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Metabolism of Lithocholate in Healthy Man
GASTROENTEROLOGY 69:59-66, 1975 Copyright © 1975 by The Williams & Wilkins Co.
Vol. 69, No. I Printed in U.S.A .
METABOLISM OF LITHOCHOLATE IN HEALTHY MAN I. Biotransformation and biliary excretion of intravenously administered lithocholate, lithocholylglycine, and their sulfates ALISTAIR E . COWEN, M.R.A.C.P., MELVYN G. KOHMAN, M.R.A.C .P ., ALAN F. HOFMANN, M.D ., AND OLIVER w. CASS
M.B.B.S.,
PH.D.,
Gastroenterology Unit, Mayo Clinic and Mayo Foundation, Rochester, Minnesota
The metabolism of intravenously injected radiolabeled lithocholate, lithocholylglycine, and their 3a-sulfate esters was characterized in healthy subjects. Lithocholate radioactivity was excreted rapidly and predominantly in bile; the excreted radioactivity had the chromatographic properties of glycine and taurine conjugates of lithocholate, of which 60% were sulfated. Lithocholylglycine also was excreted rapidly and predominantly in bile, and 60% of excreted radioactivity was sulfated. Sulfolithocholate radioactivity was only partially conjugated (about 60%) in association with biliary excretion. Sulfolithocholylglycine was excreted unchanged in bile. Neither sulfated derivative showed appreciable excretion in urine, although both were excreted more slowly in bile than unsulfated free or conjugated lithocholate. The data suggest that unconjugated lithocholate which is absorbed is completely conjugated and partially sulfated before excretion which occurs exclusively in bile. Since sulfation is not complete, some unsulfated lithocholate is always present in bile. This conjugated but unsulfated lithocholate, if reabsorbed, would be again partially sulfated during its next enterohepatic circulation. Thus, the end result of these biotransformations would be for absorbed lithocholate to be excreted in bile mostly, but not entirely as the sulfated conjugates. 5% lithocholate when analyzed by chromatography after alkaline saponification, 5 • 6 it has generally been held that in healthy man little lithocholate is absorbed from the intestine. 7-9 Lithocholate is unequivocally hepatotoxic when administered orally in a variety of experimental animals, 9 and
Lithocholic acid (lithocholate), which is formed in the distal intestine by bacterial 7 a-hydroxylation of chenodeoxycholic acid, is a major fecal bile acid in man. 2 ' 4 Because biliary bile acids contain less than Received November 11, 1974. Accepted February 10, 1975. This paper was presented in part at the 1974 Meeting of the American Association for the Study of Liver Disease and published in abstract form.' Address requests for reprints to: Alan F. Hofmann, M.D., Gastroenterology Unit, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55901. Supported in part by Research Grant AM-16770 from the National Institutes of Health and hy grants· in-aid from the Share Foundation, Mead ,Johnson & Company, Eli Lilly and Company, and Weddel Pharmaceuticals (U.K.) .
Dr. Cowen is an Eli Lilly Fellow, Australia. His present address is: Department of Medicine, Hoyal Brisbane Hospital, Queensland, Australia . Dr. Korman is an Eli Lilly Fellow, Australia. His present address is: Monash University, Department of Medicine, Prince Henry Hospital, Melbourne, Australia. Mr. Cass is a technical assistant. His present address is: School of Medicine, University of Min· nesota, Minneapolis, Minnesoa 55455. !i9
60
COWEN ETAL.
these observations led to speculation by Carey 10 that lithocholate might be involved in the initiation or perpetuation of hepatic disease. Despite this, little is known of the metabolism of lithocholate in healthy man. Carey and Williams 11 administered 14 [ C ]lithocholate to 2 patients with bile fistulas and concluded that lithocholate was conjugated with glycine and taurine but not further biotransformed during hepatic passage. This conclusion was questioned by Palmer and Bolt 12 who found that administered lithocholate could be recovered from human bile as the sulfate. Palmer and Bolt showed further that conventional techniques for biliary bile acid analysis that do not include a solvolysis step may not yield a valid recovery of lithocholate if it is present as the sulfate, which suggests that previous analyses of biliary bile acid composition may have underestimated the proportion of lithocholate in bile. 12 Because of these conflicting observations, and because of the increasing use of chenodeoxycholic acid as a specific agent for cholesterol gallstone dissolution in man, 13 we have carried out studies aimed at a more complete definition of lithocholate metabolism in healthy man. In this paper we report experiments that define the metabolism .of intravenously administered radiolabeled lithocholate, its glycine conjugate (lithocholylglycine), its sulfate (sulfolithocholate), and its sulfated glycine conjugate (sulfolithocholylglycine). A second paper 14 describes experiments aimed at elucidating the influence of sulfation on the site and efficiency of intestinal absorption in man, as well as measurements of lithocholate absorption (input) by the isotope dilution procedure of Lindstedt. The final paper in this series 15 describes the plasma disappearance of radioactivity after injection of free and conjugated lithocholate and their sulfates. Together, these studies show that lithocholate has an enterohepatic circulation of considerable magnitude in healthy man, during which it undergoes biotransformations unique among the other major primary and secondary bile acids.
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Materials and Methods Experimental design . Labeled bile acid was injected intravenously into a fasting subject, and serial sam pies of bile were collected from an indwelling duodenal tube. Complete recovery of bile was obtained by inflating an occlusive balloon in the jejunum and preventing gallbladder storage by infusing cholecystokinin intravenously throughout the course of the experiment. Three studies were carried out with lithocholate, two studies with sulfolithocholate, and a single study each with lithocholylglycine and sulfolithocholylglycine. In addition, single studies were carried out with the primary bile acids, cholic acid and chenodeoxycholic (chenic) acid, and their respective glycine and taurine conjugates. These experiments were carried out to validate complete recovery, as well as to define the time required for transit from blood to the sampling site. Subjects. Subjects participating in this study were healthy, young adult men judged to have normal liver function based on normal values for serum bilirubin, serum glutamic oxalacetic transaminase, serum alkaline phosphatase, sulfobromophthalein retention, protein electrophoresis, and fasting state serum cholic acid conjugates as determined by radioimmunoassay.'" Informed consent was obtained. Procedure. A fasting volunteer was intubated with a double lumen tube with an occlusive balloon that was positioned immediately proximal to the ligament of Treitz (verified by fluoroscopy with image intensification) . Gallbladder contraction was initiated and maintained by an infusion of cholecystokinin (Karolinska lnstitutet, Stockholm, Sweden) given at 75 Ivy dog units per hr ; this dose has previously been shown to induce vigorous gallbladder contraction and to be reasonably well tolerated in man. 17 After the majority of the fasting gallbladder bile had passed distally, the duodenal balloon was inflated and continuous duodenal aspiration was begun. In addition, throughout the study, gastric contents were aspirated by intermittent suction via a separate gastric tube. Labeled bile acids (10 to 30 !J.C) were then administered by rapid intravenous injection. Bile aspirated from the duodenum by siphonage was pooled in 30-min collections for 4 hr. Aliquots of bile were counted to calculate recovery of radioactivity, both during each 1/2-hr period and as cumulative recovery. Recovery of intravenously administered labeled bile acids exceeded 80% within the 4-hr study period in all cases. The total interruption of the enterohepatic circulation by the occluding duodenal balloon was. further confirmed by the
July 1975
LITHOCHOLATE METABOLISM IN HEALTHY MAN I.
