Hepatic Cholesterol Metabolism in Patients with Gallstones

GASTROENTEROLOG¥69:676-684, 1975 Copyright© 1975 by The Williams & Wilkins Co.

Vol. 69, No.3 Printed in U.S .A .

HEPATIC CHOLESTEROL METABOLISM IN PATIENTS WITH GALLSTONES GERALD SALEN, PH.D.

M.D.,

G ·: NICOLAU, PH.D., S. SHEFER, PH.D., AND

E . H.

MOSllACH,

Section of Gastroenterology , East Orange Veterans Administration H ospital, East Orange, New Jersey , Department of Medicine, College of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey; and Public Health Research Institute of New York , Inc. New York, New York

Relative rates of cholesterol and bile acid synthesis were estimated in patients with cholesterol gallstones and biliary obstruction by determining the hepatic activities of 3-hydroxy-3-methylglutaryl-CoA reductase and cholesterol 7a-hydroxylase, the respective rate-determining enzymes for cholesterol and bile acid synthesis. As compared with eight control studies, 3-hydroxy-3-methylglutaryl-CoA reductase activity was 27 % higher in 12 gallstone subjects, but 75 % lower in 5 subjects with biliary obstruction. Cholesterol 7a-hydroxylase activity was reduced in the gallstone ( 47 % lower) and biliary obstruction (78% lower) subjects. Liver cholesterol concentrations were 56% higher in the gallstone and 53% higher in the biliary obstruction subjects than the control group. These findings suggest that the pathogenesis of gallstones is related to both increased cholesterol synthesis and decreased bile acid formation, whereas cholesterol accumulates in biliary obstruction because of defective removal since cholesterol production is low. Body cholesterol pools are regulated by a balance between the rate of formation and catabolism of cholesterol. Although cholesterol can be synthesized from acetate in virtually every body tissue, 1• 2 the main sites of formation are the liver and intestine. 3 The production of cholesterol is dependent on the formation of mevalonic Received March 25, !975. Accepted May ! , 1975. Address reprint requests to: Dr. Gerald Salen, Section of Gastroenterology, East Orange Veterans Administration Hospital, East Orange, New Jersey 07019. This work was supported in part by United States Public Health Service Grants HL17818, HL 10894, and AM 05222, and Grant GB 3191X from the National Science Foundation. The authors are indebted to Dr. Richard Kessler, Ass is tan t Chief of Surgery at the Manhattan Veterans Administration for help in obta inin g t he surgical liver specimens. The authors are also grateful to Mr. William Saltzman and the I.P.D. Corporat ion for their continued support of this work.

676

acid from 3-hydroxy-3-methylglutaryl (HMG) -CoA. This reaction is catalyzed by the microsomal enzyme HMG-CoA reductase, • and consequently, the feedback control of cholesterol biosynthesis is mediated at this step. Such factors as absorbed cholesterol and the enterohepatic circulation of bile acids are believed to influence cholesterol synthesis by affecting either the formation or degradation of hepatic microsomal HMGCoA reductase. 3 Although small amounts of cholesterol are transformed into steroid hormones by the adrenal gland and the gonads, 'the major quantitative pathway of cholesterol degradation is conversion into bile acids .5 The only tissue capable of this transformation is the liver. The key step that governs bile acid synthesis is the formation of 7 a -hydroxycholesterol from cholesterol. 6 This reaction is also catalyzed by a microsomal enzyme, cholesterol 7a-hydroxylase.

September 1975

CHOLESTEROL METABOLISM IN GALLSTONES

Unlike hepatic HMG-CoA reductase activity, which is under the feedback control of both cholesterol and bile acids, cholesterol 7a-hydroxylase activity seems to be regulated by bile acids, except in the rat, where the enzyme is induced by cholesterol. 5 Recently, the pathogenesis of human cholelithiasis has been ascribed to abnormal hepatic cholesterol metabolism. 7 • 8 An important discovery was that bile acid pools were diminished in this disease. 9 Presumably, as a consequence, hepatic bile acid secretion is reduced. In other studies carried out by Grundy and co-workers in American Indian women who manifest a very high incidence of gallstones and youn!!: Caucasian women with gallstones, cholesterol secretion into the bile was found to be extremely high and was associated with a decreased flow of biliary bile acids. 10 • 11 Thus, at least two factors, increased cholesterol secretion and a diminished bile acid pool, may contribute to the pathogenesis of lithogenic bile. In order to obtain information about the hepatic metabolism of cholesterol in patients with gallstones, the relationship between cholesterol synthesis and degradation was examined by measuring the activities of the hepatic rate-determining enzymes for cholesterol and bile acid synthesis, HMG-CoA reductase and cholesterol 7a-hydroxylase. For comparison, similar measurements were determined in patients with extrahepatic biliary obstruction who manifest large cholesterol tissue deposits and high plasma concentrations and in individuals without hepatic or lipid disorders. Our results showed that the regulation of cholesterol metabolism was abnormal in subjects with gallstones and biliary obstruction.

