sodium sulphate was purchased from the Radio- chemical Centre Ltd, Amersham, England. Radioactive 3' -phosphoadenosine-5 ' -phosphosulphate ( (35S)PAPS) was biosyn- thesised and purified as earlier described (10). Radioactive samples were counted in 5 ml LumagelB in a Packard Tricarb 3000 liquid scintillation coun- ter. Total serum bile salts were determined by a ready made enzymatic kit (Sterognost-3 CI Flu, Nygaard & Co, O s l o , Norway). 2 Tissue p repa rat ion The percutaneous liver biopsy specimens were divided into two parts, one for histological examination and the other for analysis of sulphotransferase activity. The latter specimen was immedeatly transported to the laboratory in ice cold 0.15M KC1 containing 0.02 M Tris/HCl (pH 7.5), 0.001 M, EDTA and 0 . 0 0 1 M 2-mercaptoethanol. Unless otherwise specified all further procedures were carried out at + 4 O C . The tissue was homogenised in 0 . 5 ml of the aforementioned buffer and after addition of another 0.5 ml of the buffer the homogenate was centrifuged for 60 min. at 105000 x g in a Beckman L2-65B ultracentrifuge. The clear supernatant was pipetted off and stored at -7OOC in 0.2-0.3 ml portions until used. Sulphotransferase assay A modification (10) of the sulphotransferase assay described previously ( 8 ) was used. The incubation mixture contained: a) 2.5 m o l glycolithocholate added as an ethanolic solution and evaporated to dryness in vacuo; b) 7.5 nmol (35S)PAPS, specific activity 50 cpm/pmol, in 100 pl 0.15 M K C 1 containing 0 . 0 2 M Tris/HCl (pH 7.5), 0 . 0 0 4 M EDTA and 0.001 M 2-mercaptoethanol; c) 50 1-11 of liver cytosol containing 10-40 1-18 of protein. Incubations were carried out in duplicate for 30 min. at 37OC followed by butanol extraction and quantification of radioactive sulphate esters by liquid scintillation (8). One enzyme unit was defined as the amount of enzyme neces- sary to catalyze the formation of one pmol glycolithocholate sulphate per 3 minute. Specific enzyme activity is expressed as units/mg cytosol protein. The protein content of liver cytosol was determined by the method of Lowry (7). Statistics Students t-test was used for testing the statistical significance between two means. Means (x) and standard deviation (S.D.) were calculated by conven- tional methods (2). RESULTS Liver bile salt sulphotransferase activity towards glycolithocholate in patients with PBC, Table I, varied between 42 and 193 units/mg cytosol protein. No obvious difference with respect to the sulphotransferase activity was found between drug treated patients and patients without drugs. The lowest enzyme activities belonged to the two men included in the PBC group. No correlation was found between bile salt sulphotransferase activity and serum concentration o f bilirubin, total bile salt and alkaline phosphatases or to the intravenous 3 P T a b le 1 . P ri m ar y b il ia ry c ir rh o s is . P a ti e n t d a ta . P a ti e n t A ge S ex L iv e r S -b il ir u b in S -a lk . S -b il e AM A I .v . g a la k to se L iv e r b il e s a lt N o. y e a rs h is to lo g y p m o l/ l p h o sp h . s a lt ti tr e to le ra n c e su lp h o tr a n sf e ra se st a g e p k a t/ l p rn o ll l (n e g ) te s t u n it sl m g c y to so l I- IV (4 - 2 1 )+ < (0 .8 -4 .8 ) (2 .7 -7 .3 ) T + m in p ro te in (< 1 7 m in ) 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 15 1 6 74 0 I1 5 2 0 I1 1 43 0 I1 1 48 0 I1 1 6 4 6 11 1 47 6 I1 1 5 7 0 IV 6 6 0 IV 5 6 0 IV 6 2 0 IV 5 6 0 IV 39 0 IV 42 0 IV 36 0 IV 5 9 0 IV 59 0 IV 7 19 9 82 50 27 7 8 1 7 4 1 4 20 19 29 15 1 3 4 3 1 2 3 4 1 1 .5 1 4 .0 1 6 .7 4 2 .0 6 .0 1 7 .5 1 1 .7 9 .3 1 1 .6 1 0 .4 2 9 .0 2 5 .0 3 5 .0 1 9 .2 3 7 .0 1 2 .8 2 .0 2 2 .0 7 .0 1 7 .0 1 7 .0 2 2 .0 3 9 .3 5 4 .0 4 .6 3 1 .6 1 7 .0 1 2 .0 5 9 .0 2 7 0 .0 2 5 .0 1 0 .0 M ea nk SD 5 4 2 1 1 ~ _ _ _ _ ~ 66 ?8 9 1 9 .3 2 1 1 .O 3 8 2 6 4 .0 1 1 4 00 11 40 0 11 40 0 1 /1 0 0 11 40 0 1 /1 0 0 1 / 4 00 11 10 0 11 40 0 1 /4 0 0 11 40 0 1 /2 5 11 10 0 11 40 0 1 /4 0 0 11 40 0 2 1 1 3 18 1 8 19 1 5 2 3 44 2 3 1 3 17 11 1 6 28 2 1 9 4 11 7 10 0 12 6 42 76 18 8 79 14 6 1 2 1 13 3 1 2 5 72 12 2 1 2 4 1 9 3 20 28 11 6+ 40 ;‘i N or m al ra n g e o f se ru m v a lu e s i s g iv e n i n p a ra n th e si s. galactose tolerance test. Moreover, no correlation was found between the bile salt sulfotransferase level and the known duration of the disease. The bile salt sulphotransferase activity in patients with alcoholic liver