Apolipoprotein E polymorphism and gallstones

Apolipoprotein E polymorphism and gallstones

GASTROENTEROLOGY 1996;111:1603–1610 Apolipoprotein E Polymorphism and Gallstones ´ N,* MARI´A VELA,‡ ROSA M. PE´REZ–AYUSO,‡ ANTONIA BERTOMEU,* EMILIO...

325KB Sizes 6 Downloads 145 Views

GASTROENTEROLOGY 1996;111:1603–1610

Apolipoprotein E Polymorphism and Gallstones ´ N,* MARI´A VELA,‡ ROSA M. PE´REZ–AYUSO,‡ ANTONIA BERTOMEU,* EMILIO ROS,* DANIEL ZAMBO EDUARDO TARGARONA,§ MANUEL TRI´AS,§ CAROLINA SANLLEHY,\ ELENA CASALS,\ ´Ø and JOSEP M. RIBO *Lipid Clinic, Nutrition and Dietetics Service, ‡Gastroenterology Service, §Surgical Department, and \Clinical Biochemistry Service, Hospital ClıB nic i Provincial, and ØDepartment of Organic Chemistry, Barcelona School of Chemistry, University of Barcelona, Barcelona, Spain

See editorial on page 1764. Background & Aims: Apolipoprotein (apo) E is a genetically polymorphic protein influencing lipoprotein metabolism and the risk of both atherosclerosis and Alzheimer’s disease. As opposed to common apo E3, apo E2 decreases and apo E4 increases hepatic lipoprotein uptake; hence, apo E4 could promote gallstone formation by increasing hepatic and biliary cholesterol concentrations. This study was designed to evaluate whether apo E polymorphism is related to gallstone risk. Methods: apo E phenotype was determined in subjects older than 40 years of age (160 with and 125 without gallstones) and in 61 patients with cholesterol gallstones who underwent cholecystectomy. Bile composition, nucleation time, and gallstone features were analyzed in surgical patients. Results: The E4/3 phenotype was enriched in both patients with gallstones and those who underwent cholecystectomy, with significantly (P õ 0.006) higher e4 allele frequencies than in gallstone-free subjects (odds ratio, 2.67 [95% confidence limits, 1.23–5.93] and 3.62 [95% confidence limits, 1.49–8.91], respectively); women, but not men, accounted for these differences. The prevalence of the e4 allele increased with age in patients with gallstones, whereas the opposite occurred in gallstone-free subjects. Biliary lipid and gallstone cholesterol content tended to increase in the sequence E4 ú E3 ú E2 in patients who underwent cholecystectomy. Conclusions: Carrying the apo E4 isoform is a genetic risk factor for cholelithiasis in humans, thus adding another adverse effect of apo E polymorphism on health.

A

polipoprotein (apo) E is a polymorphic protein associated with plasma lipoproteins. Human apo E is genetically controlled by three alleles (e2, e3, and e4) at a single gene locus in chromosome 19; these code for three isoforms (E2, E3, and E4) and thus determine the six phenotypes resulting from the combination of any two of them.1 The ancestral isoform of the protein is apo E3, and this has a cysteine at residue 112 and arginine at residue 158, whereas arginine in apo E4 and cysteine / 5e14$$0003

11-13-96 21:28:36

gasa

in apo E 2 are present at both sites.1 apo E isoforms are important in cholesterol metabolism and atherosclerosis. The apo E protein acts as the ligand between triglyceriderich lipoprotein particles and hepatic low-density lipoprotein (LDL) and chylomicron remnant receptors, and lipoproteins with the E4 isoform are taken up with greater affinity than those with the E2 isoform.2 Homozygous and heterozygous E4 subjects have been shown to both absorb3 and deliver4 to the liver more intestinal cholesterol than subjects with E2/E2, E3/E2, or E3/E3. Accelerated chylomicron remnant clearance leads to down-regulation of hepatic LDL receptors,2 which underlies the well-known hypercholesterolemic effect of the e4 allele, with its attendant high risk of atherosclerosis.5 Thus, the e4 allele has been reported to increase the risk of preclinical atherosclerosis in young people,6 for exercise-induced myocardial ischemia,7 for coronary heart disease mortality,8,9 and for cerebrovascular disease.10 Moreover, the e4 allele is one major factor responsible for the familial predisposition to ischemic heart disease.11 As a lipid transport protein, apo E also plays a part in normal neurobiology and the pathogenesis of Alzheimer’s disease.12 Indeed, there is a remarkable association between the apo E4 protein variant and both familial lateonset13 and sporadic Alzheimer’s disease.14 Because the efficiency of lipoprotein uptake by the liver increases in the sequence E4 ú E3 ú E2, the polymorphism of apo E influences hepatic cholesterol content2,4 and, possibly, cholesterol gallstone formation. The presence of bile supersaturated with cholesterol is a necessary prerequisite in the formation of gallstones, and current evidence indicates that hypersecretion of biliary cholesterol is the most common cause of lithogenic bile in humans.15,16 Trafficking of cholesterol in hepatocytes is complex. Hepatic cholesterol is acquired by de novo synthesis as well as the uptake of both LDL and triglycerAbbreviations used in this paper: apo, apolipoprotein; HDL, highdensity lipoprotein; LDL, low-density lipoprotein; VLDL, very-low-density lipoprotein. q 1996 by the American Gastroenterological Association 0016-5085/96/$3.00