Methods Clinical Liver tissue was obtained at surgery or by percutaneous liver biopsy from 26 hospitalized persons who were patients at the Manhattan Veterans Administration Hospital or East Orange Veterans Administration Hospital. The diagnosis for each subject is listed in table 1. Twelve patients had cholesterol gallstones (The gallstones that were removed at surgery con-

677

tained over 80 % cholesterol (w/w).) demonstrated at surgery or suggested by oral cholecystography; 5 patients had extrahepatic biliary obstruction proven at surgery that was caused by either carcinoma of the head of the pancreas or a pseudocyst of the pancreas; 6 patients were treated for chronic duodenal ulcer disease; and no diagnosis could be established in :l patients . The last group of 9 patients showed no evidence of lipid or liver abnormalities and were studied for comparative purposes. All subjects were in good nutritional states and were fed the regular hospital diet for at least 1 week prior to liver biopsy. The per cent of ideal weight" (table 1) did not differ significantly between the three groups, although the weights of the galbtone and control subjects were higher than those of patients with extrahepatic biliary obstruction. In order to minimize variations in hepatic enzyme activity, liver specimens were obtained between 10 AM and 12 PM, after fasting from the preceding evening. Consent to perform the liver biopsies was obtained after the protocol had been explained to each subject. The ex peri mental protocol was reviewed and approved by the human studies committee at the College of Medicine and Dentistry of New ,Jersey, New Jersey Medical School, and East Orange Veterans Administration Hospital. With the exception of the patients with biliary obstruct ion, liver function tests and plasma lipid levels were normal (table 1). As expected, the subjects with biliary obstruction showed increased serum hilirubin concentration, elevated levels of serum alkaline phosphatase and SGOT, and sig-nificantly higher levels of plasma cholesterol. Surgical liver biopsies were obtained during cholecystectomy for cholesterol gallstones in 7 subjects; during subtotal g-astrectomy with vag-otomy in 6 patients with chronic peptic ulcer disease and during- cholecystojejunostomy for relief of biliary obstruction in fi patients. Prior to operation, the patients were premedicated with Droperidol and atropine. The general anesthesia included the administration of sodium hexabarbitol, nitrous oxide, succinylcholine, and Ethrane (Enflurance, Ohio Medical Products, Madison, Wis.). Percutaneous liver biopsies were performed with a 16-gauge Menghini needle in 5 subjects with gallstones and in :3 subjects without evidence of liver disease. Twenty to 40 mg of liver tissue were removed by percutaneous biopsy and 44 to 700 mg of liver tissue were obtained during abdominal surgery. Liver morphology was normal in the gallstone and control subjects and was consistent with extrahepatic biliary obstruction in those 5 subjects with obstructive jaundice. No evidence of

678

SALEN ETAL. T ABLE

Patient

Sex

Age

Vol. 69, No . 3

1. Clinical features Per cen t idea l

P lasma

Diagnosisa

wei~:ht

Cholesterol

yr

K. S." J. T . I. K. E.M. R. p • A.S . T.S." J.G. A.D . W.M." D. C. J.P."

F M M F M M F M M M F M

Mean ± so F. B. J. R. J.R. J. T. R. E . J . S." A. S." J.R." L.Y."

M M M M M M M M M

Mean ± so J.W. F.K. W. S.

c. R.

J. c.

Mean ± so

M M M M M

Triglyccridcs

mii/I OOm l

26 58 66 55 54 60 44 21 58 53 54 64

96 109 122 120 110 93 108 104 102 102 105 103

51 ± 14

107 ± 8

58 60 62 44 51 55 53 62 58

103 114 119 101 93 126 113 96 104

57 ± 4

108 ± 11

68 66 62 48 74

84 93 102 109 104

59± 13

98 ± 10

G.S. G.S. G.S. G.S. G.S . G.S . G.S. G.S. G.S. G.S. G.S. G.S.

