Biochem. J. (1976) 156, 339-345 Printed in Great Britain
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Synthesis of Bile Acid Monosulphates by the Isolated Perfused Rat Kidney By JOHN A. SUMMERFIELD, JOHN L. GOLLAN and BARBARA H. BILLING Department of Medicine, The Royal Free Hospital, Pond Street, Hampstead, London NW3 2QG, U.K. (Received 20 November 1975)
Perfusion of an isolated rat kidney with labelled bile acids, in a protein-free medium, resulted in the urinary excretion of the labelled bile acid, 3 % being converted into polar metabolites in 1 h. These metabolites were neither -glycine nor taurine conjugates, nor bile acid glucuronides, and on solvolysis yielded the free bile acid. On t.l.c. the metabolite of [24-14C]lithocholic acid had the mobility of lithocholate 3-sulphate. The principal metabolite of [24-14Cjchenodeoxycholic acid had the mobility of chenodeoxycholate 7-sulphate; trace amounts appeared as chenodeoxycholate 3-sulphate. [35S]Sulphate was incorporated into chenodeoxycholic acid by the kidney, resulting in a -similar pattern of sulphation. No disulphate salt of chenodeoxycholic acid was detected. These findings lend support to the hypothesis that renal synthesis may account for some of the bile acid sulphates present in urine in the cholestatic syndrome in man. l3ile acid sulphates were identified first in low concentrations in human bile (Palmer & Bolt, 1971). They are now considered to play an important role in the disturbed bile acid metabolism in cholestasis, and are excreted in large amounts in the urine (Stiehl, 1974; Makino et al., 1974). It has generally been accepted that bile acid sulphates are formed in the liver and that the discrepancy between the low proportion of bile acids present as sulphates in the serum and the high proportion in the urine is due to the high renal clearance of these compounds (Palmer, 1971; Stiehl, 1974; Makino et al., 1974). Recent studies (J. A. Summerfield, J. Cullen, S. Barnes & B. H. Billing, unpublished work) have, however, shown that an increasing bile acid sulphate output in the urine is not accompanied by a rise in the serum bile acid sulphate concentration, and it has been postulated that renal synthesis may account for some of the bile acid sulphates in urine. To test this hypothesis the ability of the isolated perfused rat kidney (Gollan & Billing, 1975) to synthesize bile acid sulphates was investigated. The present paper reports experiments that demonstrate that bile acid monosulphates can be synthesized by the isolated rat kidney.
Materials and Methods Reagents
All solvents were of analytical grade, and deionized distilled water was used throughout. Glucaro-1,4lactone was purchased from Calbiochem, San Diego, CA, U.S.A., and ,8-glucuronidase (bacterial, type II) from Sigma Chemical Co., St. Louis, MO, U.S.A. Chenodeoxycholic acid (3ar,7a-dihydroxy-5fi-cholan24-oic acid) was purchased from Weddel PharmaVol. 156
ceuticals, Wrexham, Clwyd LL13 9PX, U.K. Sodium [24-14C]chenodeoxycholate was obtained from The Radiochemical Centre, Amersham, Bucks., U.K., and sodium [24_14Cllithocholate (3a-hydroxy5fi-cholan-24-oate) from Amersham-Buchler G.m.b.H. and Co. K.G., D-3300 Braunschweig, W. Germany,. The purity of the labelled bile acids sodium [24-'4C]chenodeoxycholate and sodium [24-14C]lithocholate was checked by t,l,c. on silica-gel plates (0.25 mm thick; DC-Fertigplatten Kieselgel 60; Merck, Darmstadt, W. Germany) in a solvent system of butan-I-ol/acetic acid/water (10:1 :1, by vol.) (Gdnshirt et al., 1960). The labelled bile acids were co-chromatographed with standards of chenodeoxycholic acid and lithocholic acid. More than 98 % of each labelled compound had the mobility of the authentic bile acid standard. H235SO4 was purchased from The Radiochemical Centre and neutralized with Na2CO0 before use. Synthe,vis ofreference bile acid sulphates Diammonium lithocholate 3-sulphate was prepared by the method of Palmer & Bolt (1971). Sulphate salts of chenodeoxycholic acid, prepared by the methods of Mumma (1966) and Palmer & Bolt (1971), yielded mixtures of products which probably included the 3a-monosulphate, 7a-monosulphate and 3a,7a-disulphate. It was necessary therefore to individual sulphate standards by specific methods, by using derivatives of chenodeoxycholic acid. prepare
Disodium chenodeoxycholate 3-sulphate. This
was
gift from Professor G.A.D. Haslewood (Guy's Hospital Medical School, London SEI 9RT, U.K.) (prepared from methyl chenodeoxycholate 7-acetate by the method of Haslewood & Haslewood, 1976).
a
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J. A. SUMMERFIELD, J. L. GOLLAN AND B. H. BILLING
Disodium chenodeoxycholate 7-sulphate. The 3hemisuccinate of chenodeoxycholic acid (7a-hydroxy3a-succinoxy-5,B-cholan-24-oic acid) was prepared by the method of Schwenk et al. (1943). Succinic anhydride (13g) was dissolved with warming in anhydrous pyridine (50ml) and 5g of chenodeoxycholic acid was added. The mixture was left at room temperature (21°C) overnight. The reaction mixture was added slowly to 200ml of HCI (sp.gr. 1.18) on ice and filtered. The white granular precipitate was washed with water, dried over P205, and then dissolved in ether (which had been dried over anhydrous Na2SO4) and filtered. The ether was removed by evaporation under a stream of N2 and the residue dissolved in a small volume of warm ethanol/ methanol (1:1, v/v) and cooled on ice, resulting in the formation of crystals. The crystals of 7a-hydroxy3a-succinoxy-511-cholan-24-oic acid were filtered and air-dried at 37°C. By the method of Haslewood & Haslewood (1976), this compound (about g) was sulphated in the 7a position, the hemisuccinate cleaved by alkaline hydrolysis and the product finally crystallized. Professor Haslewood independently made disodium chenodeoxycholate 7-sulphate from chenodeoxycholic acid 3-hemisuccinate, m.p. 210-212'C, prepared as above and crystallized from benzene. His disodium chenodeoxycholate 7-sulphate, crystallized from ethanol/ether as white sugary solvated crystals, had the same m.p. (2240C), i.r. spectrum and RF on t.l.c. as our compound. (Found, by flame photometry: Na+, 8.0; C24H3807Na2S,C2H5OH requires Na+, 8.2%.) Trisodium chenodeoxycholate 3,7-disulphate. This was prepared by the method of Haslewood & Haslewood (1976). Professor Haslewood independently prepared chenodeoxycholic acid disulphate by this method, and the sodium salt was crystallized by evaporating a filtered methanolic solution, with replacement of methanol by ethanol, until the appearance of crystals seemed maximal. The product was refrigerated, collected, washed with cold ethanol and dried in vacuo over CaCl2. Chenodeoxycholate 3,7-disulphate (trisodium salt) formed colourless solvated crystalline globules, m.p. 197-199°C, with decomposition. (Found, by flame photometry, Na+, 9.7; C24H37010S2Na3,2C2H50H requires Na+, 9.7 %.) This compound readily dissolved in water, 0.1 M-HCI or methanol; it was sparingly soluble in ethanol. Analysis of our material by Professor Haslewood yielded similar results. Analysis of reference standards of the sulphate salts of chenodeoxycholic acid These compounds each gave one spot on t.l.c. in a solvent system of chloroform/methanol/acetic acid/water (65:24:15:9, by vol.) (Cass et al.,1975). In