WBS-Gastro

1604 BERTOMEU ET AL.

GASTROENTEROLOGY Vol. 111, No. 6

ide-rich lipoproteins such as chylomicron remnants. Within hepatocytes, cholesterol has several fates: incorporation into membranes, conversion into cholesteryl esters, catabolism to bile acids, or direct secretion into bile.17 Much work has focused on whether different pools of preformed or newly synthesized cholesterol are preferentially used for various metabolic fates, but the mechanisms that regulate cholesterol homeostasis are still poorly understood.17 – 24 Whether derived from internalized lipoprotein cholesterol or from de novo synthesis, the amount of free cholesterol available in liver cells seems to regulate biliary cholesterol secretion; hence, any increase in the intrahepatic pool of the steroid can induce biliary cholesterol supersaturation.16 Precipitation of cholesterol crystals is another important factor in the development of gallstones.25,26 By way of increased hepatic uptake of cholesterol absorbed from the intestine and enhanced biliary cholesterol secretion, the presence of the e4 allele could predispose to gallstone formation. To test this hypothesis, we compared the frequency distribution of the apo E alleles in a sample of patients with gallstones and gallstone-free subjects. We also studied the chemical composition of gallstones and gallbladder biles as well as the bile nucleation times in relation to apo E polymorphism in a consecutive series of patients undergoing elective cholecystectomy.

Materials and Methods The studies were approved by the institutional review board, and informed consent was obtained from all subjects.

Patients With Gallstones and Controls The patients with gallstones included 160 consecutive adult patients referred to the outpatient gastroenterology clinic who received a diagnosis of cholelithiasis between April 1992 and May 1994. Patients were accepted into the study regardless of symptomatic status only when having unequivocal evidence of stones on an oral cholecystogram showing a well-opacified gallbladder or an ultrasonogram disclosing either mobile intraluminal echoes with distal acoustic shadowing or a scarred gallbladder containing echogenic structures. The control group included 125 unrelated subjects, matched closely for age, sex, and body mass index, with a strictly normal gallbladder by ultrasound examination. They were drawn from the same clinic register and had minor gastrointestinal complaints as well as a diagnosis other than gallstones (usually nonspecific dyspepsia or irritable bowel syndrome) that was reached after appropriate diagnostic tests were performed. Gallstones are infrequent in the young,27 probably because the influence of risk factors takes time to develop, which is a reason why only subjects at least 40 years of age and older were accepted into the study. All patients with gallstones as

/ 5e14$$0003

11-13-96 21:28:36

gasa

well as controls lived in or near the city of Barcelona. During the first visit, before apo E phenotype was known, all participants were asked about gallstone disease in first-degree relatives. Other factors known to influence gallstone risk,27 such as body mass index (weight in kilograms divided by height in centimeters squared) as well as parity and estrogen use in women, were also recorded. Gallstone calcification was assessed in cholecystograms, which were available for 115 patients with gallstones.

Patients Who Underwent Cholecystectomy Between October 1994 and March 1995, 100 consecutive patients with gallstones scheduled for elective cholecystectomy at the surgical department of our institution were evaluated with oral cholecystography. In patients whose gallbladder was well opacified (n Å 70), bile was retrieved for chemical analyses and nucleation studies by needle puncture of the gallbladder wall before cystic duct ligation and after manual mixing of gallbladder contents to avoid stratification. After cholecystectomy was performed, all of the stones were collected, dried in a desiccator, and stored at room temperature for subsequent analysis.