recovery, in duodenal aspirates, of more than 90 % of a marker ( ["C ]polyethylene glycol, New England Nuclear Corp., Boston, Mass.) infused via the gastric tube . Isotopes. [24- "C ]Lithocholate (specific activity 4.2 me per mmole) was obtained from New England Nuclear or Mallinckrodt Chemical Co. (St. Louis, Mo.). [11,12- 3 H]Lithocholate (specific activity 1500 me per mmole) was prepared by reduction of ~ 11 -lithocholenic acid as described. 18 Lithocholylglycine (specific activity 750 me per mmole) was prepared by the mixed carboxylic carbonic anhydride method of Norman 19 and purified by thin layer chromatography (TLC). 20 Sulfolithocholate and its glycine conjugate were prepared from [11, 12- 'H ]lithocholate and [11 ,12-'H ]li thocholylglycine, respectively, as described by Palmer and Bolt. 12 All compounds were purified by preparative TLC; when analyzed by zonal scanning of thin layer chromatograms, purified compounds had a radiopurity greater than 98%. In addition, the radiopurity of (11, 12- 3 H ]lithocholate was confirmed by crystallization to a constant 'H: "C ratio after addition of [24- ••c ]lithocholate . Analysis of bile samples by TLC. The chemical form of radioactivity in duodenal samples was determined by TLC on Silica Gel H containing calcium sulfate ( 10%, w/w) and a solvent system of chloroform-methanol-acetic acid-water (65:24:15:9, v/v) 20 followed by zonal scanning of the plates. This system resolves lithocholate, its glycine and taurine conjugates, and the 3a-sulfate esters of each. All samples were also analyzed qualitatively by using the butanol-3 system of Palmer and Bolt" in which lithocholate and its derivatives have different relative mobilities. Identity of compounds was further confirmed in selected instances by cochromatography with reference substances tagged with another isotope. Nomenclature. Lithocholic acid will be referred to throughout as lithocholate, since it is assumed to exist as the anionic species in vivo. Its glycine conjugate will be referred to as lithocholylglycine (this was administered as the sodium salt and is likely to be present in vivo as the ionized form which might be termed a "glycinate"; this term has not achieved wide usage, however). The sulfate ester of lithocholylglycine will be referred to as sulfolithocholylglycine (which is not ideal because it does not indicate clearly that the sulfate group is fully ionized).
Results Lithocholate and its conjugates. When lithocholate was administered, radioactiv-
61
ity was excreted predominantly in bile; urinary excretion was less than Wi'o of the administered dose. All recovered radioactivity had the TLC mobility of glycine- or taurine-conjugated lithocholate or sulfated conjugated lithocholate . During the 1st hr, the majority of conjugated lithocholate was unsulfated, but the proportion that was sulfated increased progressively over the course of the 4-hr study so that virtually all of the 4-hr sample was both conjugated and sulfated (fig. 1 and table 1) . Of the total radioactivity recovered, 60% was conjugated and sulfated, and 40% was conjugated but unsulfated. When lithocholylglycine was injected, radioactivity was again excreted predominantly in bile, and the chemical form of biliary radioactivity showed a similar pattern and time course, except that taurine conjugates were not present (table 2). Again, early samples were excreted predominantly in unsulfated form, and samples obtained later were predominantly sulfated. At the end of the 4-hr period, 60% of the radioactivity had been sulfated, whereas 40% was excreted without sulfation . When the primary bile acids, cholic acid or chenic acid, were injected, radioactivity was excreted more rapidly in bile than after lithocholate or sulfolithocholate (fig. 2). When cholylglycine or chenodeoxycholylglycine was injected, radioactivity again appeared more rapidly than after 40
0
Conjugated, unsulfated
Bl Conjugated, sulfated
0
2
3
4
Hours
FIG. 1. Change, with time, in chemical form of radioactivity in bile after intravenous injection of labeled lithocholate. Results, expressed as percentage of administered dose, are mean of three experiments.
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COWEN ETAL.
TABLE
Vol. 69, No. I
1. Chemical form of radioactivity recovered in proximal intestine followinf{ intravenous injection of 3 [ H ]lithocholate" Distribution of radioactivity
Time interval
lit-gly
sul-lit-gly
Iit-tau
min
Recovered radioactivity' sul -l it-tau
Interval
Cumulative
%total
%
Subject 1
0-30 30-60 60-90 90- 120 120- 150 150- 180 180-210 210-240
44 47 25 14 2 3 3 4
42 44 24 14 4 8 12 15
<1 17 42 63 84 77
11
71 65
13 14
59 52 8 12 16
23 17 3 2 2 1 1 <1
3 28 76 73 71 69 67 65
<1 3 14
<1 <1 9 9 10
11.7 22.2 14.6 13.8 17.3 4.5 8.6 7.1
11.7 33.9 48.5 62 .3 79.6 84.1 92.7 99 .8
9.6 44.1 10.3 22.5 3.6 4.0 3.2 2.6
9.6 53.7 64 .0 86.5 90.1 94.5 97.7 100.3
Subject 2
0-30 30-60 60-90 90-120 120-150 150-180 180- 210 210-240
11
6 <1
11
9 18 26 35
a lit-gly, lithocholylglycine; Iit-tau, lithocho lyltaurine ; sul-lit-gly, sulfolithocholylglycine; sui -lit- tau, sulfo. lithocholyltaurine. Chemical identity was inferred from mobility during thin layer chromatograph y using two solvent systems. • Recovery of radioactivity was 91 % of administered dose for subject 1 and 82% for subject 2.
2. Chemical form of radioactivity recovered in proximal intestine following intravenous injec tion of [' H]litho cholylf{lycinea
TABLE
Time interval
Distribution of radioactivity lit-gly
min
30-60 60-90 90-120 120-150 150-180 180-210 210- 240
sul-lit-gly
Recove red radioactivity' Interval
80 60 37 26 16 16 16
Cumulative
%total
%
20 40 63 74 84 84 84
9.9 14.9 22.4 16.1 19.2 7.9 9.4
9.9 24.8 47.2 63.3 82.5 90.4 99.8
a lit-gly , lithocholylglycine ; su l-lit-gly, sulfolithocholylglycine. Chemical identity was inferred from mobility during thin layer chromatography using two solvent systems. • Insufficient radioactivity for zonal scanning was recovered during 0 to 30 min . No appreciable radioactivity appeared in lithocholyltaurine or sulfolithocholyltaurine. Sixty per cent of administered radioactivity was recovered.