eight experiments, the 3-monosulphate of chenodeoxycholic acid had RF 0.68±0.03 (mean±s.E.M.), the 7-monosulphate had RF 0.56±0.03 and the 3,7disulphate had RF 0.33 ±0.02. In this solvent system lithocholate 3-sulphate had the same RF as chenodeoxycholate 3-sulphate. In the solvent system of Ganshirt et al. (1960), the 3,7-disulphate of chenodeoxycholic acid had RF 0.22, but the 3-monosulphate (RF 0.50) was not separated from the 7-monosulphate (RF 0.46). The individual sulphate salts were isolated from the mixtures prepared by the methods of Mumma (1966) and Palmer & Bolt (1971) by preparative t.l.c. on silica-gel plates in the solvent system of Cass et al. (1975). Although the method of Palmer & Bolt (1971) yielded predominantly the 3,7-disulphate, a large proportion of the products from the method of Mumma (1966) was the 3-monosulphate and the 7-monosulphate. The plates were air-dried and sprayed lightly with a methanolic solution of 12 (3.5g/100ml) to detect the compounds. The I was allowed to sublime, and the silica gel containing the bile acid sulphates was scraped off and eluted with methanol, which was evaporated to dryness under reduced pressure at 60'C. The residue was resuspended in 1 ml of methanol and centrifuged to remove traces of silica gel. The clear supernatant was decanted, and the bile acid sulphates were precipitated by the addition of 9ml of dry ether. The supernatant was discarded after centrifugation and the product dried under a stream of N2. This resulted in the production of three compounds, which gave separate spots on t.l.c. in the solvent system of Cass et al. (1975) and had the same mobility as the reference sulphates. To characterize the three sulphate salts of chenodeoxycholic acid, derivatives were prepared by the method of Cronholm (1969). The bile acid sulphates (about 2mg) were methylated with freshly prepared ethereal diazomethane and the free hydroxyl groups were then acetylated in acetic anhydride (1 ml) and anhydrous pyridine (1 ml) at room temperature overnight. The reagents were removed under a stream of N2 and the residues were hydrolysed by solvolysis (see under 'Methods'). The samples were evaporated to dryness under reduced pressure at 60°C, the remaining hydroxyl groups converted into the trimethylsilyl ethers (Makita & Wells, 1963), and submitted to combined g.l.c.-mass spectrometry on columns of OV1 (Applied Science Laborttories, State College, PA, U.S.A.) at 290°C in a Varian Mat 731 instrument. The predominant derivative formed from trisodium chenodeoxycholate 3,7-disulphate had the mass spectrum of methyl chenodeoxycholate 3,7-bis-(trimethylsilyl) ether. The spectrumshowedthe base peak at m/e 370 [M-(2 x 90)] and peaks at mle 460 (M-90) and m/e 255 (ABCD rings). The peak present at mle 243 is characteristic of a 3,7-bis(trimethylsiloxy) structure (Sj6vall et al., 1971). This 1976
RENAL SYNTHESIS OF BILE ACID MONOSULPHATES spectrum was consistent with the original compound being a disulphate salt of chenodeoxycholic acid. The mass spectra of the derivatives of both disodium chenodeoxycholate 3-sulphate and disodium chenodeoxycholate 7-sulphate were similar but not identical. Both had a base peak at mle 370 [M(60+90)] and a prominent peak at mle 460 (M-60); in addition peaks were present at mle 315 [M(115+90)], mle 255, mle 228 and mle 213. There was no peak at mle 243. These mass spectra were consistent with a structure of methyl chenodeoxycholate monoacetate monotrimethylsilyl ether, indicating that the original two compounds were monosulphate salts of chenodeoxycholic acid.
Methods Measurement of radioactivity. Portions of urine and perfusate were prepared for scintillation counting by incubation with 1 ml of NCS solubilizer (Amersham/Searle Corp., Arlington Heights, IL, U.S.A.) at 38°C for 1 h. Radioactivity was measured by adding portions of solubilized urine or perfusate and eluates from Sephadex LH 20 or silica gel to 10ml of a scintillant mixture consisting of 2,5diphenyloxazole (4g), 1,4-bis-(5-phenyloxazol-2-yl)benzene (50mg) and Triton X-100 [Rohm and Haas (U.K.), Ltd., Croydon CR9 3NB, U.K.] (500ml) in toluene (1 litre) and counting in a Philips liquidscintillation spectrometer. Quench correction was by external standardization. Isolatedperfused rat kidney. Male Sprague-Dawley rats weighing 350-450g were maintained on a standard laboratory diet (modified 41B; Oxoid Ltd., Southwark Bridge Rd., London S.E.1, U.K.) for these experiments. A modification ofthe perfusion technique of Nishiitsutsuji-Uwo et al. (1967) was used. Under ether anaesthesia the ureter was catheterized (PP1O; Portex Ltd., Hythe, Kent, U.K.) and the right kidney was prepared for perfusion by inserting a polyethylene cannula (internal diameter 2mm) into the inferior vena cava, distal to the renal vein for the renal-venous effluent. A curved metal cannula was placed in the mesenteric artery and advanced across the aorta into the renal artery, with the perfusate flowing, so that renal plasma flow was unimpaired. The kidney was dissected from the animal, and the venous cannula connected into the circuit. The protein-free perfusion medium consisted of a Krebs improved Ringer 1 solution (Krebs, 1950) modified by substituting 6.0 % (w/v) dextran (mol.wt. 70000) in 0.154M-NaCl (Lomodex 70; Fisons Ltd., Loughborough, Leics., U.K.) for the 0.9 % NaCl and adding 5mM-urea. The medium (120ml) was maintained at 38°C, equilibrated at pH7.4 with 0.245MNaHCO3 and gassed with 02+CO2 (95:5). It was recirculated by pulsatile flow at 32-34ml/min and Vol. 156
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continuously filtered by an in-line nylon filter, 14pm pore size (Millipore S.A., Buc, France). The function of the isolated kidney perfused with a protein-free medium has been evaluated and shown to remain stable over a perfusion period of I h (Gollan & Billing, 1975). The values obtained for the glomerular filtration rate and effective renal plasma flow were within the normal range for the intact rat (Spector, 1956). A 10min control period was allowed for mixing of the added bile acid, and recirculation of the perfusate continued for a 1 h test period. Urine was collected over 10min intervals and midpoint samples of perfusate were taken to calculate clearance values. After perfusion for 1 h, the glomerular filtration rate and effective renal plasma flow were estimated simultaneously during a further 10min collection period by the addition of 5'Cr-EDTA and sodium [I251]iodohippurate respectively. Radioactivity was measured with a dual-channel y spectrometer (GTL 300; Wallac, South Croydon, Surrey, U.K.), and the counts were corrected for spill-over. The two nuclides were readily distinguished by their different radiation energies. The values were comparable with those previously reported for this kidney preparation. In seven experiments, 5cCi of sodium (24-14C]chenodeoxycholate with 10mg of chenodeoxycholic acid as carrier (final perfusate concentration approx. 200pM) was dissolved in 2 ml of 0.245 M-NaHCO3 and added to the perfusate. In two experiments, sodium [24-14C]chenodeoxycholate and crystallized bovine serum albumin (1 g/100ml) (Armour Pharmaceutical Co., Eastbourne, Sussex, U.K.) were dissolved in the perfusate. In another two experiments with sodium [24-14C]chenodeoxycholate, kidneys were obtained from rats whose bile ducts had been ligated 14 days or 21 days before the perfusion. In one experiment, 1 mCi of Na235SO4 was administered subcutaneously to a rat, 24h later the kidney was perfused with 0.5mCi of Na235SO4, and 10mg of chenodeoxycholic acid was added to the medium. In three experiments, 50Ci of sodium [24-14C]lithocholate (final perfusate concentration approx. 0.8pM) was dissolved in 2ml of 0.245M-NaHCO3 and added to the perfusate. The low solubility of lithocholic acid in the perfusate precluded the addition of carrier. Fractionation of bile acids. The urine (about 20ml) was adjusted to pH10 with 1 M-NaOH, and 5g of Amberlite XAD-7 [Rohm and Haas (U.K.) Ltd.] was added (Summerfield et al., 1976). After mixing for I h, the urine was decanted and the resin washed with water adjusted to pH 10 with 1 M-NaOH. The water was decanted and the bile acids were eluted from the resin with 3 x 10ml of methanol. The methanol extracts were pooled and evaporated to dryness under reduced pressure at 60°C. The residue was dissolved with ultrasonic agitation in 1.5ml of chloroform/
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J. A. SUMMERFIELD, J. L.- GOLLAN AND B. H. BILLING