Lipoprotein Analysis and apo E Phenotyping Blood samples for lipoprotein separation and apo E phenotyping were obtained after an overnight fast and processed as described previously.28 Briefly, serum cholesterol levels were measured by a high-performance enzymatic assay (CHOD-PAP; Boehringer Mannheim, Mannheim, Germany) adapted to a Cobas-Mira automated analyzer (Hoffmann-LaRoche, Basel, Switzerland) with periodic standardization (World Health Organization–approved laboratory for lipid analysis). The high-density lipoprotein (HDL) cholesterol level was determined after precipitation of apo B–containing lipoproteins with phosphotungstate/magnesium. For the separation of lipoproteins, 2-mL aliquots of serum were covered with 2 mL of sodium chloride (d Å 1.006) and centrifuged at 105,000g for 18 hours at 157C in an ultracentrifuge (Kontron Instruments, Zurich, Switzerland) using a fixed angle rotor (50.3 Ti). The supernatant fraction containing the very-low-density lipoproteins (VLDLs) was separated by aspiration of 1 mL from the top for measurement of its cholesterol concentration and apo E phenotyping. LDL cholesterol level was obtained by subtraction of HDL and VLDL cholesterol levels from total cholesterol levels. apo E phenotypes were determined in a delipidated aliquot of the VLDL fraction using a modified acrylamide gel isoelectric focusing method.28 The procedure was performed in a flatbed apparatus using an ultrathin (300 mm) layer of freshly prepared polyacrylamide gel (T5, C6) containing 6 mol/L urea, 8% ampholytes (Pharmalyte 4-6.5; Pharmacia Fine Chemicals, Uppsala, Sweden), and 1.55 mol/L glycerol. After a 30-minute prefocusing (3 W), samples of apo VLDL redissolved on 0.01 mol/L Tris at a pH of 8.2 containing 25% vol/vol of nonionic detergent (Nonidet P-40; LKB, Bromma, Sweden), 8 mol urea,

WBS-Gastro

December 1996

APO E POLYMORPHISM AND GALLSTONES 1605

and 65 mmol/L dithiothreitol to a soluble protein concentration of 5 g/L were applied (5 mL) near the cathode. The gels were electrofocused at 6 W for 2 hours at 47C, fixed on 1.22 mol/L trichloroacetic acid for 30 minutes, equilibrated in alcohol/acetic acid/water (20/6/74, vol/vol), stained with Coomassie brilliant blue G-250 at 607C for 30 minutes, destained in alcohol/acetic acid/water (24/9/67) overnight, dried at ambient temperature, and assessed by densitometry.

Microscopy, Nucleation Time, and Biochemistry of Bile Immediately after fresh gallbladder bile was obtained, 10 mL was examined at 377C under a polarizing microscope fitted with a heating stage for the presence of microcrystalline and other solids. Birefringent cholesterol monohydrate crystals were identified as previously described.29,30 One milliliter of fresh bile was filtered (0.22-mm Millex filter; Millipore, Molsheim, France) into a capped glass tube and incubated at 377C in the dark. To determine the nucleation time,25 a 10-mL aliquot of bile was observed microscopically each day for up to 30 days or until cholesterol crystals were detected. Duplicate aliquots of uncentrifuged, unextracted bile samples were used to measure bile acid, phospholipid, and cholesterol composition by enzymatic methods.29 Compositional data were converted to molar percentage for each lipid component to calculate the percentage saturation of cholesterol in bile according to total lipid concentration by using Carey’s critical tables.31

Analysis of Gallstones The number and maximum diameter of the gallstones obtained at cholecystectomy were recorded. The composition of gallstones was determined by quantitative infrared spectroscopy29 using a Perkin-Elmer model 681 infrared spectrophotometer (Perkin-Elmer Corp., Norwalk, CT). The cholesterol, calcium bilirubinate, and calcium carbonate content of gallstones were expressed as a percentage of the dry weight. Stones with cholesterol content between 50% and 100% were classified as cholesterol stones and those with cholesterol content of õ50% as noncholesterol stones.32

Statistical Methods Corrected x2 statistics were used to evaluate differences between apo E allelic frequencies of patients with gallstones and controls. Mantel–Haenszel odds ratios with 95% confidence intervals were also estimated. The interaction between the apo E phenotypes and lipoprotein cholesterol level, compositional data of bile and gallstones, and bile nucleation times was tested by one-way analysis of variance (ANOVA), with Bartlett–Box and Cochran’s tests for homogeneity of variance. SPSS/PC/ software33 was used for statistical analyses.