injection of lithocholylglycine and sulfolithocholylglycine (fig. 3). Free primary bile acids were conjugated but not sulfated; the glycine conjugates were excreted without further biotransformation. Sulfolithocholate and its conjugates. In contrast to ·free lithocholate, cholic acid, or chenodeoxycholic acid, sulfolithocholate was only conjugated in part; 35% of radioactivity in bile had the mobility of unconjugated sulfolithocholate (fig. 4). The remainder appeared as sulfolithocholylglycine and sulfolithocholyltaurine. No radioactivity appeared in lithocholylglycine or lithocholyltaurine in any experiment, which indicates that net removal of the sulfate group did not occur in association with biliary excretion. When sulfolithocholylglycine was injected, it was excreted without modification, and its excretion had a time course similar to that observed for lithocholylglycine radioactivity (fig. 3). Thus, this bile
July 1975
LITHOCHOLATE METABOLISM IN HEALTHY MAN I. Cholic 60
63
Chenic
-
' Litho ch olic
40
Sulfa! ithochol ic
-
20
0
2
4
0
2
4
2
0
n 4
0
2
4
Hours FIG. 2 . Time course of excretion of radioactivity in bile after intravenous injection of unconjugated bile ac ids. Note t hat the prim ary b ile acids, cholic a nd chenodeoxycho li c (chenic) acids, are excreted more rapid ly than lithocholic acid or its sulfated derivatives. Points represent me a n s of t hree ex periments for lithocholate and of two experiments for sulfolithocholate. 80
Cho lylglyci ne ~
Chenylglycine
r:-
60
Lithocholylglycine
"tl'
~
'b
8"'
40
~
--
'b
~"'
20
'
I
~;;·
ll 0
2
4
r--
'
~ 0
2
4
0
2
n 4
Sulfolithocholylglycine
0
2
n 4
Hours FIG. 3. Cumulative excretion of radioactivity, ex pressed as percentage of adm inistered dose, after intravenous injection of labeled conjugated primary bile acids (chenodeoxycholylg lycine a nd cholylglyc ine) or labeled lithoc holylglycine and its sulfate. Points represent a s ingle experiment with each bile acid.
40
~ '<::>'
c=J 30
~
~ Uncanjugatt•d ,
'b
:.. tl
IJ
Conjugated, sulfated
sulfated
20
~
'b
"' tl
t::)
10
0
2
3
4
Hours FIG. 4. Change, with time, in che mical form of radioactivity in bile after intravenous injection of labeled sulfolithocholate into a healthy subject. Resu lts a re expressed as percentage of admin istered dose.
acid resembled cholylglycine and chenodeoxycholylglycine in undergoing biliary excretion without biotransformation.
Discussion Methodology . The method used in this study appears to be a novel and useful technique for defining biotransformations of drugs during their initial excretion in bile. The completeness of recovery indicated that gallbl adder storage did not occur and that intestinal aspiration was complete proximal to the occlusive balloon. Other alternatives, such as use of a patient with conventional or Baldwin Ttube , 21 are more difficult to arrange, and normal hepatic function cannot always be
64
COWEN ETAL.
guaranteed. An alternative to the use of a marker and a single balloon would be a three-lumen tube containing proximal and distal balloons inflated above and below the ampulla of Vater, respectively, 22 and this has been modified by the addition of an infusion port below the distal balloon. 23 Metabolism of lithocholate in man. These data provide additional evidence in support of the pioneering report by Palmer and Bolt 12 that lithocholate, whether free or conjugated, is largely sulfated before hepatic excretion in man. They suggest that lithocholate, which enters the bile acid pool from the intestine, will be completely conjugated and partially sulfated in association with biliary excretion. Conjugated but unsulfated lithocholate was also sulfated in association with biliary excretion. When lithocholylglycine is infused into the jejunum, it also . appears in bile predominantly as the sulfated conjugate. 14 Together, these experiments suggest that any conjugated but unsulfated lithocholate, if absorbed from the small intestine, will be progressively sulfated as it circulates enterohepatically. However, because some free lithocholate is likely to be entering the pool continuously and because sulfation was not complete in any study during secretion in bile, a fraction of biliary lithocholate should always be unsulfated. Sulfolithocholylglycine and probably sulfolithocholyltaurine appear to be end products of hepatic metabolism. The extent to which these compounds circulate enterohepatically is not defined by the present study. Our data suggest that the sulfate group is not removed during hepatic passage and that urine is not an important excretory route for sulfated lithocholate derivatives in healthy man in contrast to the rat. 24 Our data show further that sulfolithocholate can pass through the liver without complete conjugation. In the rat and in vitro, sulfolithocholate may be formed by bacterial deconjugation without desulfation, 24 but whether such occurs in man is uncertain. Our data confirm the early in vivo studies by Carey and Williams, 11 which indicated that additional hydroxylation of lithocholate does not occur in healthy
Vol. 69, No. 1
man although this is well documented for the rat, 25 hamster, 26 chicken, 27 and pig. 28 Hydroxylation of lithocholyltaurine has been observed in in vitro preparations of human hepatic microsomes, 29 but recent studies have shown that biotransformations of steroids observed during in vitro incubations with liver cell fractions may not occur during perfusion of the whole organ. 30 Our data cannot conclusively establish the liver as the site of sulfation or even conjugation, but sulfation and conjugation of lithocholate by the isolated perfused liver haye recently been demonstrated. 31 After this work had been completed, Norman and Strandvik 32 reported that radiolabeled lithocholate was excreted largely as sulfolithocholylglycine and sulfolithocholy l taurine when administered to 2 children with extrahepatic biliary atresia. They detected traces of a more polar derivative, as well as an unsaturated derivative, in agreement with earlier studies of Norman and Palmer, 33 as well as the initial report of Carey and Williams. 11 In our analytical procedure, we did not examine the chromatographic behavior of radioactivity after solvolysis and hydrolysis, and if such biotransformations of the lithocholate nucleus occurred, they would not have been detected. Time course of excretion of litfiocholate and its derivatives. The slower appearance of radioactivity in bile after administration of lithocholate or any of its derivatives was a consistent finding, but the method used in these studies does not permit mechanistic interpretations. The initial portion of the plasma disappearance curve of lithocholate is similar to that of cholic and chenodeoxycholic acids, 15 and it may be speculated that subsequent transit through the hepatocyte is slower for the monohydroxy derivatives . However, plasma disappearance of sulfolithocholate and sulfolithocholylglycine was considerably slower than that of free and conjugated cholic and chenodeoxycholic acids, suggesting that hepatic uptake of sulfated derivatives is less rapid or that reflux is occurring, or both. Lithocholate and its glycine and taurine conjugates (even as the sodium
July 1975
LITHOCHOLATE METABOLISM IN HEALTHY MAN I.