methanol (1: 1, W/v) containing 0.01 M-NaCl and centrifuged at 300g for 5mim. Then 1 ml of the supernatant was loaded, via a polytetrafluoroethylene rotary valve (Jobling Laboratory Division, Stone, Staffs. ST15 OBG, U.K.) on to a 6g column (1 cmx45cm) of Sephadex LH 20 (Pharmacia, Uppsala, Sweden) which had been equilibrated with this solvent. The column was eluted with chloroform/ methanol (1:1, v/v) containing 0.01 M-NaCl, in 4ml fractions; the fractions between 0 and 52 ml contained the free and glycine- and taurine-conjugatedbileapids. The eluent was changed, to methanol and the fraction between 53 and lOOml contained the, bile acid sulphates (Sjovall & Vihko, 1966). When the labelled bile acids were submitted to chromatography on Sephadex LH 20, all the radioactivity was eluted between 0 and 52ml (non-sulphate fraction). Solvolysis.. Solvolysis was -performed by first dissolving the samples, with ultrasonic agitation, in acetone/ethanol/2M-HCl (18,:2:1, by vol.) and then heating for 48h at 37°C (Burstein & Lieberman, 1958; Palmer & Bolt, 1971). The reaction mixture was evaporated to dryness under reduced pressure at 60°C. fi-Glucuronidase treatment. Solutions of bacterial /)-glucuronidase (200 Fishman units/ml) and glucaro1,4-actone (136mM) were prepared in 66mm-sodium phosphate buffer (pH6.8) and passed through filters, 0.45gm pore size (Swinnex 13; Millipore S.A.) to remove viable bacteria, which could give rise to artifact formation (Manson et al., 1972). One,portion of the dried sample under test was dissolved-in 1 ml of Il-glucuronidase solution and 1 ml of 66mM-phosphate buffer (pH6.8), and another portion was dissolved in 1 ml of f-glucuronidase solution and 1 ml of glucaro-1,4-lactone solution added to inhibit the enzyme (Levvy, 1952). The mixtures were incubated for 24h at 37°C and then evaporated to dryness under reduced pressure at 60°C. Results and Discsion The renal clearance of bile acids after 30min perfusion was calculated from the ratio UV/P, where U and P represent the urine and perfusate radioactivity per unit volume and V the urine flow (ml/rnin) The clearance of chenodeoxycholic acid (mean 0.134ml/min, n=7) was five times greater than of lithocholic acid (mean 0.025ml/min, n=3). Bileduct ligation had no effect on the clearance of chenodeoxycholic acid. As expected, the addition of bovine serum albumin- markedly decreased the clearance (0.006m/min), presumably because binding of bile acid to protein decreases the free fraction available for glomerular filtration. The use of a protein-free perfusate was therefore important in the dqign, of the ex,periments.
Fractionation ofurine bile acids
Perfusion of the kidney with sodium [24-14C]chenodeoxycholate resulted in the excretion of the compound together with two polar metabolites (A and B), which were detected in the fraction eluted with methinol on Sephadex LH 20 (Fig. 1). In six experiments these Ipolar metabolites comprised 2.42 ±0.43 o/ (mean±s.E.M.) of the total urine radioactivity, the greatest amount being 5.06 %. On average, the radioactivity in peak B (1.84± 0.28% of total urine radioactivity) was three times as great as in peak A (0.58±0.2% of total urine radioactivi'ty), but in three experiments only traces of peak A were present. The relative size of each peak did not appear to be related to the function of the isolated kidney. The two polar metabolites of chenodeoxycholic acid found in the urine were also present in the dextran perfusate at the end of the experiment. The addition of bovine serum albumin to the perfusate in two experiments resulted in a 20-fold
'0 .-
-0
;...
C4
01 0 o
50
100
Eluate (ml) Fig. 1. Fractionation ofurine from an isolated rat kidney, perfused with sodium [24-14CJchenodeoxycholate, on a column of Sephadex LH 20 Elution with chloroform/methanol (1:1, v/v) containing 0.01 m-NaCl, in 4ml fractions, yielded a major peak, representing sodium f24-14C]chenodeoxycholate. When the eluent was changed to methanol after 52ml, two polar metabolites (A and B) of sodium [24-14C]chenodeoxycholate appeared. 1976
RENAL SYNTHESIS OF BILE ACID MONOSULPHATES decrease in the renal clearance of radioactive cheno-
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ment. The conversion of chenodeoxycholic acid into more polar compounds by kidneys from rats with chronic bile-duct obstruction was similar to that observed in the normal rats (3.2 and 1.2% of total urine radioactivity), suggesting that cholestasis in itself did not enhanoe the conversion. In three experiments sodium [24-14C]lithocholate was added to the dextran perfusate, and Sephadex LH 20 fractionation revealed that in addition to the peak of radioactivity of sodium [24-14C]lithocholate in the non-sulphate fraction, a polar peak occurred in the fraction eluted with methanol. This compound had an elution volume similar to that of peak B
polar metabolites of sodium [24-14C]chenodeoxycholate, from an experimnent in which predominantly peak B was formed, were co-chromatographed on t.l.c. plates with standard sulphate salts of chenodeoxycholic acid (Fig. 2). Most of the radioactivity (71 %) had the mobility of chenodeoxycholate 7-sulphate, and only 2 % of the radioactivity was in the chenodeoxycholate 3-sulphate. Radioactivity was also present in the position of chenodeoxycholic acid (26 %), indicating partial degradation as discussed above. No radioactivity was present as chenodeoxycholate 3,7-disulphate. The polar metabolite of sodium [24- C]lithocholate was co-chromatographed with authentic diammonium lithocholate 3-sulphate and lithocholic acid on t.l.c. Most of the radioactivity (91 %) had the mobility of lithocholate 3-sulphate. The remainder (9%) was present in the lithocholic acid fraction, probably owing to breakdown of the sulphate salt during the procedure.
Identification ofpolar metabolites The polar metabolites of 'sodium [24-14C]chenodeoxycholate (peaks A and B) from six experiments were combined separately and evaporated to dryness. One portion (about 3 x 1 03d.p.m.) of each peak was hydrolysed by solvolysis, another portion was incubated with p8-glucuronidase and a -third was incubated with Ii-glucuronidase and glucaro-1,4lactone. After drying, the residues were applied to columns of Sephadex LH 20. The samples of peaks A and B, hydrolysed- by solvolysis, were eluted in the non-sulphate fraction, having thesameelutionvolume as chenodeoxycholate. After treatment with a1glucuronidase, some radioactivity was eluted in the non-sulphate fraction, with the same elution volume as the chenodeoxycholate, but most of the sample was eluted in the sulphate fraction. Similar results were obtained after incubation with f8-glucuronidase and glucaro-1,4-lactone. The breakdown of these compounds during incubation with fl-glucuronidase was therefore a non-specific effect, since it was- not abolished by glucaro-1,4-lactone, a specific inhibitor of f8-glucuronidase (Lewy, 1952). It was unlikely to be due to artifact formation by viable bacteria in the enzyme preparation, since this was filtered before use (Manson et al., 1972). Further, when a portion of peak-B material, without any further treatment, was applied to a Sephadex LH 20 column, the polar metabolite decomposed to a similar extent. This apparent lability of the compounds was probably due to degradation on the column, but was not investigated further. It was concluded that peaks A and B consisted of conjugates which were hydrolysed by solvolysis and did not contain bile acid glucuronides. Similar results were obtained with the .polar metabolite of sodium [24-14C]lithocholate. To determine the nature of these conjugates, the Vol. 156
Incorporation of [35S]sulphate into chenodeoxycholic acid To confirm the 14C studies, an experiment was performed to determine whether the isolated rat kidney could. incorporate [35S]sulphate into chenodeoxycholic acid. The rat received Na235SO4 24h before the experiment in order to label the sulphate donor, 3'-phosphoadenosine 5'-sulphatophosphate. Theurine bile acids from the isolated kidney were fractionated on Sephadex LH 20 and the polar fraction was co-chromatographed on t.l.c. plates with reference sulphate salts of chenodeoxycholic acid and chenodeoxycholic acid (Fig. .2). Most of the "5S (83%) had the mobility of chenodeoxycholate 7-sulphate and only 2 % was present in the position of chenodeoxycholate 3-sulphate. The radioactivity (1.2 O) remaining At the origin was probably inorganic sulphate. Some radioactivity (4.7%O) had the mobility of chenodeoxycholate 3,7-disulph4te. Radioactivity was also present in a more polar position (8.8 %O) and probably represented incorporation of [35S]sulphate into other organic compounds excreted by the kidney into the urine. Fig. 2 illustrates the similar pattern of incorporation of both the 14C and 35S labels into the sulphate salts of chenodeoxycholic acid. This is the first demonstration that urine bile acid sulphates can be synthesized in the kidney and is consistent with the preliminary report by Chen et al. (1974) of an enzyme present in the rat kidney that sulphates bile acids. We have extended their observations by demonstrating that the predominant compounds formed in the kidney from lithocholic acid and chenodeoxycholic acid are the 3-monosulphate and 7-monosulphate respectively. Further studies are needed to determine why the predominant monosulphate of chenodeoxycholic acid is sulphated
deoxycholic acid; no polar metabolites were detected in the une or the perfusate at the end of the experi-
obtained with sodium [24-14C]chenodeoxycholate and accounted for 2.98, 3.60 and 4.00% of the total radioactivity recovered from the urine.