D.U. D.U. D. U. D.U. D.U. D.U. N N N

142 220 150 330 180 248 220 140 258 225 260 250

72 84 110 140 210 180 120 60 110 76 84 80

215 ± 58

113 ± 47

222 260 230 190 188 240 235 260 240

110 148 140 238 100 140 110 140 130

199 ± 26

I

C.P. C.P. P.P. P.P. C.P.

600 240 310 280 290 344 ± 45

127

±

17

160 llO 150 180 210 162

±

37

a G.S., gallstones; D.U., duodenal ulcer; N, no diagnosis; C.P., carcinoma of pancreas; P.P., pancreatic pseudocyst . • Percutaneous biopsy.

liver m etast as is was found in the 3 patients with carcinom a of the pancreas. After removal, the liver tissue was immediately placed in ice-cold homogenizing solution and trans ported to the Ia boratory in an ice bucket within 15 to 20 min for processing.

Preparation of Liver Microsome ' 3 The liver tissue was blotted dry on sterile gauze, weighed, and put into a 15-ml centrifuge tube (Corning No. 8441) in the amount of homogenizing medium necessary to obtain a solution of 4 to 10% concentration (w/v). Tern-

perature was maintain ed between 0 and 5°C durin g preparation of the microsomes, either by means of an ice bath or by working in a cold room (4°C) . The homogenizing medium con tained: sucrose, 0.3 M; nicotin amide, 0.075 M; EDTA, 0.002 M; and mercapt oethanol, 0.02 M . The t issue was homogenized with a loose fitt ing Teflon pestle and the whole homogenate was centrifuged at 11,000 g for 10 min to sediment nuclei, cell debris, a nd the m itochondrial fraction. T he supernatant solution was cent rifuged at 100,000 g for 60 min to obt a in t he microsomal pellet. After the supernatant solution was de-

September 1975

CHOLESTEROL METABOLISM IN GALLSTONES

canted, the sedimented microsomal pellet was dispersed in the same centrifuge tube with a volume of homogenizing medium to give a solution of 4 to 10% concentration . These microsomal suspensions were used for the assay of cholesterol 7a-hydroxylase and HMG-CoA reductase activities. The microsomal protein concentration was determined by the method of Lowry et al. 14 A typical preparation had a concentration of 1 to 3 mg of protein per mi. This procedure, performed with a single transfer of the 11,000 g supernatant fraction into the centrifuge tube, was used both for centrifugation and homogenization of the microsomes and permitted the isolation of 25 to 30 mg of microsomes per g of liver tissue (wet weight) .

679

action was stopped with 15 volumes of methylene chloride-ethanol (5 : 1, v/v), and the reaction product, (4-"C) hydroxycholesterol, was extracted, separated by thin layer chromatography, and determined by scintillation coun ting. Enzyme activity was expressed as picomoles of 7a-hydroxycholesterol formed per mg of protein per min. When sufficient microsomes are available the mass of 7n-hyd roxycholesterol that was formed was also measured directly by an isotope derivative method with comparable results. 16 Determination of Liuer and Microsomal Cholesterol