salts) are poorly soluble in aqueous solutions at body temperature, 3 • and sulfation is known to increase their aqueous solubility greatly. However, sulfation may concomitantly decrease the affinity of lithocholate or its conjugates for the micelle, and in principle this could significantly diminish its rate of biliary secretion. Other explanations are possible. Sulfation of bile acids in health and disease. Bile acid sulfation is now increasingly recognized as an important biotransformation in disease. 35 • 36 In cholestasis, the primary bile acid conjugates may be sulfated and excreted in urine. 37 • 38 The subcellular site and enzymes responsible for bile acid sulfation have not been identified. Further studies seem indicated to determine to what extent enhanced sulfation facilitates bile acid excretion or impaired sulfation promotes bile acid retention. The latter conceivably could be important in the initiation or perpetuation of liver disease. REFERENCES 1. Cowen AE, Korman MG, Hofmann AF: Metabolism of lithocholic acid in man (abstr). Gastroen· terology 67:785, 1974 2. Danielsson H, Eneroth P, Hellstrom K, eta! : On the turnover and excretory products of cholic and chenodeoxycholic acid in man. J Bioi Chern 238:2299-2304, 1963 3. Eneroth P, Hellstrom K, Sjovall J : A method for quantitative determination of bile acids in human feces. Acta Chern Scand 22:1729-1744, 1968 4. Connor WE, Witiak DT, Stone DB, et a!: Cholesterol balance and fecal neutral steroid and bile acid excretion in normal men fed dietary fats of diffe rent fatty acid composition. J Clin Invest 48:1363-1375,1969 5. Sjovall J: Bile acids in man under normal and pathological conditions: Bile acids and steroids 73. Clin Chim Acta 5:33-41, 1960 6. Dam H, Kruse I, Prange I, et al: Studies on human bile. III. Composition of duodenal bile from healthy young volunteers compared with composition of bladder bile from surgical patients with and without un complicated gallstone disease. Z Ernaehrungswiss 10:160-177, 1971 7. Bergstrom S, Danielsson H: Formation and metabolism of bile acids. In Handbook of Physiology, sect 6: Alimentary Canal, vol 2. Edited by CF Code. Washington DC, American Physiological Society, 1968, p 2391-2407 8. Hofmann AF: The function of bile in the alimen-
65
tary canal. In Handbook of Physiology, sect 6: Alimentary Canal, vol 2. Edited by CF Code, Washington DC, American Physiological Society, 1968, p 2507-2533 9. Palmer RH: Bile acids, liver injury and liver disease. Arch Intern Med 130:606-617, 1972 10. Carey JB Jr: Bile acids, cirrhosis and human evolution. Gastroenterology 46:490-493, 1964 11. Carey JB Jr, Williams G: Metabolism of lithocholic acid in bile fistula patients. J Clin Invest 42:450-455, 1963 12. Palmer RH, Bolt MG: Bile acid sulfates. I. Synthesis of lithocholic acid sulfates and their identification in human bile . J Lipid Res 12:671-679, 1971 13. Thistle JL, Hofmann AF: Efficacy and specificity of chenodeoxycholic acid therapy for dissolving gallstones. N Eng! J Med 289:655-659, 1973 14. Cowen AE, Korman MG, Hofmann AF, et al: Metabolism of lithocholate in healthy man. II. En terohepatic circulation. Gastroenterology 69:67-76, 1975 15. Cowen AE, Korman MG, Hofmann AF, et al: Metabolism of lithocholate in healthy man . III. Plasma disappearance of radioactivity after intravenous injection of labeled lithocholate and its derivatives. Gastroenterology 69:77-82, 1975 16. Simmonds WJ, Korman MG, Go VLW, et a!: Radioimmunoassay of conjugated cholyl bile· acids in serum . Gastroenterology 65:705-711, 1973 17. Malagelada JR, Go VLW, Summerskill WHJ : Differing sensitivities of gallbladder and pancreas to cholecystokinin-pancreozymin (CCK-PZ) in man. Gastroenterology 64:950-954, 1973 18. Cowen AE, Hofmann AF, Thomas PJ, eta!: An improved preparation of tritium and deuterium labeled bile acids: synthesis and validation (abstr). Gastroenterology 66:679, 1974 19. Norman A: Preparation of conjugated bile acids using mixed carboxylic acid anhydrides. Bile acids and steroids 34. Ark Kemi 8:331-342, 1955 20. Cass OW, Cowen AE, Hofmann AF, eta!: Thin layer chromatographic separation of sulfated and unsulfated lithocholic acid and its glycine and taurine conjugates. J Lipid Res 16:159-160, 1975 21. Soloway RD, Carlson HC, Schoenfield LJ: A balloon-occludable T-tube for cholangiography and quantitative collection and reinfusion of bile in man . J Lab Clin Med 79:500-504, 1972 22. Bartelheimer H: Quantitative fraktionierte pancreas- und gallensaftuntersuchungen durch anwendung einter dreilaufigen doppelballonsonee. Dtsch Med Wschr 78:993-995, 1953 23. Klapdor R: Verteilungs- und ausscheidungskinetik von 24- "C-cholsaure beim menschen mit nackweis eines multi-compartment-stoffwechselmodells. Klin Wschr 49:159-163, 1971 24. Palmer RH : Bile acid sulfates. II. Formation,
66
25.
26.
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COWEN ETAL. metabolism, and excretion of lithocholic acid sulfates in the rat. J Lipid Res 12:680- 687, 1971 Thomas PJ, Hsia SL, Matschiner JT, et al : Bile acids. XIX. Metabolism of lithocholic acid-24- 14C in the rat. J Bioi Chern 239:102-105, 1964 Emerman S, Javitt NB: Metabolism of taurolithocholic acid in the hamster. J Bioi Chern 242:661-664, 1967 Johansson G: On the metabolism of lithocholic acid in chicken and rabbit. Acta Chern Scand 20:240, 1966 Kurata Y: Stereo-bile acids and bile sterols. LXI. Metabolism oflithocholic acid in hog liver preparation. J Biochem 55:.415, 1964 Trulzsch D, Roboz J, Greim H, eta!: Hydroxylation of taurolithocholate by isolated human liver microsomes. I. Identification of metabolic product. Biochem Med 9:158-166, 1974 Kuss E, Fernbacher S, Sies H: Metabolism of estradiol in isolated perfused rat liver. Acta Endocrinol (suppl173):1, 1973 Liersch M, Stiehl A: Bildung von Gallensauresulfatestern in perfundierten rattenlebern nach gal-
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34. 35.
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lengangverschluss. Z Gastroenterol 12:131-134, 1974 Norman A, Strandvik B: Metabolism of lithocholic acid-24- 14C in extrahepatic biliary atresia. Acta Paediat Scand 63:92- 96, 1974 Norman A, Palmer RH : Metabolites of lithocholic acid-24-"C in human bile and feces . J Lab Clin Med 63:986-1001, 1964 Small DM, Admirand WH: Solubility of bile salts. Nature 221:265-266, 1969 Makino I, Shinozaki K, Nakagawa S, et a!: Measurement of sulfated and nonsulfated bile acids in human serum and urine. J Lipid Res 15:132-138, 197 4 Back P: Die primare hepatische synthese von mono-hydroxy-gallensaure bei extrahepatischer gallengangatresie . Klin Wschr 51:926-932, 1973 Stiehl A, Earnest DL, Admirand WH: Sulfation and renal excretion of bile salts in patients with cirrhosis of the liver. Gastroenterology 68:534544, 1975 Stiehl A: Bile salt sulphates in cholestasis. Eur J Clin Invest 4:59-63, 1974
Vol. 69, No. I Printed in U.S.A .