J. A. SUMMERFIELD, J. L. GOLLAN ANT) B. H. BILLING
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(a),, 3,
7
3
Free
10 >
(b)
8
ky6
0
3-
2-
x
Fig. 2. Incorporation of radioactivity into chenodeoxycholate sulphates in rat urine The polar metabolites of chenodeoxycholic acid, eluted with methanol from Sephadex LH 20, were co-chromatographed on t.l.c. plates with standard sulphate salts of chenodeoxycholic acid in the solvent system of Cass et al. (1975). (a) shows the chromatogram of trisodium chenodeoxycholate 3,7-disulphate (3,7), disodium chenodeoxycholate 7-sulphate (7), disodium chenodeoxycholate 3-sulphate (3) and chenodeoxycholic acid (free). The bile acids were detected by the method of Usui (1963). (b) shows the distribution of radioactivity after t.l.c. of the polar metabolites from an experiment in which a kidney was perfused with sodium [24-14CJchenodeoxycholate. (c) shows the distribution of radioactivity in the polar metabolites from an experiment in which a kidney was perfused with Na235SO4 and chenodeoxycholic acid. Most of the radioactivity has the mobility of disodium chenodeoxycholate 7-sulphate; trace amounts appear as disodium chenodeoxycholate 3-sulphate.
at C-7, and not at C-3 as Makino et al. (1974) had proposed. It now seems probable that there are several bile acid sulphokinases with different specificities in kidney tissue. In this regard, it is pertinent that monosulphated Clg steroid diols with a 171?hydroxyl group are sulphated at C-3 whereas the 17a epimers are sulphated at C-17 (Ruokonen & Vihko, 1974). The observation that the isolated rat kidney can synthesize bile acid monosulphates lends support to
the concept that some of the bile acid sulphates in human cholestatic urine may originate in the kidney. Makino et al. (1974) and Stiehl et al. (1975) have shown that sulphate salts of chenodeoxycholic acid are the predominant components of the bile acid sulphate fraction in the urine of patients with the cholestatic syndrome and about 80% of these are monosulphates (Stiehl, 1974). However, in man both the nature of the bile acid monosulphates and the role of the kidney in the disturbed bile acid metabolism of cholestasis remain to be established. Note Added in Proof (Received 26 January 1976) Since this paper was submitted for publication, Parnentier et al. (1975) have reported that cholate 7-sulphate is a major component of the faecal bile acids of the mouse, which suggests that sulphation at C-7 may be the usual pattern of sulphation for dihydroxy and trihydroxy bile acids. We thank Mr. M. Chu and Dr. K. Setchell (MRC Clinical Research Centre, Harrow, Middx. HAl 3UJ, U.K.) for the mass spectrometry and Mr. E. Shaw for assistance with the animal experiments. We also thank Dr. P. Back (Medizinische Klinik der Universitait Freiburg/Brsg., West Germany) and Dr. S. Barnes for helpful advice. We are especially grateful to Professor G. A. D. Haslewood (Guy's Hospital Medical School, London, SE1 9RT, U.K.) for the help and advice that made this study possible and for allowing the use of his unpublished work. J.L.G. was supported by the Wellcome Trust and J.A.S. by a Medical Research Council Clinical Research Fellowship. References Burstein, S. & Lieberman, S. (1958) J. Am. Chem. Soc. 80, 5235-5239 Cass, 0. W., Cowen, A. E., Hofmann, A. F. & Coffin, S. B. (1975) J. Lipid Res. 16, 159-160 Chen, L.-J., Bolt, R. & Admirand, W. H. (1974) Gastroenterology 67, Abstr. 782 Cronholm, T. (1969) Steroids 14, 285-296 Ganshirt, H., Koss, F. W. & Morianz, K. (1960) Arzneim.Forsch. 10, 943-947 Gollan, J. L. & Billing, B. H. (1975) Clin. Sci. Mol. Med. 49,28 Haslewood, E. S. & Haslewood, G. A. D. (1976) Biochem. J. 155, 401-04 Krebs, H. A. (1950) Biochim. Biophys. Acta 4, 249-269 Levvy, G. A. (1952) Biochem. J. 52,464-472 Makino, I., Shinozaki, K., Nakagawa, S. & Mashimo, K. (1974) J. Lipid Res. 15, 132-138 Makita, M. & Wells, W. W. (1963) Anal. Biochem. 5, 523530 Manson, M. E., Nocke-Fink, L., Gustafsson, J.-A. & Shackleton, C. H. L. (1972) Clin. Chim. Acta 38, 45-49 Mumma, R. 0. (1966) Lipids 1, 221-223 Nishiitsutsuji-Uwo, J. M., Ross, B. D. & Krebs, H. A. (1967) Biochem. J. 103, 852-862
1976
RENAL SYNTHESIS OF BILE ACID MONOSULPHATES Palmer, R. H. (1971) J. Lipid Res. 12, 680-687 Palmer, R. H. & Bolt, M. G. (1971) J. Lipid Res. 12, 671679 Parmentier, G., Mertens, J. & Eyssen, H. (1975) in Advances in Bile Acid Research (Matern, S., Hackenschmidt, J., Back. P. & Gerok, W., eds.), pp. 139-143, F. K. Schattauer Verlag, Stuttgart and New York Ruokonen, A. & Vihko, R. (1974) Steroids 23, 1-16 Schwenk, E., Riegel, B., Moffett, R. B. & Stahl, E. (1943) J. Am. Chem. Soc. 65, 549-551 Sjovall, J. & Vihko, R. (1966) Acta Chem. Scand. 20, 1419-1421
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Sjovall, J., Eneroth, P. & Ryhage, R. (1971) in The Bile Acids: Chemistry, Physiology and Metabolism (Nair, P. P. & Kritchevsky, D., eds.) vol. 1, pp. 209-248, Plenum Press, New York and London Spector, W. S. (1956) Handbook ofBiological Data, p. 341, W. B. Saunders Co., Philadelphia and London Stiehl, A. (1974) Eur. J. Clin. Invest. 4, 59-63 Stiehl, A., Earnest, D. L. & Admirand, W. H. (1975) Gastroenterology 68, 534-544 Summerfield, J. A., Billing, B. H. & Shackleton, C. H. L. (1976) Biochem. J. 154, 507-516 Usui, T. (1963) J. Biochem. (Tokyo) 54, 283-286
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Synthesis of Bile Acid Monosulphates by the Isolated Perfused Rat Kidney By JOHN A. SUMMERFIELD, JOHN L. GOLLAN and BARBARA H. BILLING Department of Medicine, The Royal Free Hospital, Pond Street, Hampstead, London NW3 2QG, U.K. (Received 20 November 1975)
Perfusion of an isolated rat kidney with labelled bile acids, in a protein-free medium, resulted in the urinary excretion of the labelled bile acid, 3 % being converted into polar metabolites in 1 h. These metabolites were neither -glycine nor taurine conjugates, nor bile acid glucuronides, and on solvolysis yielded the free bile acid. On t.l.c. the metabolite of [24-14C]lithocholic acid had the mobility of lithocholate 3-sulphate. The principal metabolite of [24-14Cjchenodeoxycholic acid had the mobility of chenodeoxycholate 7-sulphate; trace amounts appeared as chenodeoxycholate 3-sulphate. [35S]Sulphate was incorporated into chenodeoxycholic acid by the kidney, resulting in a -similar pattern of sulphation. No disulphate salt of chenodeoxycholic acid was detected. These findings lend support to the hypothesis that renal synthesis may account for some of the bile acid sulphates present in urine in the cholestatic syndrome in man. l3ile acid sulphates were identified first in low concentrations in human bile (Palmer & Bolt, 1971). They are now considered to play an important role in the disturbed bile acid metabolism in cholestasis, and are excreted in large amounts in the urine (Stiehl, 1974; Makino et al., 1974). It has generally been accepted that bile acid sulphates are formed in the liver and that the discrepancy between the low proportion of bile acids present as sulphates in the serum and the high proportion in the urine is due to the high renal clearance of these compounds (Palmer, 1971; Stiehl, 1974; Makino et al., 1974). Recent studies (J. A. Summerfield, J. Cullen, S. Barnes & B. H. Billing, unpublished work) have, however, shown that an increasing bile acid sulphate output in the urine is not accompanied by a rise in the serum bile acid sulphate concentration, and it has been postulated that renal synthesis may account for some of the bile acid sulphates in urine. To test this hypothesis the ability of the isolated perfused rat kidney (Gollan & Billing, 1975) to synthesize bile acid sulphates was investigated. The present paper reports experiments that demonstrate that bile acid monosulphates can be synthesized by the isolated rat kidney.