Results Patient Characteristics Age, sex, and body mass index were similar in patients with and without gallstones (Table 1). Parity / 5e14$$0003

11-13-96 21:28:36

gasa

Table 1. Demographic and Clinical Data as Well as Serum Lipid and Lipoprotein Cholesterol Levels in Patients With Gallstones, Gallstone-free Subjects (Controls), and Patients With Cholesterol Gallstones Who Underwent Cholecystectomy

No. Sex (F/M) Age ( yr ) Body mass index (kg/cm2) Total cholesterol LDL cholesterol VLDL cholesterol HDL cholesterol Triglycerides

Patients with gallstones

Controls

Patients who underwent cholecystectomy

160 105/55 59 { 1

125 82/43 58 { 1

61 51/10 56 { 2

26.8 243 168 21 50 133

{ { { { { {

0.2 3.9a 3.6b 1.1a 1.0 5.8

26.4 228 156 17 53 126

{ { { { { {

0.3 4.1 4.6 1.0 1.5 7.1

27.3 235 156 20 53 138

{ { { { { {

0.5 6.0 5.5 1.5a 2.1 8.9

NOTE. Lipid and lipoprotein data are expressed as milligrams per deciliter (mean { SEM). a P õ 0.01 vs. controls. b P õ 0.05 vs. controls by unpaired t test.

(average of two pregnancies for women in both groups) and estrogen use (10 women each with and without gallstones) were also well matched. Seventy-one patients with cholelithiasis (44%) had a history of biliary pain, whereas 89 (56%) had silent gallstones. Of 115 patients in whom oral cholecystography had been performed, 30 (26%) had gallstone calcification, which was unrelated to apo E phenotypes. The patients with gallstones submitted to cholecystectomy included 55 women and 15 men, with ages ranging from 40 to 77 years. Cholesterol stones were present in 61 patients; their age, sex, and body mass index are shown in Table 1. Noncholesterol stones were present in 9 patients (4 women and 5 men; age, 58 { 4 years; body mass index, 27.4 { 1.5). Serum Lipids and Lipoproteins Patients with gallstones had total cholesterol, LDL cholesterol, and VLDL cholesterol serum levels greater than those of gallstone-free subjects (Table 1). As shown in Table 1, VLDL cholesterol levels were also higher in patients who underwent cholecystectomy than in controls. Figure 1 shows LDL cholesterol levels for each of the three main apo E phenotypes in patients with gallstones, patients with cholesterol stones who underwent cholecystectomy, and controls. As expected,5 apo E polymorphism had a significant (P õ 0.05) influence on LDL cholesterol levels in patients with gallstones but not in patients who underwent cholecystectomy, although there was a similar trend for an E4 ú E3 ú E2 concentration WBS-Gastro

1606 BERTOMEU ET AL.

GASTROENTEROLOGY Vol. 111, No. 6

Figure 1. LDL cholesterol concentrations in patients with gallstones, patients with cholesterol gallstones who underwent cholecystectomy, and gallstone-free subjects according to apo E polymorphism, with exclusion of E4/2 carriers. Values are expressed as the mean { SEM. h, E2 subjects (all with phenotype E3/2); , E3 subjects (all E3 homozygous); j, E4 subjects (all with phenotype E4/3 except two E4 homozygous in the cholecystectomy group). *P õ 0.05, E2 õ E3 õ E4 in patients with gallstones by multiple range test (one-way ANOVA).

Figure 2. Allelic frequency of e4 in 160 patients with gallstones (j) and 125 controls (h) according to age. Numbers on top of bars are alleles (two per subject) for each age interval. *P Å 0.012, two-tailed Fisher’s Exact Test.

are shown in Table 2. There was enrichment of the E4/ 3 phenotype in patients with gallstones, with a significantly higher e4 allele frequency (odds ratio, 2.67; 95% confidence limits, 1.23–5.93; P Å 0.007). The differences in e4 allelic frequency between patients and controls were statistically significant (P õ 0.05) in women (0.090 vs. 0.031) but not in men (0.118 vs. 0.058). Figure 2 shows the distribution of e4 allele frequencies in patients with gallstones and controls as a function of age. The prevalence of the e4 allele was higher in patients that were 46 years of age or older with gallstones, with a peak at 66–70 years. The apo E phenotype of the 9 patients with noncholesterol gallstones was 3/3 in 8 cases and 4/3 in 1 case.

gradient. In gallstone-free subjects, LDL cholesterol levels were lower in E4 subjects than in E3 subjects, which is a deviation that can probably be attributed to chance because of the small number of E4 subjects. The lowering effect of the e2 allele and increasing effect of the e4 allele on LDL cholesterol levels were similar in men and women. No apo E–related concentration gradient was observed for VLDL cholesterol. apo E Phenotype Distribution The apo E phenotype distribution and allelic frequencies of patients with gallstones as well as controls