METABOLISM OF LITHOCHOLATE IN HEALTHY MAN I. Biotransformation and biliary excretion of intravenously administered lithocholate, lithocholylglycine, and their sulfates ALISTAIR E . COWEN, M.R.A.C.P., MELVYN G. KOHMAN, M.R.A.C .P ., ALAN F. HOFMANN, M.D ., AND OLIVER w. CASS
M.B.B.S.,
PH.D.,
Gastroenterology Unit, Mayo Clinic and Mayo Foundation, Rochester, Minnesota
The metabolism of intravenously injected radiolabeled lithocholate, lithocholylglycine, and their 3a-sulfate esters was characterized in healthy subjects. Lithocholate radioactivity was excreted rapidly and predominantly in bile; the excreted radioactivity had the chromatographic properties of glycine and taurine conjugates of lithocholate, of which 60% were sulfated. Lithocholylglycine also was excreted rapidly and predominantly in bile, and 60% of excreted radioactivity was sulfated. Sulfolithocholate radioactivity was only partially conjugated (about 60%) in association with biliary excretion. Sulfolithocholylglycine was excreted unchanged in bile. Neither sulfated derivative showed appreciable excretion in urine, although both were excreted more slowly in bile than unsulfated free or conjugated lithocholate. The data suggest that unconjugated lithocholate which is absorbed is completely conjugated and partially sulfated before excretion which occurs exclusively in bile. Since sulfation is not complete, some unsulfated lithocholate is always present in bile. This conjugated but unsulfated lithocholate, if reabsorbed, would be again partially sulfated during its next enterohepatic circulation. Thus, the end result of these biotransformations would be for absorbed lithocholate to be excreted in bile mostly, but not entirely as the sulfated conjugates. 5% lithocholate when analyzed by chromatography after alkaline saponification, 5 • 6 it has generally been held that in healthy man little lithocholate is absorbed from the intestine. 7-9 Lithocholate is unequivocally hepatotoxic when administered orally in a variety of experimental animals, 9 and
Lithocholic acid (lithocholate), which is formed in the distal intestine by bacterial 7 a-hydroxylation of chenodeoxycholic acid, is a major fecal bile acid in man. 2 ' 4 Because biliary bile acids contain less than Received November 11, 1974. Accepted February 10, 1975. This paper was presented in part at the 1974 Meeting of the American Association for the Study of Liver Disease and published in abstract form.' Address requests for reprints to: Alan F. Hofmann, M.D., Gastroenterology Unit, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55901. Supported in part by Research Grant AM-16770 from the National Institutes of Health and hy grants· in-aid from the Share Foundation, Mead ,Johnson & Company, Eli Lilly and Company, and Weddel Pharmaceuticals (U.K.) .
Dr. Cowen is an Eli Lilly Fellow, Australia. His present address is: Department of Medicine, Hoyal Brisbane Hospital, Queensland, Australia . Dr. Korman is an Eli Lilly Fellow, Australia. His present address is: Monash University, Department of Medicine, Prince Henry Hospital, Melbourne, Australia. Mr. Cass is a technical assistant. His present address is: School of Medicine, University of Min· nesota, Minneapolis, Minnesoa 55455. !i9
60
COWEN ETAL.
these observations led to speculation by Carey 10 that lithocholate might be involved in the initiation or perpetuation of hepatic disease. Despite this, little is known of the metabolism of lithocholate in healthy man. Carey and Williams 11 administered 14 [ C ]lithocholate to 2 patients with bile fistulas and concluded that lithocholate was conjugated with glycine and taurine but not further biotransformed during hepatic passage. This conclusion was questioned by Palmer and Bolt 12 who found that administered lithocholate could be recovered from human bile as the sulfate. Palmer and Bolt showed further that conventional techniques for biliary bile acid analysis that do not include a solvolysis step may not yield a valid recovery of lithocholate if it is present as the sulfate, which suggests that previous analyses of biliary bile acid composition may have underestimated the proportion of lithocholate in bile. 12 Because of these conflicting observations, and because of the increasing use of chenodeoxycholic acid as a specific agent for cholesterol gallstone dissolution in man, 13 we have carried out studies aimed at a more complete definition of lithocholate metabolism in healthy man. In this paper we report experiments that define the metabolism .of intravenously administered radiolabeled lithocholate, its glycine conjugate (lithocholylglycine), its sulfate (sulfolithocholate), and its sulfated glycine conjugate (sulfolithocholylglycine). A second paper 14 describes experiments aimed at elucidating the influence of sulfation on the site and efficiency of intestinal absorption in man, as well as measurements of lithocholate absorption (input) by the isotope dilution procedure of Lindstedt. The final paper in this series 15 describes the plasma disappearance of radioactivity after injection of free and conjugated lithocholate and their sulfates. Together, these studies show that lithocholate has an enterohepatic circulation of considerable magnitude in healthy man, during which it undergoes biotransformations unique among the other major primary and secondary bile acids.
Vol . 69, No. I
Materials and Methods Experimental design . Labeled bile acid was injected intravenously into a fasting subject, and serial sam pies of bile were collected from an indwelling duodenal tube. Complete recovery of bile was obtained by inflating an occlusive balloon in the jejunum and preventing gallbladder storage by infusing cholecystokinin intravenously throughout the course of the experiment. Three studies were carried out with lithocholate, two studies with sulfolithocholate, and a single study each with lithocholylglycine and sulfolithocholylglycine. In addition, single studies were carried out with the primary bile acids, cholic acid and chenodeoxycholic (chenic) acid, and their respective glycine and taurine conjugates. These experiments were carried out to validate complete recovery, as well as to define the time required for transit from blood to the sampling site. Subjects. Subjects participating in this study were healthy, young adult men judged to have normal liver function based on normal values for serum bilirubin, serum glutamic oxalacetic transaminase, serum alkaline phosphatase, sulfobromophthalein retention, protein electrophoresis, and fasting state serum cholic acid conjugates as determined by radioimmunoassay.'" Informed consent was obtained. Procedure. A fasting volunteer was intubated with a double lumen tube with an occlusive balloon that was positioned immediately proximal to the ligament of Treitz (verified by fluoroscopy with image intensification) . Gallbladder contraction was initiated and maintained by an infusion of cholecystokinin (Karolinska lnstitutet, Stockholm, Sweden) given at 75 Ivy dog units per hr ; this dose has previously been shown to induce vigorous gallbladder contraction and to be reasonably well tolerated in man. 17 After the majority of the fasting gallbladder bile had passed distally, the duodenal balloon was inflated and continuous duodenal aspiration was begun. In addition, throughout the study, gastric contents were aspirated by intermittent suction via a separate gastric tube. Labeled bile acids (10 to 30 !J.C) were then administered by rapid intravenous injection. Bile aspirated from the duodenum by siphonage was pooled in 30-min collections for 4 hr. Aliquots of bile were counted to calculate recovery of radioactivity, both during each 1/2-hr period and as cumulative recovery. Recovery of intravenously administered labeled bile acids exceeded 80% within the 4-hr study period in all cases. The total interruption of the enterohepatic circulation by the occluding duodenal balloon was. further confirmed by the
July 1975
LITHOCHOLATE METABOLISM IN HEALTHY MAN I.