Materials and Methods Reagents
All solvents were of analytical grade, and deionized distilled water was used throughout. Glucaro-1,4lactone was purchased from Calbiochem, San Diego, CA, U.S.A., and ,8-glucuronidase (bacterial, type II) from Sigma Chemical Co., St. Louis, MO, U.S.A. Chenodeoxycholic acid (3ar,7a-dihydroxy-5fi-cholan24-oic acid) was purchased from Weddel PharmaVol. 156
ceuticals, Wrexham, Clwyd LL13 9PX, U.K. Sodium [24-14C]chenodeoxycholate was obtained from The Radiochemical Centre, Amersham, Bucks., U.K., and sodium [24_14Cllithocholate (3a-hydroxy5fi-cholan-24-oate) from Amersham-Buchler G.m.b.H. and Co. K.G., D-3300 Braunschweig, W. Germany,. The purity of the labelled bile acids sodium [24-'4C]chenodeoxycholate and sodium [24-14C]lithocholate was checked by t,l,c. on silica-gel plates (0.25 mm thick; DC-Fertigplatten Kieselgel 60; Merck, Darmstadt, W. Germany) in a solvent system of butan-I-ol/acetic acid/water (10:1 :1, by vol.) (Gdnshirt et al., 1960). The labelled bile acids were co-chromatographed with standards of chenodeoxycholic acid and lithocholic acid. More than 98 % of each labelled compound had the mobility of the authentic bile acid standard. H235SO4 was purchased from The Radiochemical Centre and neutralized with Na2CO0 before use. Synthe,vis ofreference bile acid sulphates Diammonium lithocholate 3-sulphate was prepared by the method of Palmer & Bolt (1971). Sulphate salts of chenodeoxycholic acid, prepared by the methods of Mumma (1966) and Palmer & Bolt (1971), yielded mixtures of products which probably included the 3a-monosulphate, 7a-monosulphate and 3a,7a-disulphate. It was necessary therefore to individual sulphate standards by specific methods, by using derivatives of chenodeoxycholic acid. prepare
Disodium chenodeoxycholate 3-sulphate. This
was
gift from Professor G.A.D. Haslewood (Guy's Hospital Medical School, London SEI 9RT, U.K.) (prepared from methyl chenodeoxycholate 7-acetate by the method of Haslewood & Haslewood, 1976).
a
340
J. A. SUMMERFIELD, J. L. GOLLAN AND B. H. BILLING
Disodium chenodeoxycholate 7-sulphate. The 3hemisuccinate of chenodeoxycholic acid (7a-hydroxy3a-succinoxy-5,B-cholan-24-oic acid) was prepared by the method of Schwenk et al. (1943). Succinic anhydride (13g) was dissolved with warming in anhydrous pyridine (50ml) and 5g of chenodeoxycholic acid was added. The mixture was left at room temperature (21°C) overnight. The reaction mixture was added slowly to 200ml of HCI (sp.gr. 1.18) on ice and filtered. The white granular precipitate was washed with water, dried over P205, and then dissolved in ether (which had been dried over anhydrous Na2SO4) and filtered. The ether was removed by evaporation under a stream of N2 and the residue dissolved in a small volume of warm ethanol/ methanol (1:1, v/v) and cooled on ice, resulting in the formation of crystals. The crystals of 7a-hydroxy3a-succinoxy-511-cholan-24-oic acid were filtered and air-dried at 37°C. By the method of Haslewood & Haslewood (1976), this compound (about g) was sulphated in the 7a position, the hemisuccinate cleaved by alkaline hydrolysis and the product finally crystallized. Professor Haslewood independently made disodium chenodeoxycholate 7-sulphate from chenodeoxycholic acid 3-hemisuccinate, m.p. 210-212'C, prepared as above and crystallized from benzene. His disodium chenodeoxycholate 7-sulphate, crystallized from ethanol/ether as white sugary solvated crystals, had the same m.p. (2240C), i.r. spectrum and RF on t.l.c. as our compound. (Found, by flame photometry: Na+, 8.0; C24H3807Na2S,C2H5OH requires Na+, 8.2%.) Trisodium chenodeoxycholate 3,7-disulphate. This was prepared by the method of Haslewood & Haslewood (1976). Professor Haslewood independently prepared chenodeoxycholic acid disulphate by this method, and the sodium salt was crystallized by evaporating a filtered methanolic solution, with replacement of methanol by ethanol, until the appearance of crystals seemed maximal. The product was refrigerated, collected, washed with cold ethanol and dried in vacuo over CaCl2. Chenodeoxycholate 3,7-disulphate (trisodium salt) formed colourless solvated crystalline globules, m.p. 197-199°C, with decomposition. (Found, by flame photometry, Na+, 9.7; C24H37010S2Na3,2C2H50H requires Na+, 9.7 %.) This compound readily dissolved in water, 0.1 M-HCI or methanol; it was sparingly soluble in ethanol. Analysis of our material by Professor Haslewood yielded similar results. Analysis of reference standards of the sulphate salts of chenodeoxycholic acid These compounds each gave one spot on t.l.c. in a solvent system of chloroform/methanol/acetic acid/water (65:24:15:9, by vol.) (Cass et al.,1975). In
eight experiments, the 3-monosulphate of chenodeoxycholic acid had RF 0.68±0.03 (mean±s.E.M.), the 7-monosulphate had RF 0.56±0.03 and the 3,7disulphate had RF 0.33 ±0.02. In this solvent system lithocholate 3-sulphate had the same RF as chenodeoxycholate 3-sulphate. In the solvent system of Ganshirt et al. (1960), the 3,7-disulphate of chenodeoxycholic acid had RF 0.22, but the 3-monosulphate (RF 0.50) was not separated from the 7-monosulphate (RF 0.46). The individual sulphate salts were isolated from the mixtures prepared by the methods of Mumma (1966) and Palmer & Bolt (1971) by preparative t.l.c. on silica-gel plates in the solvent system of Cass et al. (1975). Although the method of Palmer & Bolt (1971) yielded predominantly the 3,7-disulphate, a large proportion of the products from the method of Mumma (1966) was the 3-monosulphate and the 7-monosulphate. The plates were air-dried and sprayed lightly with a methanolic solution of 12 (3.5g/100ml) to detect the compounds. The I was allowed to sublime, and the silica gel containing the bile acid sulphates was scraped off and eluted with methanol, which was evaporated to dryness under reduced pressure at 60'C. The residue was resuspended in 1 ml of methanol and centrifuged to remove traces of silica gel. The clear supernatant was decanted, and the bile acid sulphates were precipitated by the addition of 9ml of dry ether. The supernatant was discarded after centrifugation and the product dried under a stream of N2. This resulted in the production of three compounds, which gave separate spots on t.l.c. in the solvent system of Cass et al. (1975) and had the same mobility as the reference sulphates. To characterize the three sulphate salts of chenodeoxycholic acid, derivatives were prepared by the method of Cronholm (1969). The bile acid sulphates (about 2mg) were methylated with freshly prepared ethereal diazomethane and the free hydroxyl groups were then acetylated in acetic anhydride (1 ml) and anhydrous pyridine (1 ml) at room temperature overnight. The reagents were removed under a stream of N2 and the residues were hydrolysed by solvolysis (see under 'Methods'). The samples were evaporated to dryness under reduced pressure at 60°C, the remaining hydroxyl groups converted into the trimethylsilyl ethers (Makita & Wells, 1963), and submitted to combined g.l.c.-mass spectrometry on columns of OV1 (Applied Science Laborttories, State College, PA, U.S.A.) at 290°C in a Varian Mat 731 instrument. The predominant derivative formed from trisodium chenodeoxycholate 3,7-disulphate had the mass spectrum of methyl chenodeoxycholate 3,7-bis-(trimethylsilyl) ether. The spectrumshowedthe base peak at m/e 370 [M-(2 x 90)] and peaks at mle 460 (M-90) and m/e 255 (ABCD rings). The peak present at mle 243 is characteristic of a 3,7-bis(trimethylsiloxy) structure (Sj6vall et al., 1971). This 1976