L iver total cholesterol. Since the amount of liver tissue was limited, the concentration of The procedure was similar to t hat described liver cholesterol was determined in an aliquot in a recent publication 13 and was performed (0.1 ml) of the whole homogenate used for the under optimal conditions . The standard assay preparation of the microsomal fraction . This system contained, in a volume of 0.85 ml: aliquot was refluxed for 3 hr with 2fi% KOHphosphate buffer, pH 7 .4, 100 mM; MgC1 2 , 3 ethanol; the nonsaponifiable fraction was exmM; NADP, 3 mM; glucose-6-phosphate, 10 tracted with hexane. The cholesterol content was determined by gas-liquid chromatography mM, glucose-6-phosphate dehydrogenase (EC (Barber-Colman Selecta 5000 gas chromato1.1.1.49), 3 enzyme units; mercaptoethanol, graph, with hydrogen flame detector). The col20 mM; R,S- [3- "C]HMG-CoA (2600 dpm per umn (4-mm inside diameter, lilO em long) was mmole), 0.4 mM; microsomal protein, 0.1 to packed with 3% QF-1 on 80 to 100-mesh Gas 0.4 mg. Incubation was carried out at 37°C for 20 min. The reaction product, mevalonic acid, Chrom Q (Applied Science Laboratories, Inc., State College, Pa.). was lactonized with 1 N sulfuric acid and exMicrosomal free ch olesterol. This was dete rtracted exhaustively with ether, after adding mined by an isotope derivative micromethod as unlabeled mevalonolactone as carrier. After ,8- [3 H ]acetoxycholesterol as follows. An aliquo t evaporation of the ether, the residue was puriof the microsomal s uspension was extracted fied by thin layer chromatography on Silica twice with 15 times its volume of dichloroGel G plates, developed with acetone-benzene methane-ethanol (5: 1, v / v); the organic solvent (1:1, v/v) and counted as described previously.'' was evaporated under n itrogen and acetylated Enzyme activity was expressed as picomoles of with a solution of 5% [3 H ]acetic anhydride mevalonolactone formed per mg of protein per in pyridine at l00°C for 1 hr. J 3 H JAcetoxymin. cholesterol was separated by thin layer chromatography (Alumina Plates, Analtech, Inc., NewAssay of Cholesterol ?a-Hydroxylase Activity ark, Del.) and counted in a liquid scintillation Incubations of the microsomal enzyme with spectrometer (Beckman LS-200B, Beckman In[4, 14 C]cholesterol) (6 x 10' dpm, 5 x 6o-• M) struments, Fullerton , Calif.). The amount of were performed under optimal conditions ac- cholesterol (in nmoles) is calculated by dividing cording to Nicolau et al. 15 The substrate- the disintegrations per min in the cholesteryl was solubilized with cutscum (isooctylphenox- acetate by the specific radioactivity of the ypolyethylene-ethanol, Fisher Scientific Co., [ 3 H ]acetic anhydride, taking into account t hat Fair Lawn N.J.). The assay system contained, 1 mole of cholesterol requires 1 mole of acetic in a volume of 0.5 ml: 0.15 ml of an NADPH- anhydride for acetylation. Corrections for losses generating system (potassium phosphate during acetylation and extraction are made by buffer, pH 7.4, 70 mM; MgCl,, 4.5 mM; utilizing a known amount of [4-"C ]cholesterol N ADP, 1.25 mM; glucose-6-phosphate, 2.5 (approximately 0.1 nmole, 11 x 10 7 dpm) added mM; glucose-6-phosphate dehydrogenase, 5 to the microsomal suspension before extraction. enzyme units; 0.15 ml of medium containing Microsomal total cholesterol was determined the solubilized substrate; and 0.2 ml of micro- by the same procedure, after hydrolysis of an somal protein. Incubation was carried out at aliquot of the microsomal suspension with 25% 37 ac for 20 min, with exclusion of light; the re- KOH-ethanol and extraction with hexane . Assay of HMG-CoA reductase Activity

680

SALEN ETAL .

Results Hepatic HMG-CoA reductase actwLty. The values for hepatic HMG- CoA redu~­ tase activity are presented for 12 subjects with ga llstones, 5 subjects with extrahepatic biliary obstruction, and 8 control subjects in table 2 and figure 1. The results show that t he mean hepatic HMG-CoA reductase activity was 27 % greater in the gallstone subjects than the control subjects, whereas in t he subjects

Vol. 69, No.3

0

Control

-

Gollston~s

~ %~':;::clio" (

J No. of Obs~rvot lons

FIG. 1. Hepatic HMG-CoA red uctase activity. Bars around the mean represent ± 1 SD. Statistical significance was determined by Student's t-test.

TABLE 2. H epatic cholesterol metabolism Patient

HMG-CoA Reductase

Cholest erol ?a-hydroxylase

pmoles/mf, protein/min

K.8 .' J. T.' I. K.' R. M.' R. P.' A. 8 .' T. 8.' J .G.' A.D.' W. M.' D. C.' J.P.'

77

49 80 75

7.2 5.3 8.5 10.3

77.3 ± 10.6

9.6 ± 4.4

63 64 65

23.5 20.1 12.8 20.0 21.3 13.4 19.2 18.8 15.0

77

50 73 64 55 54

Mean ± so J . W! F. K! W.8 .' C. R.' J . C.'