recovery, in duodenal aspirates, of more than 90 % of a marker ( ["C ]polyethylene glycol, New England Nuclear Corp., Boston, Mass.) infused via the gastric tube . Isotopes. [24- "C ]Lithocholate (specific activity 4.2 me per mmole) was obtained from New England Nuclear or Mallinckrodt Chemical Co. (St. Louis, Mo.). [11,12- 3 H]Lithocholate (specific activity 1500 me per mmole) was prepared by reduction of ~ 11 -lithocholenic acid as described. 18 Lithocholylglycine (specific activity 750 me per mmole) was prepared by the mixed carboxylic carbonic anhydride method of Norman 19 and purified by thin layer chromatography (TLC). 20 Sulfolithocholate and its glycine conjugate were prepared from [11, 12- 'H ]lithocholate and [11 ,12-'H ]li thocholylglycine, respectively, as described by Palmer and Bolt. 12 All compounds were purified by preparative TLC; when analyzed by zonal scanning of thin layer chromatograms, purified compounds had a radiopurity greater than 98%. In addition, the radiopurity of (11, 12- 3 H ]lithocholate was confirmed by crystallization to a constant 'H: "C ratio after addition of [24- ••c ]lithocholate . Analysis of bile samples by TLC. The chemical form of radioactivity in duodenal samples was determined by TLC on Silica Gel H containing calcium sulfate ( 10%, w/w) and a solvent system of chloroform-methanol-acetic acid-water (65:24:15:9, v/v) 20 followed by zonal scanning of the plates. This system resolves lithocholate, its glycine and taurine conjugates, and the 3a-sulfate esters of each. All samples were also analyzed qualitatively by using the butanol-3 system of Palmer and Bolt" in which lithocholate and its derivatives have different relative mobilities. Identity of compounds was further confirmed in selected instances by cochromatography with reference substances tagged with another isotope. Nomenclature. Lithocholic acid will be referred to throughout as lithocholate, since it is assumed to exist as the anionic species in vivo. Its glycine conjugate will be referred to as lithocholylglycine (this was administered as the sodium salt and is likely to be present in vivo as the ionized form which might be termed a "glycinate"; this term has not achieved wide usage, however). The sulfate ester of lithocholylglycine will be referred to as sulfolithocholylglycine (which is not ideal because it does not indicate clearly that the sulfate group is fully ionized).
Results Lithocholate and its conjugates. When lithocholate was administered, radioactiv-
61
ity was excreted predominantly in bile; urinary excretion was less than Wi'o of the administered dose. All recovered radioactivity had the TLC mobility of glycine- or taurine-conjugated lithocholate or sulfated conjugated lithocholate . During the 1st hr, the majority of conjugated lithocholate was unsulfated, but the proportion that was sulfated increased progressively over the course of the 4-hr study so that virtually all of the 4-hr sample was both conjugated and sulfated (fig. 1 and table 1) . Of the total radioactivity recovered, 60% was conjugated and sulfated, and 40% was conjugated but unsulfated. When lithocholylglycine was injected, radioactivity was again excreted predominantly in bile, and the chemical form of biliary radioactivity showed a similar pattern and time course, except that taurine conjugates were not present (table 2). Again, early samples were excreted predominantly in unsulfated form, and samples obtained later were predominantly sulfated. At the end of the 4-hr period, 60% of the radioactivity had been sulfated, whereas 40% was excreted without sulfation . When the primary bile acids, cholic acid or chenic acid, were injected, radioactivity was excreted more rapidly in bile than after lithocholate or sulfolithocholate (fig. 2). When cholylglycine or chenodeoxycholylglycine was injected, radioactivity again appeared more rapidly than after 40
0
Conjugated, unsulfated
Bl Conjugated, sulfated
0
2
3
4
Hours
FIG. 1. Change, with time, in chemical form of radioactivity in bile after intravenous injection of labeled lithocholate. Results, expressed as percentage of administered dose, are mean of three experiments.
62
COWEN ETAL.
TABLE
Vol. 69, No. I
1. Chemical form of radioactivity recovered in proximal intestine followinf{ intravenous injection of 3 [ H ]lithocholate" Distribution of radioactivity
Time interval
lit-gly
sul-lit-gly
Iit-tau
min
Recovered radioactivity' sul -l it-tau
Interval
Cumulative
%total
%
Subject 1
0-30 30-60 60-90 90- 120 120- 150 150- 180 180-210 210-240
44 47 25 14 2 3 3 4
42 44 24 14 4 8 12 15
<1 17 42 63 84 77
11
71 65
13 14
59 52 8 12 16
23 17 3 2 2 1 1 <1
3 28 76 73 71 69 67 65
<1 3 14
<1 <1 9 9 10
11.7 22.2 14.6 13.8 17.3 4.5 8.6 7.1
11.7 33.9 48.5 62 .3 79.6 84.1 92.7 99 .8
9.6 44.1 10.3 22.5 3.6 4.0 3.2 2.6
9.6 53.7 64 .0 86.5 90.1 94.5 97.7 100.3
Subject 2
0-30 30-60 60-90 90-120 120-150 150-180 180- 210 210-240
11
6 <1
11
9 18 26 35
a lit-gly, lithocholylglycine; Iit-tau, lithocho lyltaurine ; sul-lit-gly, sulfolithocholylglycine; sui -lit- tau, sulfo. lithocholyltaurine. Chemical identity was inferred from mobility during thin layer chromatograph y using two solvent systems. • Recovery of radioactivity was 91 % of administered dose for subject 1 and 82% for subject 2.
2. Chemical form of radioactivity recovered in proximal intestine following intravenous injec tion of [' H]litho cholylf{lycinea
TABLE
Time interval
Distribution of radioactivity lit-gly
min
30-60 60-90 90-120 120-150 150-180 180-210 210- 240
sul-lit-gly
Recove red radioactivity' Interval
80 60 37 26 16 16 16
Cumulative
%total
%
20 40 63 74 84 84 84
9.9 14.9 22.4 16.1 19.2 7.9 9.4
9.9 24.8 47.2 63.3 82.5 90.4 99.8
a lit-gly , lithocholylglycine ; su l-lit-gly, sulfolithocholylglycine. Chemical identity was inferred from mobility during thin layer chromatography using two solvent systems. • Insufficient radioactivity for zonal scanning was recovered during 0 to 30 min . No appreciable radioactivity appeared in lithocholyltaurine or sulfolithocholyltaurine. Sixty per cent of administered radioactivity was recovered.