RENAL SYNTHESIS OF BILE ACID MONOSULPHATES spectrum was consistent with the original compound being a disulphate salt of chenodeoxycholic acid. The mass spectra of the derivatives of both disodium chenodeoxycholate 3-sulphate and disodium chenodeoxycholate 7-sulphate were similar but not identical. Both had a base peak at mle 370 [M(60+90)] and a prominent peak at mle 460 (M-60); in addition peaks were present at mle 315 [M(115+90)], mle 255, mle 228 and mle 213. There was no peak at mle 243. These mass spectra were consistent with a structure of methyl chenodeoxycholate monoacetate monotrimethylsilyl ether, indicating that the original two compounds were monosulphate salts of chenodeoxycholic acid.
Methods Measurement of radioactivity. Portions of urine and perfusate were prepared for scintillation counting by incubation with 1 ml of NCS solubilizer (Amersham/Searle Corp., Arlington Heights, IL, U.S.A.) at 38°C for 1 h. Radioactivity was measured by adding portions of solubilized urine or perfusate and eluates from Sephadex LH 20 or silica gel to 10ml of a scintillant mixture consisting of 2,5diphenyloxazole (4g), 1,4-bis-(5-phenyloxazol-2-yl)benzene (50mg) and Triton X-100 [Rohm and Haas (U.K.), Ltd., Croydon CR9 3NB, U.K.] (500ml) in toluene (1 litre) and counting in a Philips liquidscintillation spectrometer. Quench correction was by external standardization. Isolatedperfused rat kidney. Male Sprague-Dawley rats weighing 350-450g were maintained on a standard laboratory diet (modified 41B; Oxoid Ltd., Southwark Bridge Rd., London S.E.1, U.K.) for these experiments. A modification ofthe perfusion technique of Nishiitsutsuji-Uwo et al. (1967) was used. Under ether anaesthesia the ureter was catheterized (PP1O; Portex Ltd., Hythe, Kent, U.K.) and the right kidney was prepared for perfusion by inserting a polyethylene cannula (internal diameter 2mm) into the inferior vena cava, distal to the renal vein for the renal-venous effluent. A curved metal cannula was placed in the mesenteric artery and advanced across the aorta into the renal artery, with the perfusate flowing, so that renal plasma flow was unimpaired. The kidney was dissected from the animal, and the venous cannula connected into the circuit. The protein-free perfusion medium consisted of a Krebs improved Ringer 1 solution (Krebs, 1950) modified by substituting 6.0 % (w/v) dextran (mol.wt. 70000) in 0.154M-NaCl (Lomodex 70; Fisons Ltd., Loughborough, Leics., U.K.) for the 0.9 % NaCl and adding 5mM-urea. The medium (120ml) was maintained at 38°C, equilibrated at pH7.4 with 0.245MNaHCO3 and gassed with 02+CO2 (95:5). It was recirculated by pulsatile flow at 32-34ml/min and Vol. 156
341
continuously filtered by an in-line nylon filter, 14pm pore size (Millipore S.A., Buc, France). The function of the isolated kidney perfused with a protein-free medium has been evaluated and shown to remain stable over a perfusion period of I h (Gollan & Billing, 1975). The values obtained for the glomerular filtration rate and effective renal plasma flow were within the normal range for the intact rat (Spector, 1956). A 10min control period was allowed for mixing of the added bile acid, and recirculation of the perfusate continued for a 1 h test period. Urine was collected over 10min intervals and midpoint samples of perfusate were taken to calculate clearance values. After perfusion for 1 h, the glomerular filtration rate and effective renal plasma flow were estimated simultaneously during a further 10min collection period by the addition of 5'Cr-EDTA and sodium [I251]iodohippurate respectively. Radioactivity was measured with a dual-channel y spectrometer (GTL 300; Wallac, South Croydon, Surrey, U.K.), and the counts were corrected for spill-over. The two nuclides were readily distinguished by their different radiation energies. The values were comparable with those previously reported for this kidney preparation. In seven experiments, 5cCi of sodium (24-14C]chenodeoxycholate with 10mg of chenodeoxycholic acid as carrier (final perfusate concentration approx. 200pM) was dissolved in 2 ml of 0.245 M-NaHCO3 and added to the perfusate. In two experiments, sodium [24-14C]chenodeoxycholate and crystallized bovine serum albumin (1 g/100ml) (Armour Pharmaceutical Co., Eastbourne, Sussex, U.K.) were dissolved in the perfusate. In another two experiments with sodium [24-14C]chenodeoxycholate, kidneys were obtained from rats whose bile ducts had been ligated 14 days or 21 days before the perfusion. In one experiment, 1 mCi of Na235SO4 was administered subcutaneously to a rat, 24h later the kidney was perfused with 0.5mCi of Na235SO4, and 10mg of chenodeoxycholic acid was added to the medium. In three experiments, 50Ci of sodium [24-14C]lithocholate (final perfusate concentration approx. 0.8pM) was dissolved in 2ml of 0.245M-NaHCO3 and added to the perfusate. The low solubility of lithocholic acid in the perfusate precluded the addition of carrier. Fractionation of bile acids. The urine (about 20ml) was adjusted to pH10 with 1 M-NaOH, and 5g of Amberlite XAD-7 [Rohm and Haas (U.K.) Ltd.] was added (Summerfield et al., 1976). After mixing for I h, the urine was decanted and the resin washed with water adjusted to pH 10 with 1 M-NaOH. The water was decanted and the bile acids were eluted from the resin with 3 x 10ml of methanol. The methanol extracts were pooled and evaporated to dryness under reduced pressure at 60°C. The residue was dissolved with ultrasonic agitation in 1.5ml of chloroform/
342
J. A. SUMMERFIELD, J. L.- GOLLAN AND B. H. BILLING
methanol (1: 1, W/v) containing 0.01 M-NaCl and centrifuged at 300g for 5mim. Then 1 ml of the supernatant was loaded, via a polytetrafluoroethylene rotary valve (Jobling Laboratory Division, Stone, Staffs. ST15 OBG, U.K.) on to a 6g column (1 cmx45cm) of Sephadex LH 20 (Pharmacia, Uppsala, Sweden) which had been equilibrated with this solvent. The column was eluted with chloroform/ methanol (1:1, v/v) containing 0.01 M-NaCl, in 4ml fractions; the fractions between 0 and 52 ml contained the free and glycine- and taurine-conjugatedbileapids. The eluent was changed, to methanol and the fraction between 53 and lOOml contained the, bile acid sulphates (Sjovall & Vihko, 1966). When the labelled bile acids were submitted to chromatography on Sephadex LH 20, all the radioactivity was eluted between 0 and 52ml (non-sulphate fraction). Solvolysis.. Solvolysis was -performed by first dissolving the samples, with ultrasonic agitation, in acetone/ethanol/2M-HCl (18,:2:1, by vol.) and then heating for 48h at 37°C (Burstein & Lieberman, 1958; Palmer & Bolt, 1971). The reaction mixture was evaporated to dryness under reduced pressure at 60°C. fi-Glucuronidase treatment. Solutions of bacterial /)-glucuronidase (200 Fishman units/ml) and glucaro1,4-actone (136mM) were prepared in 66mm-sodium phosphate buffer (pH6.8) and passed through filters, 0.45gm pore size (Swinnex 13; Millipore S.A.) to remove viable bacteria, which could give rise to artifact formation (Manson et al., 1972). One,portion of the dried sample under test was dissolved-in 1 ml of Il-glucuronidase solution and 1 ml of 66mM-phosphate buffer (pH6.8), and another portion was dissolved in 1 ml of f-glucuronidase solution and 1 ml of glucaro-1,4-lactone solution added to inhibit the enzyme (Levvy, 1952). The mixtures were incubated for 24h at 37°C and then evaporated to dryness under reduced pressure at 60°C. Results and Discsion The renal clearance of bile acids after 30min perfusion was calculated from the ratio UV/P, where U and P represent the urine and perfusate radioactivity per unit volume and V the urine flow (ml/rnin) The clearance of chenodeoxycholic acid (mean 0.134ml/min, n=7) was five times greater than of lithocholic acid (mean 0.025ml/min, n=3). Bileduct ligation had no effect on the clearance of chenodeoxycholic acid. As expected, the addition of bovine serum albumin- markedly decreased the clearance (0.006m/min), presumably because binding of bile acid to protein decreases the free fraction available for glomerular filtration. The use of a protein-free perfusate was therefore important in the dqign, of the ex,periments.
