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Altered metabolism of bile alcohol and bile acid in complete extrahepatic cholestasis: qualitative and quantitative aspects

Hitoshi Ichimiya, Jiro Yanagisawa, and Fumio Nakayama

Department of Surgery I, Kyushu University Faculty of Medicine, Fukuoka, Japan

Abstract Urinary excretion of bile alcohols and bile acids in patients with complete extrahepatic cholestasis before and after the release by external biliary drainage was studied. Following extraction, isolation, and hydrolysis, bile alcohols were determined by capillary gas-liquid chromatography-mass spectrometry as dimethylethylsilyl derivatives. During cholestasis, 8.89 pmollday of bile alcohol and 140.4 pmol/day of bile acid were excreted in urine. The amount of bile alcohol excreted was 6.1% of that of bile acid. Positive correlation between excretion of bile alcohols and bile acids was observed. The major bile alcohols excreted were also present in urine from healthy individuals but in much smaller amounts. After the release of extrahepatic cholestasis, urinary excretion of bile acid decreased rapidly, but that of bile alcohol decreased only gradually. The latter often increased again and remained high. The results indicate that the increased excretion of bile alcohols in complete extrahepatic cholestasis may reflect the expansion of a normally existing pathway of bile alcohol synthesis and excretion leading to the modification of bile alcohols for their efficient urinary elimination. It is also suggested that the rate of synthesis of bile alcohols is determined partly by the size of the substrate pool available.- Ichimiya, H., J. Yanagisawa, and F. Nakayama. Altered metabolism of bile alcohol and bile acid in complete extrahepatic cholestasis: qualitative and quantitative aspects. J Lipid RCS. 1987. 28: 1028 - 1037.

Supplementary key words bile alcohol gas-liquid chromatography-mass spectrometry extrahepatic cholestasis biliary drainage

Bile alcohols form important constituents of bile, feces, and urine in cerebrotendinous xanthomatosis (CTX) (1-4). A block in the mitochondrial 26-hydroxylation of intermediates in the biosynthetic pathway from cholesterol to bile acids is responsible for their occurrence (5). Bile alcohols different from those found in CTX have also been detected in urine from patients with cholestatic liver diseases (6-9). In a previous publication, we described the occurrence of 5P-cholestane-3a, 701, 1201, 26, 27-pent01 (50- cyprinol) and 50-cholestane-Sa, 7 a , 12a,26-tetrol (27-, deoxy-50-cyprinol) in addition to 27-nor-50-cholestane- 3a, 7a, 12a, 24, 25-pent01 and 50-cholestane-Sa, 7a, 12a, 25, 26-pent01 (50-bufol), in the urine of a patient

with obstructive jaundice (10). Considerable attention is focussed on their significance as a possible reflection of altered metabolism or subcellular function of the damag- ed liver (6-10). However, these bile alcohols are known to be present in healthy subjects (11, 12). Therefore, quantitative rather than qualitative change is of more interest. Little is known about the rate of synthesis and the possible enterohepatic circulation of these bile alcohols in the normal as well as in the diseased state. In patients with complete extrahepatic cholestasis, bile alcohols and bile acids are excreted solely in urine. Therefore, the study of urinary bile acids and bile alcohols in such patients would provide complete qualitative and quantitative data on their metabolism. A follow-up study of urinary bile acid and bile alcohol excretion after the release of extrahepatic cholestasis may be useful in monitoring the recovery of bile acid and bile alcohol metabolism compared to that which occurred in extrahepatic cholestasis.

MATERIALS AND METHODS

Reagents

All solvents were of analytical grade and were distilled before use. Diethylaminohydroxypropyl Sephadex LH-20 (Lipidex-DEAP) from Packard Instrument Go., Downers Grove, IL, and SP-Sephadex C25 from Pharmacia Fine Chemicals, Uppsala, Sweden were prepared as described by AlmC et al. (13). Piperidinohydroxypropyl Sephadex LH-20 (PHP-LH-20) was prepared according to the procedure described by Goto et al. (14). 0-Glucuronidase

Abbreviations: CTX, cerebrotendinous xanthomatosis; GLC, gas-liquid chromatography; GLC-MS, gas-liquid chromatography-mass spectrometry; DMES, dimethylethylsilyl; RRT, relative retention time; Lipidex-DEAP, diethylaminohydroxypropyl Sephadex LH-20; PHP-LH- 20, piperidinohydmxypropyl Sephadex LH-20.

1028 Journal of Lipid Research Volume 28, 1987

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(EC 3.2.1.31, not containing sulfatase activity) was pur- chased from Sigma Chemical Co., St. Louis, MO. Several authentic bile alcohols were kindly donated by Prof. T. Hoshita, Hiroshima University, Hiroshima, Japan. Nor- cholic acid was obtained from Reseach Plus Steroids Laboratories Inc., Denville, NJ and hyocholic acid was from Canada Packers, Notredame, Montreal, Canada. Tauro-[11,11,12,12,-~H4]chenodeoxycholic acid and glyco- [6,6,7,7-2H4]lithocholic acid 3-sulfate were synthesized according to the methods described previously (15, 16) from the corresponding deuterated bile acids used in previous studies (17, 18). Other reagents, authentic and deuterated bile acids used were the same as in previous studies (10, 17).

Patients

Seven patients with complete extrahepatic cholestasis were studied. Diagnoses were based on radiological findings, laparotomy, and histological examination. The release of extrahepatic cholestasis was effected by external biliary drainage in the form of either external cholecystostomy or percutaneous transhepatic cholangio- drainage. Complete biliary obstruction was confirmed with repeated cholangiograms after the release of extrahepatic cholestasis. Urine samples were collected on the day before the release of extrahepatic cholestasis; on days 1, 3, 5, 7, 14, and 21 after the release, 24-hr urine and bile samples were collected and stored at - 2OoC until analyzed. Seven controls without liver dysfunction were also studied. Clinical and laboratory data are summarized in Table 1.