61.0 ± 7.4 29 .0 19.4 13.3 10.4 13.0

SD

17.0

±

m{<(f,

10.4 3.0 5.5

77

F. B.' J. R.' J. R.' ,J. T.' R. E.' J . 8' A.8.' J. R.' L. Y.'

I

Hepatic Cholesterol

7.2 13.2 19.7

90 78

Mean ± so

Mean ±

I

86 70 79 89

7.8 6.9

I 1

7.5

a Gallstones. ' Duodenal u lcer. ' No diagnosis. "Carcinoma pancreas. 'Pancreatic pseudocyst.

18.2 ± 3.7 2.0 2.8 4.3 3.5 7.2 4.0 ± 2.0

3.0 5.4 4.9 4.6 5.5 5.3

5.3

±

2.2

3.9 2.7 3.5 3.9 3.5 3.0

3.4

±

0.5

10.5 2.2 3.4 3.5 6.4 5.2

±

3.3

with extrahepatic biliary obstruction, mean hepatic HMG-CoA reductase activity was 75% lower t han in the controls. Both differences were statistically significant. T hese findin gs are consistent with elevated hepatic cholesterol production in gallstone subjects. In contrast , patients with biliary obstruction probably have diminished rates of hepatic cholesterol synthesis. The large differences in hepatic HMG-CoA reductase activity between the patients with gallstones a nd biliary obstruction and the control group were not reflected in their plasma cholesterol levels (table 1). In fact, the plasma cholesterol concentrations were highest in t he subjects with biliary obstruction where H MG-CoA reductase activity was lowest; a finding that suggested cholesterol accumulated because of retention rather t han overproduction. Hepatic cholesterol 7a-h ydroxylase activity. T he potential capacity of t he liver to produce bile acid from cholesterol was estimated indirectly by measuring the activity of hepatic cholesterol ?a-hydroxylase, and the results are presented in table 2 and figure 2. In 9 gallstone subjects, mean hepatic cholesterol ?a-hydroxylase activity was barely one-ha lf t h at of 9 control subjects. The difference between the groups is highly significant. Similarly, hepatic cholesterol 7a -hydroxylase activity was severely depressed in the 5 subjects with extrahepatic biliary obstruction as compared with the 9 control subjects. The reduction in hepatic ch o lesterol 7a· hydroxylase activity in both t he subjects with gallstone and biliary obstruction is consistent with lower bile acid synthesis.

September 1975

Hepatic cholesterol concentration. Hepatic cholesterol concentrations were determined on a portion of the liver biopsy specimen used for enzyme analysis. The results are presented in table 2 and figure 3. The mean concentration of cholesterol in the livers of the gallstone subjects was 56 % higher than the controls. The difference was of marginal statistical significance (0.06 > P > 0.05), and larger numbers of observations will be required to substantiate this point. In those subjects with biliary obstruction, hepatic cholesterol content was higher in 2 of 5 subjects than the mean values in the controls. However, the wide variation in hepatic cholesterol concentration in the subjects with biliary obstructions probably reflects the duration of extrahepatic bile obstruction; i.e., the longer the obstruction, the greater the retention of hepatic cholesterol. Relationship between the level of cholesterol in the liver and hepatic microsomes. In order to eliminate the possibility that

0

Control

-

Gollston~s

~ ~~:~:~clion (

J No. of Obs~rvotions

FIG. 2. Hepatic cholesterol7a-hydroxylase activity. Bars around the mean represent ± 1 so . Statistical significance was determined by Student 's t-test.

6 !5

CJ

Control

-

Gollston~s

~ g;'::::ction (

J No. of Observations

e; 4

~

3

~

2

681

CHOLESTEROL METABOLISM IN GALLSTONES

FIG. 3. Hepatic cholesterol concentrations . Bars around the mean represent ± 1 SD. Mean differences were of marginal statistical significance as determined by Student's t-test.