disease, Table 11, varied between 70 and 136 units/mg cytosol protein. Also in this group the lowest values were found in the male patients. When the mean liver bile salt sulphotransferase activity in patients with PBC (116 2 40 units/mg cytosol protein) and patients with alcoholic liver disease (106 % 26 units/mg cytosol protein) were compared no significant diffe- rence was found. D I S C U S S I O N In the present work liver bile salt sulphotransferase activity was measured in primary biliary cirrhosis. The patients had varying degree of cholestasis as indicated by the serum concentrations of bilirubin and bile salts. The enzyme levels differed by 500 % when the highest and lowest values were compared. However, the variations in bile salt sulphotransferase activity were not correlated to the degree of cholestasis. Furthermore no correlation was found between the bile salt sulphotransferase activity and liver function as evidenced by the intravenous galactose tolerance test. We have not measured the amounts of bile salt sulphates excreted in the urine from these patients. However, other investigations have shown that patients with P B C constantly have an increased urinary elimination of bile salts which mainly appear in the sulphated form ( 1 5 ) . Moreover, the magnitude of the bile salt sulphate eli- mination in the urine seem to correlate well with the serum bile salt concen- tration (15, 2 6 ) . Thus, the present data suggest that patients with PBC, a clinical condition characterized mainly by the symptoms of a chronic chole- stasis, do not increase their sulphation capacity by induction of liver bile salt sulphotransferase when the cholestasis progresses. This assumption is further supported by the observation that the mean bile salt sulphotransferase activities in P B C and in alcoholic liver disease with no clinical signs of cholestasis, respectively, do not differentiate significantly. These results also agree with the reported absence of bile salt sulphotransferase induction in the livers from bile duct ligated hamsters ( 4 ) . The individual data of the male patients in the P B C group and in the alcohol liver disease group with respect to bile salt sulphotransferase activity had a tendency to be lower than the corresponding enzyme activity in females. We have not observed a sex diffe- rence with respect to human bile salt sulphotransferase activity in the liver in previous studies (9). However, steroid sulphotransferase activity, including bile salt sulphotransferase, in the rat and hamster seem to be significantly influenced by sex hormones ( 3 , 17, 18, 1 9 , 2 0 ) . Female rat and hamster livers 5 Ta bl e 2. A lc oh ol ic l iv er d is ea se . Pa ti en t da ta . Pa ti en t Ag e Se x Li ve r S- bi li ru bi n S- al k. I . V . Li ve r bi le sa lt S- bi le N o . ye ar s hi s t o l og y pm ol /l ph os ph . sa lt ga la kt os e su lp ho tr an sf er as e pk at /l p m o l/ l to le ra nc e un it s/ mg cy to so l (4 -2 1) (0 .8 -4 .8 ) (2 .7 -7 .3 ) te st T k m in pr ot ei n (< 17 m in ) 6 7 8 54 41 38 45 73 63 22 44 d 0 0 _ _ n or ma l st ea to si s st ea to si s ci rr ho si s in fl am ma ti on , si de ro si s no rm a 1 no rm al s t ea to si s 8 4. 8 1. 5 15 6 5. 3 4. 5 12 20 2. 1 6. 5 14 11 2. 0 3. 5 23 4 3. 3 4. 5 31 9 2. 8 2. 5 20 7 2. 2 2. 5 11 19 3. 5 10 .0 16 70 78 12 7 10 6 12 9 80 11 8 13 6 Me an tS D 48 t 16 11 t 6 3. 3t l. 2 4 . 4 f 2. 7 18 f 7 10 6 t 26 contain two to six times the activity in males (17, 20). Even if such a sex difference might be present in man and is taken into consideration because of the uneven sex distribution between the PBC and alcoholic liver group it would rather support the observed absence of bile salt sulphotransferase induction in primary biliary cirrhosis. The increased biosynthesis of bile salt sulphates as observed in PBC (15, 26) may instead be caused by a better substrate availability for bile salt sulphotransferase when the bile salt concentration increases in the cholestatic liver as suggested by Barnes (4). In addition extrahepatic sulphation, e.g. in the small instestine or adrenals (9) may contribute to the enhanced sulphation of bile salts in primary biliary cirrhosis. ACKNOWLEDGEMENTS This work was supported by grants from the Swedish Medical Research Council, Project n o . B 81-03P-5727-02 and B81-03x-5449-03. REFERENCES 1 2. 3. 4. 5 6 7. 8. 9. 10. 11. 12. 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