Analysis of urinary bile alcohol Three to ten ml of urine was applied to a Bond-Elut

C18 cartridge (500 mg of octadecylsilane-bonded silica, Analytichem International, Harbor City, CA) and bile alcohols were extracted with 5 ml of 90% ethanol. The extract was evaporated to dryness under reduced pressure. The residue was dissolved in 4 ml of 0.075 M phosphate buffer (pH 7.0) and unconjugated bile alcohol was extracted from the buffer solution with three 4-ml portions of ethyl acetate. To the remaining aqueous phase, 1000 units of @-glucuronidase was added and the solution was incubated at 37OC for 24 hr. Liberated bile alcohols were extracted from the solution with ethyl acetate. Bile alcohol sulfates were extracted from the final remaining aqueous phase using a Bond-Elut C18 cartridge and the extract was evaporated to dryness. Solvolysis was carried out in equilibrated ethyl acetate (with 2 M H2S04)-ethanol 9:l (v/v). After 24 hr incubation at 37OC, the mixture was neutralized with 20% sodium hydroxide and evaporated. The residue was redissolved in 4 ml of 0.075 M phosphate buffer (pH 7.0) and applied to a Bond-Elut cartridge. The liberated bile alcohols were eluted with 5 ml of 90% ethanol. After addition of ethyl cholate as internal stand-

ard in each fraction, unconjugated and deconjugated bile alcohols thus obtained were analyzed by gas-liquid chromatography-mass spectrometry (GLC-MS) as their dimethylethylsilyl (DMES) ether derivatives (10, 17).

In preliminary recovery experiments using authentic 27-nor-50-cholestane-3a, 7a, 12a, 24, 25-pent01 and 50-cholestane-3a, 7a 12a, 25, 26-pentol, complete extraction of bile alcohols from buffer solution was obtained with two 4-ml portions of ethyl acetate. The recovery in extraction with a Bond-Elut C18 cartridge was 93.4% for 27-nor-5@-cholestane-3a, 7a, 12a, 24, 25-pent01 and 94.2% for 50-cholestane-Sa, 7a, 12a, 25, 26-pentol. To examine the efficacy of the fractionation, an unconjugated bile alcohol fraction was treated with 0-glucuronidase and subjected to solvolysis, but no significant increase of bile alcohols was observed.

Analysis of urinary bile acid The methods described by Almt (13) and by Takigawa

et al. (19) were combined and modified. Briefly, [2H4]chen~de~xycholic acid, [2H3]glycocholic acid, [ 2H4]taurochenodeoxychooxycholic acid, and [ *H4] glycolithocholic acid 3-sulfate were added to 2-10 ml of urine as internal standards. The bile acids were extracted with a Bond-Elut C18 cartridge, converted to H' form on SP-Sephadex C25, and separated according to the mode of conjugation on Lipidex-DEAP. As bile acid glucuronides were eluted from the Lipidex-DEAP column together with the fractions containing sulfated and taurine-conjugated bile acids (20), both fractions were combined, and after the enzymatic cleavage of amino acid conjugates (21), bile acids were separated into unconjugated (originally taurine conjugates), glucuronidated, and sulfated bile acids on PHP-LH-20 (19). Unconjugated bile acids and deconjugated bile acids obtained after enzymatic cleavage of amino acid conjugates (21), @-glucuronidase treatment, solvolysis (22), and purification were analyzed by GLC-MS as their ethyl ester-DMES ether derivatives (17).

Biliary bile acids

Total biliary bile acid concentration was determined by an enzymatic procedure using Sa-hydroxysteroid dehy- drogenase (23), and biliary bile acid composition was determined by high performance liquid chromatography (24).

Gas-liquid chromatography-mass spectrometry

Equipment and operating conditions of GLC-MS were the same as those in previous studies (10, 17). Identifica- tion of individual bile acid and bile alcohol derivatives was based on retention times of peaks and on complete mass spectra. Quantitation of bile alcohols and bile acids was based on comparison of peak areas given by the individual compound derivatives with peak areas given by

Ichimiya, Yanagisawa, and Nakayama Bile alcohol in extrahepatic cholestasis 1029


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TABLE 1 . Clinical and laboratory data'

Case Duration of

No. Age Sex Diagnosis Jaundice T-Bil D-BII ALP S-GOT S-GPT

Patient 1 60 F 2 75 F 3 70 M 4 71 M 5 66 M 6 56 M 7 76 F

Control 8 69 M 9 73 F

10 58 F 11 56 M 12 59 M 13 76 F 14 35 M

Normal range

Carcinoma (duodenal papilla) Carcinoma (common bile duct) Carcinoma (common bile duct) Lymphnode metastasis (liver hilum) Carcinoma (pancreas) Carcinoma (pancreas) Carcinoma (liver hilum)

Cholelithiasis Cholelithiasis Gastric ulcer Cholelithiasis Carcinoma (stomach) Carcinoma (stomach) Health

mg/dl mg/dl u/L

4 weeks 19.9 17.0 > 700 3 weeks 29.3 21.1 > 700 4 weeks 21.3 18.3 627 1 week 9.8 7.3 > 700 10 days 26.7 20.3 > 700 2 weeks 20.3 14.5 328 6 weeks 31.9 24.5 226

0.3 0 68 0.5 0 105 0.3 0.1 66 0.8 0.2 82 1.1 0.1 68 0.3 0.1 80 0.3 0.1 65

0.2-1.2 <0.4 30-110

7J/L b/L

128 237 198 328 229 260 162 242 255 489 62 192 56 23

19 11 36 21 24 23 19 20 21 25 25 20 20 16

<40 <40

"T-Bil, serum total bilirubin; D-Bil, serum direct bilirubin; ALP, alkaline phosphatase; S-GOT, serum glutamic oxalo-acetic transaminase; S-GPT, serum glutamic pyruvic transaminase.

the internal standards in selected fragment ion current chromatograms. The ions used for quantitation of individual bile alcohol and bile acid are listed in Tables 2 and 3.

RESULTS

Components of urinary bile alcohol and bile acid

A typical chromatogram obtained by GLC-MS of the urinary glucuronidated bile alcohol fraction is shown in Fig. 1. The following bile alcohols were identified by direct comparison with reference compounds, and their sum was expressed as total bile alcohols: 27-nor-5P-cholestane-3a, 7a, 12a, 24, 25-pentol, 5P-cholestane-3a, 7a, 12a, 24, 26-pent01 (5P-chimaerol), 5P-cholestane-3a, 7a , 12a, 25, 26-pent01 (50-bufol), 5P-cholestane-Sa, 7a, 12a, 26, 27-pent01 (5P-cyprinol), and 5P-cholestane-3a, 7a, 12a, 26-tetrol (27-deoxy-5P-cyprinol) (Table 2). The mass spectrum of peak B was quite similar to that of 27-nor-5P-cholestane-3a, 7a, 12a, 24, 25-pentol; it con- tained a series of fragment ions at mlz 737, 633, 529, 425, 321 which resulted from the scission of the bond between C24 and C25 and successive loss of one to four molecules of dimethylethylsilanol. These data suggest that peak B is an isomer of 27-nor-5P-cholestane-3a, 7a, 12a, 24, 25- pentol.