Fractionation ofurine bile acids
Perfusion of the kidney with sodium [24-14C]chenodeoxycholate resulted in the excretion of the compound together with two polar metabolites (A and B), which were detected in the fraction eluted with methinol on Sephadex LH 20 (Fig. 1). In six experiments these Ipolar metabolites comprised 2.42 ±0.43 o/ (mean±s.E.M.) of the total urine radioactivity, the greatest amount being 5.06 %. On average, the radioactivity in peak B (1.84± 0.28% of total urine radioactivity) was three times as great as in peak A (0.58±0.2% of total urine radioactivi'ty), but in three experiments only traces of peak A were present. The relative size of each peak did not appear to be related to the function of the isolated kidney. The two polar metabolites of chenodeoxycholic acid found in the urine were also present in the dextran perfusate at the end of the experiment. The addition of bovine serum albumin to the perfusate in two experiments resulted in a 20-fold
'0 .-
-0
;...
C4
01 0 o
50
100
Eluate (ml) Fig. 1. Fractionation ofurine from an isolated rat kidney, perfused with sodium [24-14CJchenodeoxycholate, on a column of Sephadex LH 20 Elution with chloroform/methanol (1:1, v/v) containing 0.01 m-NaCl, in 4ml fractions, yielded a major peak, representing sodium f24-14C]chenodeoxycholate. When the eluent was changed to methanol after 52ml, two polar metabolites (A and B) of sodium [24-14C]chenodeoxycholate appeared. 1976
RENAL SYNTHESIS OF BILE ACID MONOSULPHATES decrease in the renal clearance of radioactive cheno-
343
ment. The conversion of chenodeoxycholic acid into more polar compounds by kidneys from rats with chronic bile-duct obstruction was similar to that observed in the normal rats (3.2 and 1.2% of total urine radioactivity), suggesting that cholestasis in itself did not enhanoe the conversion. In three experiments sodium [24-14C]lithocholate was added to the dextran perfusate, and Sephadex LH 20 fractionation revealed that in addition to the peak of radioactivity of sodium [24-14C]lithocholate in the non-sulphate fraction, a polar peak occurred in the fraction eluted with methanol. This compound had an elution volume similar to that of peak B
polar metabolites of sodium [24-14C]chenodeoxycholate, from an experimnent in which predominantly peak B was formed, were co-chromatographed on t.l.c. plates with standard sulphate salts of chenodeoxycholic acid (Fig. 2). Most of the radioactivity (71 %) had the mobility of chenodeoxycholate 7-sulphate, and only 2 % of the radioactivity was in the chenodeoxycholate 3-sulphate. Radioactivity was also present in the position of chenodeoxycholic acid (26 %), indicating partial degradation as discussed above. No radioactivity was present as chenodeoxycholate 3,7-disulphate. The polar metabolite of sodium [24- C]lithocholate was co-chromatographed with authentic diammonium lithocholate 3-sulphate and lithocholic acid on t.l.c. Most of the radioactivity (91 %) had the mobility of lithocholate 3-sulphate. The remainder (9%) was present in the lithocholic acid fraction, probably owing to breakdown of the sulphate salt during the procedure.
Identification ofpolar metabolites The polar metabolites of 'sodium [24-14C]chenodeoxycholate (peaks A and B) from six experiments were combined separately and evaporated to dryness. One portion (about 3 x 1 03d.p.m.) of each peak was hydrolysed by solvolysis, another portion was incubated with p8-glucuronidase and a -third was incubated with Ii-glucuronidase and glucaro-1,4lactone. After drying, the residues were applied to columns of Sephadex LH 20. The samples of peaks A and B, hydrolysed- by solvolysis, were eluted in the non-sulphate fraction, having thesameelutionvolume as chenodeoxycholate. After treatment with a1glucuronidase, some radioactivity was eluted in the non-sulphate fraction, with the same elution volume as the chenodeoxycholate, but most of the sample was eluted in the sulphate fraction. Similar results were obtained after incubation with f8-glucuronidase and glucaro-1,4-lactone. The breakdown of these compounds during incubation with fl-glucuronidase was therefore a non-specific effect, since it was- not abolished by glucaro-1,4-lactone, a specific inhibitor of f8-glucuronidase (Lewy, 1952). It was unlikely to be due to artifact formation by viable bacteria in the enzyme preparation, since this was filtered before use (Manson et al., 1972). Further, when a portion of peak-B material, without any further treatment, was applied to a Sephadex LH 20 column, the polar metabolite decomposed to a similar extent. This apparent lability of the compounds was probably due to degradation on the column, but was not investigated further. It was concluded that peaks A and B consisted of conjugates which were hydrolysed by solvolysis and did not contain bile acid glucuronides. Similar results were obtained with the .polar metabolite of sodium [24-14C]lithocholate. To determine the nature of these conjugates, the Vol. 156
Incorporation of [35S]sulphate into chenodeoxycholic acid To confirm the 14C studies, an experiment was performed to determine whether the isolated rat kidney could. incorporate [35S]sulphate into chenodeoxycholic acid. The rat received Na235SO4 24h before the experiment in order to label the sulphate donor, 3'-phosphoadenosine 5'-sulphatophosphate. Theurine bile acids from the isolated kidney were fractionated on Sephadex LH 20 and the polar fraction was co-chromatographed on t.l.c. plates with reference sulphate salts of chenodeoxycholic acid and chenodeoxycholic acid (Fig. .2). Most of the "5S (83%) had the mobility of chenodeoxycholate 7-sulphate and only 2 % was present in the position of chenodeoxycholate 3-sulphate. The radioactivity (1.2 O) remaining At the origin was probably inorganic sulphate. Some radioactivity (4.7%O) had the mobility of chenodeoxycholate 3,7-disulph4te. Radioactivity was also present in a more polar position (8.8 %O) and probably represented incorporation of [35S]sulphate into other organic compounds excreted by the kidney into the urine. Fig. 2 illustrates the similar pattern of incorporation of both the 14C and 35S labels into the sulphate salts of chenodeoxycholic acid. This is the first demonstration that urine bile acid sulphates can be synthesized in the kidney and is consistent with the preliminary report by Chen et al. (1974) of an enzyme present in the rat kidney that sulphates bile acids. We have extended their observations by demonstrating that the predominant compounds formed in the kidney from lithocholic acid and chenodeoxycholic acid are the 3-monosulphate and 7-monosulphate respectively. Further studies are needed to determine why the predominant monosulphate of chenodeoxycholic acid is sulphated
deoxycholic acid; no polar metabolites were detected in the une or the perfusate at the end of the experi-
obtained with sodium [24-14C]chenodeoxycholate and accounted for 2.98, 3.60 and 4.00% of the total radioactivity recovered from the urine.
