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Bile Acid Metabolism in Mammals. VIII. Biliary Secretion of Chulylarginine by the Isolated Perfused Rat Liver D e p a r t ~ n e n tqf , ~Medicine rand Pcrtholugy, Lrnivewsity of Toronto, Toronto, Ontario ,V5S IAX Received January 29, 1975

YOUSEF,I. M.,and FISHER,M. M. 1975. Bile acid metabolism in mamn~als.VBII. Biliary secretion of cholylarginine by the isolated perfused rat liver. Can. J. Physiol. Pharmacol. 53, 880-887. In st~adiesof cholic acid metabolism using the isolated perfused rat liver system, an unknown conjugate of cholic acid was observed. This conjugate comprised 15-27% of the biliary bile acids in these experiments, was less polar than cholylglycine on thin-layer chromatography using butanol, acetic acid, and water, and had an apparent molecular weight greater than that of cholyltaurine on gas-liquid chromatography. Amino acid analysis of the hydrolyzed conjugate demonstrated the presence of arginine. Perfusion studies with radioactive arginine, and mass bpec tron~etricanalysis p~ovedthat the con-jugate was ckaolylarginine. Secretion of this conjugate does not represent a deficiency sf available glycine and taurine. YOUSEF.I. M. et FISHEK,M. M. 1975. Bile acid metabolism in mammals. VIII. Biliary secretion of cholylarginine by the isolated pelfused rat liver. Can. J . Physiol. Phrmacol. 53, 880-887. Dans des etudes concernant Be rnetabolisme de l'acide choliqe~efaites a l'aide d'un systkme de perfusion du foie de rat isole, on a observe un conj~agueinconnu de l'acide cholique. Ce conjugue contient 1527% des acides biliaires, est rnoins polaire que la cholylglycine dans Ies chromatographies en couche mince faites B l'aide de butanoi, d'acide acetique et d'eau, et a nn poids moleculaire apparent plus Clevi que celrri de la cholyltaurine, tel que ditermine par chromatographie gar-liquide. L>'annlyse des amino acides du compose hydroilysi montre la presence d'arginine. kes etudes de pepfusion B I'arginine radioactive et la spectrometrie de masse prouvent que le conjugue est la cholylarginine. La secretion de ce conjugu6 n'est pas due 5 une deficience en glycine oea en taurine. [Traduit par le journal]

A considerable body of evidence supports the current dognaa that only conjugated bile acids are secreted into the bile and that conjugation of bile acids is restricted to the two amino acids, glycine and taurine. The only evidence to the contrary, in terms of the nature of the conjugate, appears to be that of PericGolia and Jones ( 1962, 1963) and of Gordon et ak. (19631, who reported the formation of cholylornithine by guinea pigs and by humans. During studies on the kinetics of cholic acid uptake and secretion by the isolated perfused rat liver we observed the biliary secretion of two conjugates other than cholylglycine and chslyltaurime. One of these two unknown con'Presented in part at the annual. meeting of the American Association for the Study of Liver Diseases, Chicago, October 1973; the Canadian Hepatic Foundation National Conference on 'Bile Acids' in Montreal., January 1974; and the HHH Bile Acid meeting in Freiburg, June 1974. 3Dr. Yousef is a Scholar of the Canadian Hepatic Foundation.

jugates has been found to be cholylarginine. This report deals with the characterization and identification of cholylarginine and with some of the phenomena associated with its for.

Materials and Methods Cholic acid (3a,4a,l2n-trihydroxy-5p-ckolanicacid) was obtained from Calbiochem (San Diego, Calif,). No trace of contaminants was found on either thinlayer or gas chromatographic analysis and the cornpound was therefore used without further purification. [24-'4C]Cholic acid, specific activity 40 mCi/ mmol and [2,4-"H]cholic acid, specific activity 3.8 Ci/rnmol, were obtained from New England Nuclear (Boston, Mass.). Aliquots of these materials were taken to dryness under nitrogen, and 4 mg of nonr,adioactive cholic acid were added. The mixture was dissolved in 2 rnl of methanol and esterified as previously described, (Yousef et ak. 1972). The methyl esters were analyzed by thin-layer chromatography using chloroform - acetic acid - methanol, 70:25:5 (v/v/v). The bands were isolated and the radioactivity was determined. Cholic acid accounted for 98% of the radioactivity which was recovered in the 14C studies and 97% in the tritium studies. On the