Bile acids identified in urine are listed in Table 3. Of all the bile acids, cholic acid, hyocholic acid, ursodeoxycholic acid, chenodeoxycholic acid, deoxycholic acid, norcholic acid, 3P, 12a-dihydroxy-5-cholenoic acid, 30-hydroxy-5-cholenoic acid, and lithocholic acid were regular-

ly present in cholestatic urine and were quantitated. The bile acid profile of control urine was more varied, probably due to the functioning enterohepatic circulation.

Urinary bile alcohol and bile acid in healthy individuals

Of five bile alcohols identified and quantitated in cholestatic urine, three bile alcohols, i.e., 27-nor-5P-cholestane-3a, 7a, 12a, 24, 25-pentol, 5P-cholestane-3a, 7a,

I , 1 I 1

0 2 0 4 0 6 0

Reten t ion t ime (min)

Fig. 1. Typical chromatogram obtained by GLC-MS of urinary glucuronidated bile alcohol fraction; IS. , ethyl cholate (internal standard); A, 5@-cholestane-3a, 7a, 12a, 26-tetrol; B', 27-nor-5/3-cholestane-3a, 7a, 12a, 24, 25-pent01 (probably an isomer of compound B); B, 27-nor-5@-cholestane-3a, 7a , 12a, 24, 25-pentol; C, 5&cholestane-3a, 7a, 12a, 24, 26-pentol; D, 5@-cholestane-3a, 7a, 12a, 25, 26-pentol; E, 5@-cholestane-3a, 7a, 12a, 26, 27-pentol.



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TABLE 2. Relative retention times of DMES ethers of bile alcohols on SE-30 and ions used for quantitation

Compounds Ions Used for Quantitative

RRT MW GLC-MS Analysis, mho

Ethyl cholate 1 .oo 694 665 (M - 29)

27-Nor-5@-cholestane-3a, 701, 1201, 24, 25-pent01 2.61 868 529 (M - 2 x 104 - 131) 5/3-Cholestane-3a, 7a, 12a, 24, 26-pent01 3.06 882 529 (M - 2 x 104 - 145) 58-Cholestane-3a, 701, 1201, 25, 26-pent01 3.50 882 765 (M - 117)

58-Cholestane-3a, 7a, 12a, 26-tetrol 1.88 7 80 469 (M - 2 x 104 + H')

58-Cholestane-3a, 7a, 12a, 26, 27-pent01 3.80 882 571 (M - 2 x 104 + H*)

RRT, retention time relative to the derivative of ethyl cholate; M W, molecular weight of individual derivative. "29 = C2H5; 104 = dimethylethylsilanol; 131 = side chain; 145 = side chain; 117 = side chain.

12a, 25, 26-pentol, and 5/3-cholestane-Sa, 7a, 12a, 24, 26-pentol, could be quantitated in normal urine. Others were present only in trace amounts. Unconjugated and sulfated bile alcohols could not be detected. Total bile alcohol excretion averaged 0.80 * 0.45 pmollday (mean * SD) (Table 4). 27-Nor-5P-cholestane-3a, 7a, 12a, 24, 25-pent01 (compound B in Table 4), the predominant bile alcohol of urine, constituted an average of 77% of total urinary bile alcohol followed by 50- cholestane-3a, 7a, 12a, 25, 26-pent01 (compound D) (15%), and 5@-cholestane-3a, 7a, 12a, 24, 26-pent01 (compound C) (8%) (Table 5). The urinary bile acid excretion was 6.02 k3.58 pnoYday (mean* SD) (Table 4). Of total bile acids, lithocholic acid (4-15% of the total), deoxycholic acid (8-26%), ursodeoxycholic acid (1-4%), ursocholic acid (1-43%), and keto bile acids (4-22%), which were considered to be formed from primary bile acids through the action of intestinal microorganisms during enterohepatic circulation, constituted 37-66%. Cholic acid constituted 2-2876 and chenodeoxycholic acid constituted 8-21%. Thus primary bile acids constituted 11-45% of total bile acids.

Urinary bile alcohol and bile acid in complete extrahepatic cholestasis

The mode of conjugation was similar among individual bile alcohols which were excreted mostly as glucuronides. The unconjugated bile alcohols and their sulfates ac- counted for less than 10% of total urinary bile alcohol excreted. In complete extrahepatic cholestasis, urinary bile alcohol excreted was 8.89 * 4.26 pmol/day (mean f SD), 10-20 times control values (P < 0.01) (Table 6). The major constituents were the same as those in controls. How- ever, the percentage distribution of an individual bile alcohol differed greatly from that in controls. 27-Nor- 5@-cholestane-3a, 7a, 12a, 24, 25-pent01 was most predominant in cholestatic urine as in normal urine, but its proportion to the total bile alcohols (62%) was less than that in controls (77%) (P < 0.05). On the other hand, the proportion of 5@-cholestane-3a, 7a, 12~1, 25, 26-pent01 (31%) was significantly higher than that in controls (15%) (P < 0.05) (Table 5). The urinary excretion of 27-nor- 5&cholestane-3a, 7a, 12a, 24, 25-pent01 was about 9 times greater and that of 50-cholestane-Sa, 7a, 12a, 25, 26-

TABLE 3. Completely and partially identified bile acids in urine

Bile Acid' Ions Used for Quantitative

RRT GLC-MS Analysis, m l z b

Lithocholic acid (5BB-3a-01) 0.534 461 (M - 29)

Allodeoxycholic acid (5aB-301, 1201-01) 0.680 563 (M - 29) Deoxycholic acid (5fiB-301,lZa-ol) 0.719 563 (M - 29) Chenodeoxycholic acid (5@B-301,7a-ol) 0.773 459 (M - 29 - 104) 12-Keto-lithocholic acid (5OB-3a-01- 1 2-one) 0.785 475 (M - 29) 23-Norcholic acid (23-nor-5/3B-3a,7a,1201-01) 0.792 651 (M - 29)