injection of lithocholylglycine and sulfolithocholylglycine (fig. 3). Free primary bile acids were conjugated but not sulfated; the glycine conjugates were excreted without further biotransformation. Sulfolithocholate and its conjugates. In contrast to ·free lithocholate, cholic acid, or chenodeoxycholic acid, sulfolithocholate was only conjugated in part; 35% of radioactivity in bile had the mobility of unconjugated sulfolithocholate (fig. 4). The remainder appeared as sulfolithocholylglycine and sulfolithocholyltaurine. No radioactivity appeared in lithocholylglycine or lithocholyltaurine in any experiment, which indicates that net removal of the sulfate group did not occur in association with biliary excretion. When sulfolithocholylglycine was injected, it was excreted without modification, and its excretion had a time course similar to that observed for lithocholylglycine radioactivity (fig. 3). Thus, this bile
July 1975
LITHOCHOLATE METABOLISM IN HEALTHY MAN I. Cholic 60
63
Chenic
-
' Litho ch olic
40
Sulfa! ithochol ic
-
20
0
2
4
0
2
4
2
0
n 4
0
2
4
Hours FIG. 2 . Time course of excretion of radioactivity in bile after intravenous injection of unconjugated bile ac ids. Note t hat the prim ary b ile acids, cholic a nd chenodeoxycho li c (chenic) acids, are excreted more rapid ly than lithocholic acid or its sulfated derivatives. Points represent me a n s of t hree ex periments for lithocholate and of two experiments for sulfolithocholate. 80
Cho lylglyci ne ~
Chenylglycine
r:-
60
Lithocholylglycine
"tl'
~
'b
8"'
40
~
--
'b
~"'
20
'
I
~;;·
ll 0
2
4
r--
'
~ 0
2
4
0
2
n 4
Sulfolithocholylglycine
0
2
n 4
Hours FIG. 3. Cumulative excretion of radioactivity, ex pressed as percentage of adm inistered dose, after intravenous injection of labeled conjugated primary bile acids (chenodeoxycholylg lycine a nd cholylglyc ine) or labeled lithoc holylglycine and its sulfate. Points represent a s ingle experiment with each bile acid.
40
~ '<::>'
c=J 30
~
~ Uncanjugatt•d ,
'b
:.. tl
IJ
Conjugated, sulfated
sulfated
20
~
'b
"' tl
t::)
10
0
2
3
4
Hours FIG. 4. Change, with time, in che mical form of radioactivity in bile after intravenous injection of labeled sulfolithocholate into a healthy subject. Resu lts a re expressed as percentage of admin istered dose.
acid resembled cholylglycine and chenodeoxycholylglycine in undergoing biliary excretion without biotransformation.
Discussion Methodology . The method used in this study appears to be a novel and useful technique for defining biotransformations of drugs during their initial excretion in bile. The completeness of recovery indicated that gallbl adder storage did not occur and that intestinal aspiration was complete proximal to the occlusive balloon. Other alternatives, such as use of a patient with conventional or Baldwin Ttube , 21 are more difficult to arrange, and normal hepatic function cannot always be
64
COWEN ETAL.
guaranteed. An alternative to the use of a marker and a single balloon would be a three-lumen tube containing proximal and distal balloons inflated above and below the ampulla of Vater, respectively, 22 and this has been modified by the addition of an infusion port below the distal balloon. 23 Metabolism of lithocholate in man. These data provide additional evidence in support of the pioneering report by Palmer and Bolt 12 that lithocholate, whether free or conjugated, is largely sulfated before hepatic excretion in man. They suggest that lithocholate, which enters the bile acid pool from the intestine, will be completely conjugated and partially sulfated in association with biliary excretion. Conjugated but unsulfated lithocholate was also sulfated in association with biliary excretion. When lithocholylglycine is infused into the jejunum, it also . appears in bile predominantly as the sulfated conjugate. 14 Together, these experiments suggest that any conjugated but unsulfated lithocholate, if absorbed from the small intestine, will be progressively sulfated as it circulates enterohepatically. However, because some free lithocholate is likely to be entering the pool continuously and because sulfation was not complete in any study during secretion in bile, a fraction of biliary lithocholate should always be unsulfated. Sulfolithocholylglycine and probably sulfolithocholyltaurine appear to be end products of hepatic metabolism. The extent to which these compounds circulate enterohepatically is not defined by the present study. Our data suggest that the sulfate group is not removed during hepatic passage and that urine is not an important excretory route for sulfated lithocholate derivatives in healthy man in contrast to the rat. 24 Our data show further that sulfolithocholate can pass through the liver without complete conjugation. In the rat and in vitro, sulfolithocholate may be formed by bacterial deconjugation without desulfation, 24 but whether such occurs in man is uncertain. Our data confirm the early in vivo studies by Carey and Williams, 11 which indicated that additional hydroxylation of lithocholate does not occur in healthy
Vol. 69, No. 1
man although this is well documented for the rat, 25 hamster, 26 chicken, 27 and pig. 28 Hydroxylation of lithocholyltaurine has been observed in in vitro preparations of human hepatic microsomes, 29 but recent studies have shown that biotransformations of steroids observed during in vitro incubations with liver cell fractions may not occur during perfusion of the whole organ. 30 Our data cannot conclusively establish the liver as the site of sulfation or even conjugation, but sulfation and conjugation of lithocholate by the isolated perfused liver haye recently been demonstrated. 31 After this work had been completed, Norman and Strandvik 32 reported that radiolabeled lithocholate was excreted largely as sulfolithocholylglycine and sulfolithocholy l taurine when administered to 2 children with extrahepatic biliary atresia. They detected traces of a more polar derivative, as well as an unsaturated derivative, in agreement with earlier studies of Norman and Palmer, 33 as well as the initial report of Carey and Williams. 11 In our analytical procedure, we did not examine the chromatographic behavior of radioactivity after solvolysis and hydrolysis, and if such biotransformations of the lithocholate nucleus occurred, they would not have been detected. Time course of excretion of litfiocholate and its derivatives. The slower appearance of radioactivity in bile after administration of lithocholate or any of its derivatives was a consistent finding, but the method used in these studies does not permit mechanistic interpretations. The initial portion of the plasma disappearance curve of lithocholate is similar to that of cholic and chenodeoxycholic acids, 15 and it may be speculated that subsequent transit through the hepatocyte is slower for the monohydroxy derivatives . However, plasma disappearance of sulfolithocholate and sulfolithocholylglycine was considerably slower than that of free and conjugated cholic and chenodeoxycholic acids, suggesting that hepatic uptake of sulfated derivatives is less rapid or that reflux is occurring, or both. Lithocholate and its glycine and taurine conjugates (even as the sodium
July 1975
LITHOCHOLATE METABOLISM IN HEALTHY MAN I.