TABLE

3 . Compartmentation of cholesterol in human liver Liver fraction

Control (3)"

Whole homogenate' 1.6 (mg Ch/g) Microsomes< 51.2 (nmoles Ch/mg protein)

Gallstones (3)"

±

0 .3

2.7

±

0.5

±

4 .8

53.6

±

5.2

• Number in parentheses is number of observations. 'Liver tissue plus homogenizing solution. Ch, cholesterol. c Free cholesterol represented 90% and esterified cholesterollO% oftbe total cholesterol in microsomes.

the differences in HMG-CoA reductase and cholesterol 7a-hydroxlase activities were related to the content of hepatic or microsomal cholesterol, measurements of cholesterol were performed on the microsomal fraction and liver homogenate from 3 gallstone and 3 control subjects. The results are presented in table 3. As expected, the mean value for the homogenate (liver plus homogenizing solution) from gallstone subjects contained more cholesterol than the control homogenate. However, the cholesterol concentration of the isolated ml crosomes were almost the same in the gallstone and control subjects. Thus, the difference in hepatic cholesterol ?ahydroxylase activity between both groups probably cannot be ascribed to an increased amount of microsomal cholesterol.

Discussion The results of this investigation clearly show abnormalities in the regulation of two key enzymes involved in hepatic cholesterol metabolism in patients with gallstones and biliary obstruction. In the gallstone group, hepatic cholesterol ?a-hydroxylase activity was diminished, suggesting that bile acid formation was low in this condition. However, quantitative measurements of bile acid production in a similar group of male gallstone subjects did not differ from control subjects , yet bile acid turnover was more rapid. 17 • 18 Thus, the small bile acid pools found in the gallstone subjects 19 may result from an inability to increase production sufficiently to compensate for intestinal losses. It is important to

682

SALEN ETAL.

emphasize that the effective bile salt pool includes the size multiplied by the number of enterohepatic circulations. Thus, a small pool can be counterbalanced by an increased number of enterohepatic circulations, and a report by Northfield and Hofmann indicates that this may be so in patients with gallstones. 19 Yet, as a consequence of a small pool of bile salts, the hepatic bile salt concentration may be reduced and leads to increased hepatic cholesterol production in an effort to supply additional substrate for conversion into bile acids. It is noteworthy that hepatic HMG-CoA reductase activity was significantly elevated (fig. 1) in the gallstone subjects. Although the quantitative measurements of cholesterol production could not be obtained in our patients, increased hepatic HMG-CoA reductase activity has been correlated with elevated cholesterol production in the rare inherited lipid storage disease, cerebrotendinous xanthomatosis. 13· 20 Nevertheless, it is important to restate that only the hepatic activities of the two rate-determining enzymes, HMG-CoA reductase and cholesterol 7ahydroxylase, were measured. The quantitative relationship of the enzyme activities to actual measurements of cholesterol and bile acid production remains to be established in man. Nevertheless, it appears that in gallstone patients, a portion of the cholesterol entering the liver, exogenously or endogenously, cannot be metabolized further to bile acids, perhaps because of reduced microsomal cholesterol 7a-hydroxylase activity, and must either be retained or excreted into the bile. Our finding that cholesterol levels were marginally higher in the gallstone subjects suggests that the cholesterol was deposited in the liver and is supported by similar observations of increased liver cholesterol levels in subjects with gallstones reported by Villa et a!. 21 Thus, we propose t hat the elevated concentration of cholesterol in the liver may be a factor in promoting the flux of cholesterol in the bile. Another interesting observation was the lack of correlation between HMG-CoA reductase activity and plasma cholesterol levels. In the gallstone subjects, cholesterol

Vol. 69, No.3

levels were not elevated and did not differ significantly from the control subjects. Therefore, despite augmented hepatic synthesis and diminished catabolism cholesterol did not accumulate in the plasma. It is also noteworthy that no relationship existed between total body weight and hepatic HMG-CoA reductase activity. Gallstone subjects were slightly overweight, but their mean per cent of ideal weight did not differ significantly from the mean per cent of ideal weight of the control subjects (table 1). Despite the evidence that obesity is associated with greater cholesterol production, 22 the increased hepatic HMG-CoA reductase activity seen in the gallstone group seems to be an intrinsic part of this condition. In contrast to the situation for cholel ithiasis where diminished cholesterol ?ahydroxylase activity was associated with augmented HMG-CoA reductase activity, patients with biliary obstruction show markedly depressed hepatic cholesterol 7ahydroxylase and HMG-CoA reductase activities . This indicated that both cholesterol and bile acid synthesis were depressed. Although the enterohepatic circulation of bile acids were interrupted by obstruction of the common bile duct, the resulting retention and hepatic accumulation of bile acids probably inhibited the microsomal production of cholesterol and bile acids. It is important to emphasize t hat the measurements of HMG-CoA reductase and cholesterol 7a-hydroxylase were performed in subjects who were blocked for relatively long periods, and differ from observations made in animals during t he acute phase of biliary obstruction. A number of investigators incuding Fredrickson et al. 23 and Dietschy and W eis, 24 have measured t he conversion of [ 14 C ]acetate into cholesterol in liver specimens from rats with acute biliary obstruction and have found high rates of cholesterol formation. Since obstructed patients (and animals) show increased levels of plasma and tissue cholesterol, it was suggested that the increased tissue cholesterol arose from this increased synthesis. However, since HMG-CoA reductase activity was so low in the pat ients with chronic