38, 12a-Dihydroxy-5-cholenoic acid (BA5-30, 12a-01)' 0.827 561 (M - 29) Ursodeoxycholic acid (5BB-3a,78-01) 0.832 563 (M - 29 Cholic acid (5@B-3a, 7a, 12a-01) 1 .00 665 (M - 29) Ursocholic acid (5flB-3a,78,12a-ol) 1.02 665 (M - 29) 7-Keto-deoxycholic acid (5PB-3a, 12a-ol-7-one) 1.08 577 (M - 29)

12-Keto-chenodeoxycholic acid (5bB-3a,7a-ol-12-one) 1.16 502 (M - 104)

3/3-Hydroxy-5-cholenoic acid (BA5-3/3-ol) 0.649 459 (M - 29)

Hyodeoxycholic acid (58B-301,6a-ol) 0.797 385 (M - 2 x 104 + H')

Hyocholic acid (5j3B-3~~ ,6a, 7a-01) 1.12 383 (M - 3 x 104 + H')

RRT, retention time relative to the derivative of cholic acid on SE-30. "As ethyl ester DMES ether; B, cholanoic acid. Configuration at C5 and of hydroxyl groups is indicated by Greek

'29 - CpH,; 104 = dimethylethylsilanol. 'Tentatively identified.

letters. Superscript denotes positions of double bond.

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TABLE 4. Urinary excretion of bile acid and bile alcohol in normal subjects

Bile Alcoholn Case No. Bile Acid A B C D E Total Bile Alcohol/Bile Acid

fimol/dy fimollday %

8 3.10 ND 0.62 0.04 0.18 Tr 0.84 27.1 9 4.20 ND 0.37 0.03 0.08 Tr 0.48 11.4

10 3.62 ND 0.25 0.03 0.08 Tr 0.36 9.9 1 1 5.99 ND 0.34 0.04 0.05 Tr 0.43 7.2 12 3.78 ND 0.84 0.06 0.09 Tr 0.99 26.2 13 13.0 ND 0.66 0.08 0.11 Tr 0.85 6.5 14 8.42 ND 1.22 0.24 0.19 Tr 1.65 19.6

Mean 6.02 0.61 0.07 0.11 0.80 15.4

'A, 5@-cholestane-3a, 7a, 12a ,26-tetrol; B, 27-nor-5fl-cholestane-3a, 7a, 12a, 24,25-pentol; C , 5j3-cholestane- 3a,7a,12a,24,26-pentol; D, 5fl-cholestane-3a,7ty, 12a,25,26-pentol; E, 5L3-cholestane-3a,7a1, 12a,26,27-pentol; Tr, trace amounts; ND, not detected

pent01 about 23 times greater than that of controls. Thus, the increase of 5P-cholestane-3a, 7a , 12a, 25, 26-pent01 was more pronounced than that of 27-nor-5@-cholestane-3a, 7a, 12a, 24, 25-pentol.

The mean urinary excretion of bile acids was 140.4 pmol daily, more than 20 times that of controls (P < 0.01). Of total bile acids, cholic acid constituted 8 4 5 % and chenodeoxycholic acid constituted 25-72%. Thus primary bile acids constituted 71-9076 of total bile acids. Of the other bile acids, 3~-hydroxy-5-cholenoic acid (2-19%) and hyocholic acid (l- l l%) constituted the major part. Secondary bile acids constituted less than 2% of the total, and only trace amounts of keto bile acids were detected. Thirty-seven to 94% of total bile acids were sulfated and 3-8% were giucuronidated. The amount of bile alcohol was 6.1% of bile acid excreted in urine in complete extrahepatic cholestasis and 15.4% in controls (Tables 4 and 6). A positive correlation between bile alcohol and bile acid excretion was observed in complete extrahepatic cholestasis (r=0.860, P < 0.05), but not in controls. No correlation between magnitude of bile alcohol excretion and other parameters of liver function listed in Table 1 was observed.

After release of extrahepatic cholestasis (Fig. 2; Table

In all but one case (case 7), sufficient recovery from cholestasis, as judged by disappearance of jaundice and

7)

the liver function test, was obtained by the external biliary drainage by means of percutaneous transhepatic cholan- giodrainage or external cholecystostomy. Complete biliary obstruction prior to surgery was confirmed by the repeated cholangiographies after biliary drainage. Biliary bile acid composition, absence or presence of only trace amounts of secondary bile acids, also indicates the per- sistence of complete biliary obstruction. In concordance with the improvement in liver function, bile output, biliary bile acid output representing net bile acid synthesis, increased gradually and even exceeded the normal range (Fig. 2). A concurrent rapid fall in the excretion of urinary bile acid was observed: 33.8% on the 7th day, 28.8% on the 14th day, and 15.9% on the 21st day. However, even on the 21st day after the release of extrahepatic cholestasis, the excretion rate remained above the normal range (P < 0.01). As for bile alcohol, the change in the urinary excretion induced by the release of extrahepatic cholestasis was markedly different from that of bile acid. In all cases, the decrease of excretion was not SO

marked as that of bile acid and usually increased again from the 5th to 7th day. In three out of six cases, excretion rates still remained higher than before the release. The prolonged rise of excretion rates continued and at the end of the study they were still about 10 times that of controls (P < 0.01). As a result, the ratio of bile alcohol relative to bile acid rose from 6.1% before the release to about 37% at the 21st day after the release which was sig-

TABLE 5. Percentage distribution of bile alcohols

Cholestasis" Normal'

Mean f SD ( R a w ) Mean t SD (Range)

27-Nor-5fl-cholestane-3a, 7a, 12a,24,25-pentol 61.9 * 8.5 (49.6-74.8) 76.5 * 4.gb (69.4-84.8) 5fl-Cholestane-3a,7a, 12a,25,26-pentol 31.2 + 8.0 (21.1-42.2) 15.1 i 5.1b (9.1-22.2) 5B-Cholestane-3a1 7a, 12a ,24,26-pentol 6 .9 f 1.6 (4.2-9.1) 8 . 4 i 3.2 (4.8-14.5)

'n - 7 . b P < 0.05.