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YOUSEF A N D FISHER: BILE ACID ME,TABOLISM IN MAMMALS. VIlI

basis of these recoveries the radioactive cholic acid was used without purification. [U-14C]Arginine, specific activity 300 mCi/mmol, was obtained from New England Nuclear (Boston, Mass.). The purity of arginine was 99% on the basis of tlc recovery experiments using N-butanol - acetic acid - water, 25:4: 10 (v/v/v) . Lecithin, bovine, was obtained from Nutritional Biochemicals Corporation (Cleveland, Ohio) ; carbenicillin (Pyopen) from Ayerst Laboratories (New York, N.Y.) and heparin sodium U.S.P. from Connaught Medical Research Laboratories (Toronto Ont.). Taurine and glycine were obtained from Calbiochem (San Diego, Calif. ) . Perf usion Experiments Wistar rats (Nigh Oak Ranch, Toronto, Ont.) of both sexes, and weighing approximately 250 g, were used as liver donors. Prior to use the animals were kept in a constant temperature environment (22 "C); were in darkness for 12 h each day (7 pm - 7 am); were allowed water ad libitum; and for 1-2 weeks, from 4 pm to 9 am daily, were given a powdered semi-synthetic diet (Teklad Mills, Madison TD-72460 basal diet with 27% casein and salt mixture USP XIV). The animals were starved 24 h before use and anesthetized with diethyl ether. The perfusion technique, which involves hepatectomy and recirculation of the perfusion medium, has been described in other communications (Fisher and Kerly 1964; Fisher et al. 1971). The perfusion medium used was whole rat blood containing heparin 7 Units/ml, carbenicillin 200 pg/rnl, and added glucose 1 mg/ml. After 2 h of perfusion, a period apparently sufficient to deplete the system of endogenous bile acids, 6 ml of saline solution containing lecithin and bile acid were added as a single bolus to the 100 ml of perfusion medium. The saline solution contained lecithin at a concentration of 7.5 mg/ml and cholic acid at a concentration of 0.17 or 5.0 pmollml. Thirty-four perfusion experiments were performed and certain details of these are listed in Table 1. Each perfusion was continued for 3 h following the addition of the bile acid to the perfusion medium and bile was collected in hourly aliquots. Isolation and Decorzjugation of Bile Acids Bile was deproteinized by the addition of 10 volumes of a hot ethanol-methanol solution, 95:5 (v/v). The tubes were capped, vibrated, and then centrifuged at 3000 rpm at 4 "C for 15 min. The alcoholic supernatant was decanted and the precipitate washed once with 2 ml of methanol. The alcoholic extracts were combined, delipidated with petroleum ether, 30-60 "C, and then evaporated to dryness under nitrogen. The residue was then dissolved in 1 ml of methanol. A 0.5 ml sample of this methanol solution was used for analysis of the conjugated bile acids, the remainder for analysis of the total bile acids. A known volume of bile acid extract was applied to thin-layer plates, 20 X 20 cm, and coated with silica gel G 0.25 mm thick. The plates were developed 4 h in butanol - acetic acid - water, 10: 1: 1 (v/v/v). The conjugates were identified by comparison with known standards of conjugated bile

881

acids obtained from Supelco Inc., Bellef onte, Pa. After drying, the spots were located by iodine vapour and scraped. The bile acids were extracted from the silica gel by 0.05 N HCl and 75% ethanol. Recovery experiments have indicated an efficiency greater than 95% for this procedure (Fisher et al. 1974). The conjugated bile acids were subjected to hydrolysis in hot NaOH. After acidification, the free bile acids were extracted with diethyl ether, taken to dryness, dissolved in methanol, and esterified. The bile acid methyl esters were converted into their trifluoroacetates or trimethylsilyl ethers and analyzed by glc as described previously (Yousef et al. 1972). Conjugated bile acids were also subjected to glc. A 2-ft (1 ft = 0.31 m ) glass column was used and the TMS derivatives of the conjugates were separated using 3% OV-1 on Gas Chrom Q 100-120 mesh (Applied Science Laboratories) as the stationary phase, a temperature of 300 "C, a flow rate of 60 cm3/min and a flame ionization detector. This technique, based on a report by Hanaineh and Brooks (1964) does not separate individual bile acids under these conditions but it does separate the glycine conjugates from the taurine conjugates and probably, at least in part, on the basis of their molecular weights. Synthesis o f Cottjugated Bile Acids Conjugation of cholic acid with the a-amino group of arginine was carried out using the method of Bergstrom and Norman (1953) and was verified by mass spectrometry (Myher et al. 1975). Amino Acid Analyses Conjugated bile acids were extracted from bile and then hydrolyzed in 6 N HCl for 24 h in a sealed tube at 110 "C. The amino acids involved in conjugation with the bile acids were measured without further treatment using a Beckman 120C amino acid analyzer. The basic elution procedures were the standard ones for physiological Auids using sodium citrate buffers (Beckman procedure manual 120 PM- 1) . Mass Spectrometry Mass spectrometric analyses of the bile acid conjugates were performed in the laboratory of Dr. Arnis Kuksis, using a Varian Mat CH-5 single focussing mass spectrometer, coupled to a Varian G20 i computer and direct inlet probe (Myher et al. 1975). Radioactive Measurements Total radioactivity in the bile was measured by direct counting of a known volume of bile after Protosol (New England Nuclear) digestion. The radioactivity of each conjugated bile acid was determined by direct counting of its silica gel scrapings after addition of 15 ml Aquasol (New England Nuclear) (Fisher et al. 1974). The samples were counted for 10 min in triplicate in a Packard liquid scintillation spectrometer, equipped with an automatic external standard. The counts were corrected for quenching and the efficiency was 80% for 'Cin the single label experiments, 20% for 14C, and 30% for 'H in the double label experiments. Experiments demonstrated that 89-9496 of the total radioactivity in the bile was recovered after thin-layer chromatography of the conjugated bile acids.

Group

n

Sex

30 30 30 30

Cholic acid, pmol

1 1

-

-

[2,4-3N]Cholic acid, pCi

E"C]Arginine, pCi

Additions to perfusion medium

Perfusion experiments

124-"C]ChoIic acid, pCi

TABLE 1.

Taurine, pmol

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Glycine, l~rnol

*76 F2

z

'd

P

2 5

z

'd

!-

r

5.

n

YOUSEF AND FISHER: BILE ACID METABOLISM IN MAMMALS. VIII

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TABLE 2. Percentage of radioactivity of biliary bile acidsa

+

Unknown compound 11 Cholylglycine Unknown compound I Cholyltaurine Origin

15.066- 1 .47 0.83k0.31 1.4320.48 82.32fa1.74 0.58k0.09

27.23 1 .73 0.77k0.16 1.34+0.07 70.36f 1.79 0.30f 0.05

Bile acid metabolism in mammals. VIII. Biliary secretion of cholylarginine by the isolated perfused rat liver.

In studies of cholic acid metabolism using the isolated perfused rat liver system, an unknown conjugate of cholic acid was observed. This conjugate co...
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