September 197.5

CHOLESTEROL METABOLISM IN GALLSTONES

683

biliary obstruction, it is possible that the pharmacological conditions. Similar reinterference with the secretion of choles- sults that confirm the validity of the isoterol and decreased catabolism of choles- tope incorporation method for the assay of terol into bile acids accounts for the hyper- hepatic microsomal cholesterol ?acholesterolemia seen in this condition. hydroxylase activity have been published One of the most interesting findings in by Bjorkhem and Danielsson. 26 Finally, the demonstration of elevated this study was the lack of an inverse correlation between hepatic HMG-CoA re- hepatic HMG-CoA reductase activity and ductase activity and the concentration of cholesterol concentrations in gallstone subhepatic cholesterol. Despite the greater jects has particular relevance to the treatliver cholesterol levels in the gallstone ment of this condition with chenodeoxvsubjects, HMG-CoA reductase activity was cholic acid . A number of papers ha~e higher in the normal subjects with less shown that the administration of the prihepatic cholesterol. This observation mary bile acid, chenodeoxycholic acid, to argues against the role of tissue cholesterol patients with cholelithiasis results in prolevels in regulating hepatic cholesterol pro- gressive diminution in gallstone size and duction and also has important implica- eventual dissolution. 28 • 29 Recently, we tions for the performance of the cholesterol have reported that chenodeoxycholic acid ?a-hydroxylase assay by the isotope incor- inhibits hepatic HMG-CoA reductase acporation method. Since cholesterol is the tivity and reduces liver cholesterol concensubstrate for microsomal cholesterol ?a- trations in gallstone subjects . 29 Similar hydroxylase, increased amounts of micro- findings have also been published by Bonsomal cholesterol may lead to spuriously orris and colleagues. 30 Thus, it appears low values of cholesterol ?a-hydroxylase that chenodeoxycholic acid may work in activity. However, as indicated, the con- gallstone subjects by restoring hepatic chocentrations of microsomal cholesterol were lesterol synthesis to normal. the same in both gallstone and control subjects despite the difference in total REFERENCES hepatic cholesterol content. This finding reduces the possibility that changes in 1. Bloch K: The biological synthesis of cholesterol. microsomal cholesterol were responsible for Science lfiO: I H··2H, I fl7 4 2. Kritchevsky D: Cholesterol. London , .John Wiley decreased cholesterol 7 a-hydroxylase ac& Son , 19fiH tivity. :3. Die tschy ,J, Wilson ,JD: Regulation of cholesterol It should be noted that Mitropoulos and metabolism. N Eng! ,J Mcd 2H2: 112H- I1:lH. Balasubramaniam have questioned the va1179-1184, 1241-1249, 1970 lidity of the isotope incorporation method 4 . Siperstein MD: Regulation of cholesterol synt heused in this study to measure microsomal sis in normal and malignant tissues . Curr Top cholesterol ?a-hydroxylase activity. 25 They Cell Regul 2:74- 76, 1970 argue that only a portion of the microsomal 5. Mosbach EH: Hepatic synthesis of bile acids. cholesterol pool is available for conversion Arch Intern Med 1:30:478-487, 1972 6. Bjorkhem I, Danielsson H, Einarsson K: On the to 7 a-hydroxycholesterol and, consemetabolism of cholesterol in rat liver hornogequently, the assay underestimates 7anates. Eur ,J Biochem 4:4fi8- 4fi:l 1968 hydroxycholesterol formation. Recently, 7. Small OM, Rapo S: Source of abnormal bile in 16 Shefer et al. have examined this question patients with cholesterol gallstones. N Eng! ,J by a newly developed isotope derivative Med 283:5:3- 57, 1970 assay that accounts for the pool of micro8. Vlahcevic ZR , Bell CC, Swell L: Significance of somal cholesterol that is available for conthe liver in the product ion of lithogenic bile in version to 7a-hydroxycholesterol. The reman. Gastroenterology fi9:62 - fi9, HJ70 sults showed that the new assay was more 9. Vlahcevic ZR, Bell CC, Huhac l, et al: Diminsensitive than the isotope incorporation ished bile acid pool size in patients with gallmethod reported here, but both methods stones. Gastroenterology fiB: 1Gfi- l7:l, lfl70 were comparable and showed the same 10. Grundy SM, Metzger AL, Adler [{f): Mechanisms of lithogenic bile formation in American Indian differences under various physiological and