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TABLE 6. Urinary excretion of bile acid and bile alcohol during complete extrahepatic cholestasis

Bile Alcohol’ CaJe No. Bile Acid A B C D E Total Bile AlcohoYBile Acid

pmoUhy pmoU&y %

1 137.4 0.05 5.57 0.31 1.57 0.13 7.63 2 112.7 0.02 3.42 0.42 2.12 0.08 6.06 3 208.4 0.30 10.05 1.12 4.30 0.40 16.17 4 140.5 0.12 6.83 0.91 2.30 0.06 10.22 5 124.7 0.10 6.88 0.70 3.19 0.14 11.01 6 170.3 0.29 4.46 0.49 3.17 0.18 8.59 7 89.0 0.13 1.14 0.19 0.97 0.14 2.57

Mean 140.4’ 0.14’ 5.48’ 0.5gb 2.52’ 0.16’ 8.89’

5.6 5.4 7.8 7.3 8.8 5.0 2.9 6.1‘

“A, 5fl-cholestane-3a,7a,l2a,26-tetrol; B, 27-nor-5@-cholestane-3a,7a, 12a,24,25-pentol; C, 519-cholestane- 3a,7a, 12a,24,26-pentol; D, 5fl-cholestane-3a,7a, 12a,25,26-pentol; E, 5fl-cholestane-3a,7ay,12a,26,27-pentol; Tr, trace amounts; ND, not detected.

’ P C 0.01 (compared to controls). ‘ P < 0.05 (compared to controls).

nificantly higher than that of controls (P < 0.05) and that before the release ( P < 0.01) (Table 7). The type of bile alcohols and the mode of conjugation of individual bile

alcohol were the same throughout the study before and during the release. However, a slight increase of 50- cholestane-3cr, 7a, 12a, 26, 27-pent01 was noted after the

Case 1

Case 4

M

*----+---- *+e.

1 3 14 21

Case 5

150

Oavs after drainage

Fig. 2. urinary bile acid; (to) urinary bile alcohol; (,!--A) biliary bile acid.

Changes in urinary bile acid and bile alcohol excretion and in biliary bile acid excretion after the release of extrahepatic cholestasis; (o--O)

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TABLE 7 . Changes in urinary excretion of bile alcohol and bile acid after the release of extrahepatic cholestad ~~ ~

Days after the Release of Extrahepatic Cholestasis

0 7 14 21

Bile acids (pnol/day) 149.0 * 34.9' 45.15 * 19.96' 39.47 f 17.47h 22.27 * 9.51h

Bile alcohol (pmol/day) 9.95 * 3.53h 12.69 + 4.87b 12.48 * 6.08* 8.73 * 51.06~ L d d L e d

L e d 27-Nor-5fl-cholestane-3a,7a, 12a,24,25-pentol ( 7 6 ) 62.1 f 7.5 59.7 f 11.4 54.5 ? 11.8 54.3 * 9.9

5fi-Cholestane-3a, 7a, 12a, 25,26-pentol (% ) 28.4 f 6.6 25.5 * 6.4 27.8 * 6.4 25.6 * 4.3

5fl-Cholestane-3a,7cy, 12a,24,26-pentol (%) 6.5 f 1.6 6.1 f 1.6 6.7 * 1.9 8.4 4 3.4

9.5 * 6.1 5fl-Cholestane-Ja, 7a, 12a, 26,27-pentol ( %) 8.4 i 4.6 1.6 f 0.7 6.7 * 5.0

5fl-Cholestane-3a, 7a, 12a, 26-tetrol ( %)

Bile alcohol/bile acid ( 7 6 )

L e d 2.1 * 0.9 1.4 i 1 . 1 2.0 * 0.7 2.7 f 1 . 1

6.7 1.5 29.2 f 7.4' 33.1 i 16.5' 37.8 f 9.3b L d d

"Expressed as mean f SD, n = 6 (case 7 was excluded) ' P < 0.01 compared to controls. ' P < 0.05 compared to controls.

' P < 0.05. d P < 0.01.

release (P < 0.05). After the release of extrahepatic cholestasis, correlation between urinary excretions of bile alcohol and bile acid gradually disappeared.

DISCUSSION

Recently, several studies have shown that increased amounts of 27-nor-5P-cholestane-3a, 7a, 12a, 24, 25- pentol and 5P-cholestane-3a, 7a , 12a, 25, 26-pent01 are excreted in urine of patients with liver diseases such as primary biliary cirrhosis (6), liver cirrhosis (7), and al- antitrypsin deficiency (8). It has been speculated that the appearance of these compounds might reflect the alter- nate pathway of biosynthesis of bile acids from cholesterol. The presence of multiple defects at different levels of the synthetic pathway of bile acid in liver diseases as suggested by Patteson et al. (25), and the reactions leading to bile alcohol formation in several subcellular compartments (26) may well be reflected in the bile alcohol profile which, in turn, may give further insight to the subcellular function of the liver. Kuwabara et al. (11) identified 27-nor-5/3-cholestane-3a, 7a, 12a, 24, 25-pentol, 5P-cholestane-3a, 7a, 12a, 25, 26-pentol, 5P-cholestane-3a, 7a, 12a, 24, 25-pentol, and SP-cholestane-3a, 701, 12a, 26, 27-pent01 in urine of healthy adults, and Kuroki et al. (12) noted 27 kinds of bile alcohols in bile of patients with gallstone diseases. They suggested that these bile alcohols are normal constituents of bile and urine and not abnormal metabolites. However, the dy- namic aspects of bile alcohol metabolism are unknown. As little or no enterohepatic circulation of bile alcohol oc-

curs in patients with complete extrahepatic cholestasis, it is possible to investigate the qualitative as well as quantitative aspect of bile alcohol metabolism in the liver by studying only one remaining excretory route, i.e., urine. In the present study, it was demonstrated that, in complete extrahepatic cholestasis, not only 27-nor-5P-cholestane-3a, 7a, 12a, 24, 25-pent01 and 5P-cholestane-Sa, 7a, 12a, 25, 26-pentol, but also 5P-cholestane-Sa, 701, 12a, 24, 26-pentol, SP-cholestane-3a, 7a, 12a, 26, 27- pentol, and 5P-cholestane-3a, 7a, 12a, 26-tetrol were excreted in increased amounts reaching about 10-20 times that seen in healthy individuals. 5P-Cholestane-3a, 7a, 12~2, 26-tetrol has not been detected in normal urine, but is usually the most predominant bile alcohol in bile (12). All pentols found in the present study were also present in normal urine, and are considered to be formed from cholesterol via 5b-cholestane-3a, 7a, 12a, 26-tetrol (11, 12). Therefore, the major bile alcohols excreted in cholestatic urine can well be considered as the metabolites of 5/3-cholestane-3a, 7a, 12a, 26-tetrol. Thus these compounds are different from those observed in CTX where large amounts of excreted bile alcohols are hydroxylated at C23, C24, and C25 (1-4) probably due to a genetic defect in the mitochondrial 26-hydroxylase required for normal bile acid biosynthesis (5). In extrahepatic cholestasis, mitochondrial 26-hydroxylase is active and supplies substrates such as 5P-cholestane-Sa, 7a, 12a, 26- tetrol for bile alcohols excreted in urine. It is difficult to define whether the patients in the present study are in a steady state. In addition, nothing is known concerning the mechanism of biliary and urinary excretion, the binding to plasma protein, hepatic uptake, and the distribution in



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the body of bile alcohols which may affect their urinary excretion. Therefore, the quantitative aspects obtained in this study may be somewhat speculative.