J. A. SUMMERFIELD, J. L. GOLLAN ANT) B. H. BILLING
344
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7
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ky6
0
3-
2-
x
Fig. 2. Incorporation of radioactivity into chenodeoxycholate sulphates in rat urine The polar metabolites of chenodeoxycholic acid, eluted with methanol from Sephadex LH 20, were co-chromatographed on t.l.c. plates with standard sulphate salts of chenodeoxycholic acid in the solvent system of Cass et al. (1975). (a) shows the chromatogram of trisodium chenodeoxycholate 3,7-disulphate (3,7), disodium chenodeoxycholate 7-sulphate (7), disodium chenodeoxycholate 3-sulphate (3) and chenodeoxycholic acid (free). The bile acids were detected by the method of Usui (1963). (b) shows the distribution of radioactivity after t.l.c. of the polar metabolites from an experiment in which a kidney was perfused with sodium [24-14CJchenodeoxycholate. (c) shows the distribution of radioactivity in the polar metabolites from an experiment in which a kidney was perfused with Na235SO4 and chenodeoxycholic acid. Most of the radioactivity has the mobility of disodium chenodeoxycholate 7-sulphate; trace amounts appear as disodium chenodeoxycholate 3-sulphate.
at C-7, and not at C-3 as Makino et al. (1974) had proposed. It now seems probable that there are several bile acid sulphokinases with different specificities in kidney tissue. In this regard, it is pertinent that monosulphated Clg steroid diols with a 171?hydroxyl group are sulphated at C-3 whereas the 17a epimers are sulphated at C-17 (Ruokonen & Vihko, 1974). The observation that the isolated rat kidney can synthesize bile acid monosulphates lends support to
the concept that some of the bile acid sulphates in human cholestatic urine may originate in the kidney. Makino et al. (1974) and Stiehl et al. (1975) have shown that sulphate salts of chenodeoxycholic acid are the predominant components of the bile acid sulphate fraction in the urine of patients with the cholestatic syndrome and about 80% of these are monosulphates (Stiehl, 1974). However, in man both the nature of the bile acid monosulphates and the role of the kidney in the disturbed bile acid metabolism of cholestasis remain to be established. Note Added in Proof (Received 26 January 1976) Since this paper was submitted for publication, Parnentier et al. (1975) have reported that cholate 7-sulphate is a major component of the faecal bile acids of the mouse, which suggests that sulphation at C-7 may be the usual pattern of sulphation for dihydroxy and trihydroxy bile acids. We thank Mr. M. Chu and Dr. K. Setchell (MRC Clinical Research Centre, Harrow, Middx. HAl 3UJ, U.K.) for the mass spectrometry and Mr. E. Shaw for assistance with the animal experiments. We also thank Dr. P. Back (Medizinische Klinik der Universitait Freiburg/Brsg., West Germany) and Dr. S. Barnes for helpful advice. We are especially grateful to Professor G. A. D. Haslewood (Guy's Hospital Medical School, London, SE1 9RT, U.K.) for the help and advice that made this study possible and for allowing the use of his unpublished work. J.L.G. was supported by the Wellcome Trust and J.A.S. by a Medical Research Council Clinical Research Fellowship. References Burstein, S. & Lieberman, S. (1958) J. Am. Chem. Soc. 80, 5235-5239 Cass, 0. W., Cowen, A. E., Hofmann, A. F. & Coffin, S. B. (1975) J. Lipid Res. 16, 159-160 Chen, L.-J., Bolt, R. & Admirand, W. H. (1974) Gastroenterology 67, Abstr. 782 Cronholm, T. (1969) Steroids 14, 285-296 Ganshirt, H., Koss, F. W. & Morianz, K. (1960) Arzneim.Forsch. 10, 943-947 Gollan, J. L. & Billing, B. H. (1975) Clin. Sci. Mol. Med. 49,28 Haslewood, E. S. & Haslewood, G. A. D. (1976) Biochem. J. 155, 401-04 Krebs, H. A. (1950) Biochim. Biophys. Acta 4, 249-269 Levvy, G. A. (1952) Biochem. J. 52,464-472 Makino, I., Shinozaki, K., Nakagawa, S. & Mashimo, K. (1974) J. Lipid Res. 15, 132-138 Makita, M. & Wells, W. W. (1963) Anal. Biochem. 5, 523530 Manson, M. E., Nocke-Fink, L., Gustafsson, J.-A. & Shackleton, C. H. L. (1972) Clin. Chim. Acta 38, 45-49 Mumma, R. 0. (1966) Lipids 1, 221-223 Nishiitsutsuji-Uwo, J. M., Ross, B. D. & Krebs, H. A. (1967) Biochem. J. 103, 852-862
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RENAL SYNTHESIS OF BILE ACID MONOSULPHATES Palmer, R. H. (1971) J. Lipid Res. 12, 680-687 Palmer, R. H. & Bolt, M. G. (1971) J. Lipid Res. 12, 671679 Parmentier, G., Mertens, J. & Eyssen, H. (1975) in Advances in Bile Acid Research (Matern, S., Hackenschmidt, J., Back. P. & Gerok, W., eds.), pp. 139-143, F. K. Schattauer Verlag, Stuttgart and New York Ruokonen, A. & Vihko, R. (1974) Steroids 23, 1-16 Schwenk, E., Riegel, B., Moffett, R. B. & Stahl, E. (1943) J. Am. Chem. Soc. 65, 549-551 Sjovall, J. & Vihko, R. (1966) Acta Chem. Scand. 20, 1419-1421
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Sjovall, J., Eneroth, P. & Ryhage, R. (1971) in The Bile Acids: Chemistry, Physiology and Metabolism (Nair, P. P. & Kritchevsky, D., eds.) vol. 1, pp. 209-248, Plenum Press, New York and London Spector, W. S. (1956) Handbook ofBiological Data, p. 341, W. B. Saunders Co., Philadelphia and London Stiehl, A. (1974) Eur. J. Clin. Invest. 4, 59-63 Stiehl, A., Earnest, D. L. & Admirand, W. H. (1975) Gastroenterology 68, 534-544 Summerfield, J. A., Billing, B. H. & Shackleton, C. H. L. (1976) Biochem. J. 154, 507-516 Usui, T. (1963) J. Biochem. (Tokyo) 54, 283-286