salts) are poorly soluble in aqueous solutions at body temperature, 3 • and sulfation is known to increase their aqueous solubility greatly. However, sulfation may concomitantly decrease the affinity of lithocholate or its conjugates for the micelle, and in principle this could significantly diminish its rate of biliary secretion. Other explanations are possible. Sulfation of bile acids in health and disease. Bile acid sulfation is now increasingly recognized as an important biotransformation in disease. 35 • 36 In cholestasis, the primary bile acid conjugates may be sulfated and excreted in urine. 37 • 38 The subcellular site and enzymes responsible for bile acid sulfation have not been identified. Further studies seem indicated to determine to what extent enhanced sulfation facilitates bile acid excretion or impaired sulfation promotes bile acid retention. The latter conceivably could be important in the initiation or perpetuation of liver disease. REFERENCES 1. Cowen AE, Korman MG, Hofmann AF: Metabolism of lithocholic acid in man (abstr). Gastroen· terology 67:785, 1974 2. Danielsson H, Eneroth P, Hellstrom K, eta! : On the turnover and excretory products of cholic and chenodeoxycholic acid in man. J Bioi Chern 238:2299-2304, 1963 3. Eneroth P, Hellstrom K, Sjovall J : A method for quantitative determination of bile acids in human feces. Acta Chern Scand 22:1729-1744, 1968 4. Connor WE, Witiak DT, Stone DB, et a!: Cholesterol balance and fecal neutral steroid and bile acid excretion in normal men fed dietary fats of diffe rent fatty acid composition. J Clin Invest 48:1363-1375,1969 5. Sjovall J: Bile acids in man under normal and pathological conditions: Bile acids and steroids 73. Clin Chim Acta 5:33-41, 1960 6. Dam H, Kruse I, Prange I, et al: Studies on human bile. III. Composition of duodenal bile from healthy young volunteers compared with composition of bladder bile from surgical patients with and without un complicated gallstone disease. Z Ernaehrungswiss 10:160-177, 1971 7. Bergstrom S, Danielsson H: Formation and metabolism of bile acids. In Handbook of Physiology, sect 6: Alimentary Canal, vol 2. Edited by CF Code. Washington DC, American Physiological Society, 1968, p 2391-2407 8. Hofmann AF: The function of bile in the alimen-
65
tary canal. In Handbook of Physiology, sect 6: Alimentary Canal, vol 2. Edited by CF Code, Washington DC, American Physiological Society, 1968, p 2507-2533 9. Palmer RH: Bile acids, liver injury and liver disease. Arch Intern Med 130:606-617, 1972 10. Carey JB Jr: Bile acids, cirrhosis and human evolution. Gastroenterology 46:490-493, 1964 11. Carey JB Jr, Williams G: Metabolism of lithocholic acid in bile fistula patients. J Clin Invest 42:450-455, 1963 12. Palmer RH, Bolt MG: Bile acid sulfates. I. Synthesis of lithocholic acid sulfates and their identification in human bile . J Lipid Res 12:671-679, 1971 13. Thistle JL, Hofmann AF: Efficacy and specificity of chenodeoxycholic acid therapy for dissolving gallstones. N Eng! J Med 289:655-659, 1973 14. Cowen AE, Korman MG, Hofmann AF, et al: Metabolism of lithocholate in healthy man. II. En terohepatic circulation. Gastroenterology 69:67-76, 1975 15. Cowen AE, Korman MG, Hofmann AF, et al: Metabolism of lithocholate in healthy man . III. Plasma disappearance of radioactivity after intravenous injection of labeled lithocholate and its derivatives. Gastroenterology 69:77-82, 1975 16. Simmonds WJ, Korman MG, Go VLW, et a!: Radioimmunoassay of conjugated cholyl bile· acids in serum . Gastroenterology 65:705-711, 1973 17. Malagelada JR, Go VLW, Summerskill WHJ : Differing sensitivities of gallbladder and pancreas to cholecystokinin-pancreozymin (CCK-PZ) in man. Gastroenterology 64:950-954, 1973 18. Cowen AE, Hofmann AF, Thomas PJ, eta!: An improved preparation of tritium and deuterium labeled bile acids: synthesis and validation (abstr). Gastroenterology 66:679, 1974 19. Norman A: Preparation of conjugated bile acids using mixed carboxylic acid anhydrides. Bile acids and steroids 34. Ark Kemi 8:331-342, 1955 20. Cass OW, Cowen AE, Hofmann AF, eta!: Thin layer chromatographic separation of sulfated and unsulfated lithocholic acid and its glycine and taurine conjugates. J Lipid Res 16:159-160, 1975 21. Soloway RD, Carlson HC, Schoenfield LJ: A balloon-occludable T-tube for cholangiography and quantitative collection and reinfusion of bile in man . J Lab Clin Med 79:500-504, 1972 22. Bartelheimer H: Quantitative fraktionierte pancreas- und gallensaftuntersuchungen durch anwendung einter dreilaufigen doppelballonsonee. Dtsch Med Wschr 78:993-995, 1953 23. Klapdor R: Verteilungs- und ausscheidungskinetik von 24- "C-cholsaure beim menschen mit nackweis eines multi-compartment-stoffwechselmodells. Klin Wschr 49:159-163, 1971 24. Palmer RH : Bile acid sulfates. II. Formation,
66
25.
26.
27.
28.
29.
30.
31.
COWEN ETAL. metabolism, and excretion of lithocholic acid sulfates in the rat. J Lipid Res 12:680- 687, 1971 Thomas PJ, Hsia SL, Matschiner JT, et al : Bile acids. XIX. Metabolism of lithocholic acid-24- 14C in the rat. J Bioi Chern 239:102-105, 1964 Emerman S, Javitt NB: Metabolism of taurolithocholic acid in the hamster. J Bioi Chern 242:661-664, 1967 Johansson G: On the metabolism of lithocholic acid in chicken and rabbit. Acta Chern Scand 20:240, 1966 Kurata Y: Stereo-bile acids and bile sterols. LXI. Metabolism oflithocholic acid in hog liver preparation. J Biochem 55:.415, 1964 Trulzsch D, Roboz J, Greim H, eta!: Hydroxylation of taurolithocholate by isolated human liver microsomes. I. Identification of metabolic product. Biochem Med 9:158-166, 1974 Kuss E, Fernbacher S, Sies H: Metabolism of estradiol in isolated perfused rat liver. Acta Endocrinol (suppl173):1, 1973 Liersch M, Stiehl A: Bildung von Gallensauresulfatestern in perfundierten rattenlebern nach gal-
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Vol. 69, No.1
lengangverschluss. Z Gastroenterol 12:131-134, 1974 Norman A, Strandvik B: Metabolism of lithocholic acid-24- 14C in extrahepatic biliary atresia. Acta Paediat Scand 63:92- 96, 1974 Norman A, Palmer RH : Metabolites of lithocholic acid-24-"C in human bile and feces . J Lab Clin Med 63:986-1001, 1964 Small DM, Admirand WH: Solubility of bile salts. Nature 221:265-266, 1969 Makino I, Shinozaki K, Nakagawa S, et a!: Measurement of sulfated and nonsulfated bile acids in human serum and urine. J Lipid Res 15:132-138, 197 4 Back P: Die primare hepatische synthese von mono-hydroxy-gallensaure bei extrahepatischer gallengangatresie . Klin Wschr 51:926-932, 1973 Stiehl A, Earnest DL, Admirand WH: Sulfation and renal excretion of bile salts in patients with cirrhosis of the liver. Gastroenterology 68:534544, 1975 Stiehl A: Bile salt sulphates in cholestasis. Eur J Clin Invest 4:59-63, 1974