The daily bile acid synthesis of about 400-600 mg in the healthy adult is reported to be suppressed to one- seventh of that rate in obstructive jaundice (27). In the present study in complete extrahepatic cholestasis, taking the daily urinary excretion as a measure of daily synthesis (28, 29), 140.4 pmol of bile acids and 8.89 Fmol of bile alcohols are synthesized. The value for bile acids agrees with that calculated by Eklund, Norlandar, and Norman (28), and indicates that the synthesis is reduced to about one-seventh of the normal rate. Because of the lack of in- formation on the rate of bile alcohol synthesis in healthy subjects, it is still unknown whether or not bile alcohol synthesis is also suppressed in cholestasis. However, the maximum synthesis rate of total bile alcohols and of 27-nor-5/3-cholestane-3a, 7a, 1201, 24, 25-pentol, the most predominant bile alcohol in normal urine, can be calculated under the assumption that the daily synthesis rate equals the loss during their enterohepatic circulation. It is known that 2 % of bile acid entering the intestine es- capes enterohepatic circulation and that newly synthesized bile acids make up for this loss (30). On the other hand, the intestinal absorption of bile alcohols has been reported to be less efficient than that of bile acids, although this was observed in animal experiments (31). Total bile alcohols and 27-nor-5&cholestane-3a, 7a, 12a, 24, 25-pent01 are reported to be 0.16% and 0.0078% of total bile acids in bile, respectively (12). Total biliary bile acid output is 30-60 mmollday (10 cycles of a 3-6 mmol pool) (32-35). Assuming no intestinal recovery, bile alcohol fecal loss is (30-60) x 0.0016 mmol/day = 48-96 ,umol/day. Similarly, for 27-nor-5/3-cholestane-3ar, 7a, 12a, 24, 25-pentol, daily fecal loss is (30-60) x 0.000078 mmollday = 2.4-4.7 pmollday. This value does not exceed the daily urinary excretion of this bile alcohol in extrahepatic cholestasis (5.48 f 2.85 pmol/day), suggesting that, despite the sup- pression of bile acid synthesis, the synthesis of 27-nor-5/3- cholestane-3a, 7a, 12a, 24, 25-pent01 is not suppressed but enhanced in complete extrahepatic cholestasis. In healthy individuals, the pattern of conjugation of bile alcohols in urine is quite different from that in bile (11, 12). Thus Karlaganis and Sjovall(36) suggested that both the state of conjugation and the number of polar groups may determine the route of excretion. Tetrols as sulfates are the major bile alcohols in bile (12), but only pentols as glucuronides have been identified in normal urine (11). Therefore, it is likely that pentols as glucuronides are the more suitable form for urinary excretion. Hence, tetrols may be hydroxylated further for efficient urinary elimination in cholestasis. Since all bile alcohols observed in cholestatic urine can be formed in healthy individuals (6, 7, 11, 12), expansion of the normally existing pathway of bile alcohol synthesis and excretion is responsible for the

increased urinary excretion of bile alcohols in extrahepatic cholestasis with the modification of bile alcohol molecules for the efficient urinary elimination resulting in the change of proportion of bile alcohols. The dissociation between urinary bile alcohol excretion and bile acid excretion was observed after the release of extrahepatic cholestasis. This could be caused by an increase. in synthesis, a delay in excretion by the liver or the kidneys, or possibly slower release of bile alcohols from tissues to blood. The release of bile alcohols from tissues to blood may be related to the binding of bile alcohols to protein. Nothing is known concerning the interaction of bile alcohols with protein. However, higher concentrations of 56- cholestane-3a, 7a, 12a-trio1, and 5/3-cholestane-3a, 7a, 12a, 25-tetrol in the liver (5) and of cholestanol in other organs (37) were shown in CTX. Therefore, a large reser- voir of bile alcohols may be present which is only slowly excreted after release of biliary obstruction. This interaction also affects the hepatic uptake (38) and renal clear- ance of bile acids (39). It is generally believed that the biliary excretion of bile acid is a carrier-mediated and saturable process (40, 41). The biliary transport maximum values for some bile acids are different from each other (42-44). Furthermore, the hepatic transport system for sulfated bile acids is less efficient (45, 46) and more easily impaired by cholestasis than that of nonsulfated bile acids (46). Similarly, renal clearances of sulfated and nonsulfated bile acids are different from each other (39). Although nothing is known concerning the mechanism both of biliary and urinary excretion of bile alcohols, it is possible that the maximum capacity for either the liver or the kidneys to excrete bile alcohols may be much lower than for bile acids. The proximal tubular reabsorption (47, 48) and tubular secretion of bile acids (39, 49) were shown to contribute to the urinary excretion of bile acids. And the competition for reabsorption between sulfated and nonsulfated bile acids was demonstrated (47). There- fore, a variety of competitive tubular mechanisms may ex- ist such that bile acids may inhibit the excretion of bile alcohols. In this event, a reduction in urinary bile acid load could be followed by a transient rise in bile alcohol excretion. The presence of a positive correlation between bile alcohol and bile acid excretion in urine during cholestasis suggests that the synthesis of bile alcohol de- pends on that of bile acids. This finding is in contrast to the result reported by Karlaganis et al. (8). The difference may be due to the complete interruption of enterohepati-c circulation in our patients andlor the difference in the type of cholestasis studied, i.e., intrahepatic versus extrahepatic cholestasis. The pool of the substrate available for the side chain cleavage, Le., the pool of 5~-cholestane-3a, 7a, 12a-triol, must be expanded when bile acid synthesis is enhanced after the release of extrahepatic cholestasis. Thus the re-increase of bile alcohol excretion after the release concomitant to the increase of bile acid synthesis

Ichimz& Yanogisawa, and Nakayama Bile alcohol in extrahepatic cholestasir 1095


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may also suggest that the formation of bile alcohols is determined partly by the available pool size of the substrate, Le., 5&cholestane-3a, 7 a , 12a-trio1, the common substrate of bile acids and bile alcohols. Therefore, it is strongly suggested that the alteration of bile alcohol metabolism induced by cholestasis persists in spite of the release of cholestasis.