684

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

SALEN ETAL .

women with cholesterol gallstones. J Clin Invest 51:3026-3043,1972 Grundy S, Duane WC, Adler RD et a!: Biliary lipid outputs in young women with cholesterol gallstones. Metabolism 23:67-73, 1974 Metropolitan Life Insurance Company, Statistical Bulletin 40 (November-December, 1959): New Weight Standards for Men and Women, New York Nicolau G. Shefer S, Salen G. et a!: Determination of hepatic 3-hydroxy-3-methylglutaryl CoA reductase activity in man . J Lipid Res 15:94-98, 1974 Lowry OH, Rosebrough NJ, Farr AL, et a!: Protein measurement with the Folin phenol reagent. J Bioi Chern 193:265- 275, 1951 Nicolau G, Shefer S, Salen G, et a!: Determination of hepatic cholesterol 7a-hydroxylase activity in man. J Lipid Res 15:146-151, 1974 Shefer S, Nicolau G, Mosbach EH : Isotope derivative assay of microsomal cholesterol ?a-hydroxylase. J Lipid Res 15:146- 151, 1975 Swell L , Gregory D, Vlahcevic ZR: Current concepts of the pathogenesis of cholesterol gallstones. Med Clin N Am 58:1449-1471, 1974 Almond HR, Vlahcevic ZR, Bell CC et a!: Bile acid pools, kinetics and biliary lipid composition before and after cholecystectomy. N Eng! J Med 289:1213- 1216, 1973 Northfield TC, Hofmann AF : Biliary lipid output during three meals and an overnight fast. 1. Relationship to bile acid pool size and cholesterol sat uation of bile in gallstone and control subjects. Gut 16:1-11, 1975 Sal en G, Grundy SM: The metabolism of cholestanol, cholesterol and bile acids in cerebrotendinous xanthomatosis. J Clin Invest 53:2822-2835, 1973

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21. Villa L, Idea G, Agostoni A, eta!: Further studies on liver metabolism in subjects with gallbladder cholesterol stones. Acta Med Scand 175:691-695, 1974 22. Miettinen TA: Cholesterol production in obesity. Circulation 44:842-850, 1971 23. Fredrickson DS, Lound A V, Kinkel man BT, eta!: The effect of ligation of the common bile duct on cholesterol synthesis in the rat. J Exp Med 99:43-53, 1954 24. Weis HG, Dietschy J: Failure of bile acids to control hepatic cholesterogenesis: evidence for endogenous cholesterol feedback. J Clin Invest 48: 2398-2408, 1969 25. Mitropoulos KA, Balasubramaniam S: Cholesterol ?a-hydroxylase in rat liver microsomal preparations. Biochem J 128:1 -9, 1972 26. Bjorkhem I, Danielsson H : Assay of liver microsomal cholesterol ?a-hydroxylase using deuterated carrier and gas chromatography-mass spectrometry. Anal Biochem 59:508-516, 1974 27 . Bell GD, Whitney BW, Dowling RH: Gallstone dissolution in man using chenodeoxycholic acid. Lancet 2:1213-1216, 1972 28. Danzinger RG, Hofmann AF, Thistle JL, et al: Effect of oral chenodeoxycholic acid on bile acid kinetics and biliary lipid composition in women with cholelithiasis. J Clin Invest 52:2809-2821, 1973 29. Salen G, Nicolau G, Shefer S: Chenodeoxycholic acid inhibits elevated hepatic HMG CoA reductase activity in subjects with gallstones (abstr) Clin Res 21:523, 1973 30. Bonorris GG, Coyne MG, Goldstein LF, et al: Chenodeoxycholic acid and phenobarbital effect on the rate-limiting enzymes of cholesterol and bile acid synthesis in man (abstr). Gastroenterology 67:780, 1974