In conclusion, the increased urinary excretion of bile alcohols in complete extrahepatic cholestasis is due to the expansion of a normally existing pathway of bile alcohol synthesis and excretion, and reflects the change of modification of bile alcohols for efficient urinary elimination. The formation of bile alcohols is determined partly by the substrate pool available. Prolonged rise of bile alcohol excretion strongly suggests that persistent alteration of the cholesterol-bile acid metabolic axis persists even after the release of extrahepatic cholestasis. I

Manuscript receiued 15 Septmbn 1986 and i n reuisedform 9 March 1987

REFERENCES

1.

2.

3.

4.

5.

6.

7.

8.

Setoguchi, T., G. S. Tint, and E. H. Mosbach. 1974. A biochemical abnormality in cerebrotendinous xanthomatosis: impairment of bile acid biosynthesis associated with in- complete degradation of the cholesterol side chain. J. Clin. Invest. 53: 1393-1401. Shefer, S., B. Dayal, G. S. Tint, G. Salen, and E. H. Mosbach. 1975. Identification of pentahydroxy bile alcohols in cerebrotendinous xanthomatosis: characterization of 5@-cholestane-3a, 7a , 12a, 246, 25-pent01 and 5P- cholestane-3a, 7a, 12a, 236, 25-pentol. J. Lipid Res. 16:

Hoshita, T., M. Yasuhara, K. Kihira, and T. Kuramoto. 1976. Identification of (23S)-5P-cholestane-3a, 7a, 12a, 23, 25-pentol. Stemidr. 27: 657-664. Yasuhara, M., T. Kuramoto, T. Hoshita, E. Itoga, and S. Kito. 1978. Identification of 5P-cholestane-Sa, 7a, 12a, 23P-tetro1, 5P-cholestane-Sa, 7a, 12a, 24a-tetrol, and 5P- cholestane-3a, 7a, 1201, 248-tetrol in cerebrotendinous xanthomatosis. Stemidr. 31: 333-345. Oftebro, H., I. Bjorkhem, S. Skrede, A. Schreiner, and J. I. Pedersen. 1980. Cerebrotendinous xanthomatosis. A defect in mitochondrial 26-hydroxylation required for normal biosynthesis of cholic acid. J. Clin. Invest. 65: 1418-1430. Karlaganis, G., B. AlmC, V. Karlaganis, and J. Sjovall. 1981. Bile alcohol glucuronides in urine. Identification of 27-nor-5P-cholestane-3a, 7 a , Ea, 24€, 25t-pentol in man. J. Stemid Biochn. 14: 341-345. Ludwig-Kohn, H., H. V. Henning, A. Sziedat, D. Mattaei, G. Spitteller, J. Reiner, and H. J. Egger. 1983. The identification of urinary bile alcohols by gas chromatography-mass spectrometry in patients with liver disease and in healthy individuals. EUT. J. Clin. Invest. 13: 91-98. Karlaganis, G., A. Nemeth, B. Hammarskjold, B. Strand- vik, and J. Sjovall. 1982. Urinary excretion of bile alcohols in normal children and patients with al-antitrypsin deficiency during development of liver disease. Eur. J. Clin. Zn-

280-286.

uwt. 12: 399-405.


9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

Karlaganis, G., V. Karlaganis, and J. Sjovall. 1984. Iden- tification of 27-nor-5B-cholestane-3a, 7a, 12a, 246, 25E. 26-hex01 and partial characterization of the bile alcohol profile in urine. J. Lipid Res. 25: 693-702. Ichimiya, H., J. Yanagisawa, and F. Nakayama. 1984. Sig- nificance of bile alcohol in urine of a patient with cholestasis: identification of 5P-cholestane-Sa, 701, 12a, 26, 27- pentol (5P-bufol) and 5P-cholestane-3a, 7a , Ea, 26-tetrol (27-deoxy-5P-cyprinol). Chem. Pharm. Bull. 32: 2874-2877. Kuwabara, M., T. Ushiroguchi, K. Kihira, T. Kuramoto, and T. Hoshita. 1984. Identification of bile alcohols in urine from healthy humans. J Lipid Res. 25: 361-368. Kuroki, S., K. Shimizu, M. Kuwabara, M. Une, K. Ki- hira, T. Kuramoto, and T. Hoshita. 1985. Identification of bile alcohols in human bile. J Lipid Res. 26: 230-240. Almt, B., A. Bremmelgaard, J. Sjovall, and P. Thomassen. 1977. Analysis of metabolic profiles of bile acids in urine using a lipophilic anion exchanger and computerized gas-liquid chromatography-mass spectrometry. J Lipid Res. 18:

Goto, J., M. Hasegawa, H. Kato, and T. Nambara. 1978. A new method for simultaneous determination of bile acids in human bile without hydrolysis. Clin. Chim. Acta. 87:

Lack, L., F. 0. Dorrity, Jr., T. Walker, andG. D. Singletary. 1973. Synthesis of conjugated bile acids by means of a pep- tide coupling reagent. J. Lipid Res. 1 4 367-370. Tserng, K. Y., and P. D. Klein. 1977. Synthesis of sulfate esters of lithocholic acid, glycolithocholic acid, and tauro- lithocholic acid with sulfur trioxide-triethylamine. J. Lipid Res. 18: 491-495. Yanagisawa, J., H. Ichimiya, M. Nagai, and F. Nakayama. 1984. Presence of monohydroxy bile acids in the urinary precipitates: a pitfall in the analysis of urinary bile acids.J. Lipid Res. 25: 750-753. Yanagisawa, J., M. Itoh, M. Ishibashi, H. Miyazaki, and F. Nakayama. 1980. Microanalysis of bile acid in human liver tissue by selected ion monitoring. Anal. Biochem. 104:

Takigawa, H., H. Otsuka, T. Beppu, Y. Seyama, and T. Yamakawa. 1982. Quantitative determination of bile acid glucuronides in serum by mass fragmentography. J Biochem. 92: 985-998. AlmC, B., and J. Sjovall. 1980. Analysis of bile acid glucuronides in urine. Identification of 3a, 6a, 12a- trihydroxy-5/3-cholanoic acid. J. Stemid Biochem. 13: 907-916. Nair, P. P., and C. Garcia. 1969. A modified gas-liquid chromatographic procedure for the rapid determination of bile acids in biological fluids. Anal. Biochem. 2 9 164-171. Kornel, L. 1965. Studies on steroid conjugates. IV. Demonstration and identification of solvolyzable corti- costeroids in human urine and plasma. Biochcmistv. 4:

Mashige, F., K. Imai, and T. Osuga. 1976. A simple and sensitive assay of total serum bile acid. Clin. Chim. Acta. 70: 79-86. Nakayama, E, and M. Nakagaki. 1980. Quantitative determination of bile acids in bile with reversed-phase high- performance liquid chromatography. J. Chmmatogz 183: 287-293. Patteson, T. E., Z. R. Vlahcevic, C. C. Schwartz, J. Gustafsson, H. Danielsson, and L. Swell. 1980. Bile acid metabolism in cirrhosis. VI. Sites of blockage in the bile acid pathways to primary bile acids. &tmentemlogy. 79:

339-362.

141-147.

75-86.

444-452.

620-628.


ww

w.jlr.org

Dow

nloaded from

http://www.jlr.org/

26. Danielsson, H. 1973. Mechanism of bile acid biosynthesis. Zn The Bile Acids. Vol. 2, P. P. Nair and D. Kritchevsky, editors. Plenum Press, New York. 1-32.

27. Danielsson, H., P. Eneroth, K. Hellstrom, S. Lindstedt, and J. SjovaU. 1963. On the turnover and excretory pro- ducts of cholic acid and chenodeoxycholic acid in man. J. Biol. Chem. 238: 2299-2304.

28. Eklund, A., A. Norlandar, and A. Norman. 1980. Bile acid synthesis and excretion following release of total extrahepatic cholestasis by percutaneous transhepatic drainage. Eur J. Clin. Invest. 10 349-355.

29. Kinugasa, T., K. Uchida, M. Kadowaki, H. Takase, Y. Nomura and Y. Saito. 1981. Effect of bile duct ligation on bile acid metabolism in rats. J. Lipid h. 22: 201-207.

30. Hofmann, A. F., and H. S. Mekhjian. 1973. Bile acids and the intestinal absorption of fat and electrolytes in health and disease. Zn The Bile Acids. Vol. 2. P. P. Nair and D. Krit- chevsky, editors. Plenum Press, New York. 103-153. Hoshita, T., N. Harada. I. Morita, and K. Kihira. 1981. In- testinal absorption of bile alcohols. J. Biochmt. 90:

32. Vlahcevic, Z. R., C. C. Bell, Jr., I. Buhac, J. T. Farrar, and L. Swell. 1970. Diminished bile acid pool size in patients with gallstones. Gastmmkrology. 5 9 165-173.

33. Danzinger, R. G., A. F. Hofmann, J. L. Thistle, and L. J. Schoenfield. 1973. Effect of oral chenodeoxycholic acid on bile acid kinetics and biliary lipid composition in women with cholelithiasis. J. Clin. Invest. 52: 2809-2821.

34. Jazrawi, R. P., and T. C. Northfield. 1986. Effects of a phar- macological dose of cholecystokinin on bile acid kinetics and biliary cholesterol saturation. Gut. 27: 355-362,

35. Hofmann, A. F. 1967. The syndrome of ileal disease and the broken enterohepatic circulation: choleretic en- teropathy. Gartmmtemlogy. 52: 752-757.

36. Karlaganis, G., and J. Sjovall. 1984. Formation and metabolism of bile alcohols in man. Hepatology. 4: 966-973.

37. Salen, G. 1971. Cholestanol deposition in cerebrotendinous xanthomatosis. Ann. Znt. Med. 75: 843-851.

38. Aldini, R., A. Roda, A. M. M. Labate, G. Cappelleri, E. Roda, and L. Barbara. 1982. Hepatic bile acid uptake:

31.

1363-1369.

effect of conjugation, hydroxyl and keto groups, and albumin binding. J. Lipid Res. 23: 1167-1173.

39. Corbett, C. L., T. C. Bartholomew, B. H. Billing, and J. A. Summerfield. 1981. Urinary excretion of bile acids in cholestasis: evidence for renal tubular secretion in man. Clin. Sci. 61: 773-780.

40. OMaille, E. R. L., T. G. Richards, and A. H. Short. 1966. Factors determining the maximum rate of organic anion secretion by the liver and hrther evidence on the hepatic site of action of the hormone secretin. Am. J. Physiol. 186:

41. Paumgartner, G., 0. K. Sauter, H. P. Schwarz, and R. Herz. Quantitative aspects of structure and function. 1973. In The Liver. G. Paumgartner and R. Preisig, editors. Karger, Basel. 337-343.

42. Kitani, K., and S. Kanai. 1981. Biliary transport maximum of tauroursodeoxycholate is twice as high as that of taurocholate in the rat. Lqe Sci. 29: 269-275.

43. Kitani, K., and S. Kanai. 1982. The choleretic effect of ur- sodeoxycholate in the rat. Lift Sci. 31: 1973-1985.

44. Hardison, G. W. M., D. E. Hatoff, K. Miyai, and R. G. Weiner. 1981. Nature of bile acid maximum secretory rate in the rat. Am. J. Physiol. 241: G337-343.

45. Bartholomew, T. C., and B. H. Billing. 1983. The effect of 3-sulfation and taurine conjugation on the uptake of chenodeoxycholic acid by rat hepatocytes. Biochim. Biophys. Acta. 745: 101-109.

46. Cleland, D. P., T. C. Bartholomew, and B. H. Billing. 1984. Hepatic transport of sulfated and non-sulfated bile acids in the rat following relief of bile duct obstruction. Hepatology.

47. Barnes, S., J. L. Gollan, and B. H. Billing. 1977. The role of tubular reabsorption in the renal excretion of bile acids. Biochem. J. 166: 65-73.

48. Weiner, I. M., J. E. Glasser, and L. Lack. 1964. Renal excretion of bile acids: taurocholic, glycocholic and cholic acids. Am. J. Physiol. 207: 964-970.

49. Zins, G. R., and I. M. Weiner. 1968. Bidirectional transport of taurocholate by the proximal tubule of the dog. Am. J. Physiol. 215: 840-845.

424-438.

4 477-485.

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