Table 2. apo E Phenotype and Allele Frequencies in Patients With Gallstones, Gallstone-free Subjects (Controls), and Patients With Cholesterol Gallstones Who Underwent Cholecystectomy Patients with gallstones All n Phenotype (%) E2/2 E3/2 E3/3 E4/2 E4/3 E4/4 Allele (frequency) 12 13 14

Women

160

105

0 8.8 71.3 1.3 18.8a 0 0.050 0.850 0.100a

Patients who underwent cholecystectomy

Controls Men

All

Women

Men

55

125

82

43

0 8.6 73.3 1.0 17.1a 0

0 9.1 67.3 1.8 21.8 0

0 5.6 86.4 1.6 6.4 0

0 4.9 89.0 2.4 3.7 0

0.048 0.862 0.090b

0.055 0.827 0.118

0.036 0.924 0.040

0.037 0.933 0.031

gasa

Men

51

0 7.0 81.4 0 11.6 0

0 9.8 67.2 1.6 18.0b 3.3

0 9.8 68.6 2.0 15.7b 3.9

0 10.0 60.0 0 30.0 0

0.035 0.907 0.058

0.057 0.812 0.131a

0.059 0.814 0.127a

0.050 0.800 0.150

P õ 0.01 and bP õ 0.05 vs. controls (x 2 test).

11-13-96 21:28:36

Women

61

a

/ 5e14$$0003

All

WBS-Gastro

10

December 1996

APO E POLYMORPHISM AND GALLSTONES 1607

Table 2 also shows the distribution of apo E phenotype and allele frequencies in the 61 patients with cholesterol gallstones that underwent cholecystectomy. The E4/3 phenotype was also more prevalent in patients with cholesterol gallstones than in controls, with a significantly higher e4 allelic frequency (odds ratio, 3.62; 95% confidence limits, 1.49–8.91; P Å 0.0005). Again, the differences in e4 allelic frequency between patients with cholesterol stones and controls were statistically significant (P Å 0.022, two-tailed Fisher’s Exact Test) among women only (0.127 vs. 0.031). Family History of Gallstone Disease Forty-five patients with gallstones (28%) and 21 gallstone-free subjects (17%) recalled that a first-degree relative (usually a parent or sibling) had been treated for gallstone disease. The patients with gallstones with known familial cholelithiasis included 12 of 32 e4 allele carriers (37.5%) and 33 of 128 e4-negative patients (26%); among the control subjects with a positive family history, there were 3 of 10 subjects (33%) with and 18 of 115 without (16%) the e4 allele. These differences are not statistically significant. In patients who underwent an operation whose gallbladder contained cholesterol stones, the frequency of a positive family history of gallstone disease was similar among e4-positive (6 of 14) and e4-negative (18 of 47) patients. Bile Composition and Nucleation Time as Well as Cholesterol Gallstone Features in Relation to apo E Polymorphism The demographic features and gallbladder bile and gallstone composition of patients with cholesterol gallstones carrying the isoforms E2 and E4 (after exclusion of the single patient with the E4/E2 phenotype) and of those homozygous for the E3 isoform are shown in Table 3. Biliary lipid concentrations increased in the order E4 ú E3 ú E2, but the differences were not statistically significant. The percent cholesterol content of gallstones followed the same pattern. The cholesterol saturation indices were similar among patients carrying the three apo E isoforms. All patients had cholesterol crystals present in fresh gallbladder bile except one each with the E2 and E3 isoforms. The median nucleation time of gallbladder bile was similar (2 days each) in the three groups. The median number of stones contained in the gallbladder was 3, 2, and 2 in respective carriers of the E2, E3, and E4 isoforms; likewise, the mean maximum stone diameter was similar (18 mm each) in the three groups. / 5e14$$0003

11-13-96 21:28:36

gasa

Table 3. Demographic Characteristics, Gallbladder Bile Composition, and Gallstone Cholesterol Content in Patients With Cholesterol Gallstones Who Underwent Cholecystectomy According to apo E Phenotype apo E isoformsa E2 (n Å 6) Demography Age ( yr ) Sex (F/M) Body mass index (kg/cm2) Bile composition Cholesterol (mmol/L) Phospholipid (mmol/L) Bile salt (mmol/L) Total lipid (g/dL) Cholesterol saturation index Gallstone composition Percent cholesterol content

E3 (n Å 41)

E4 (n Å 13)

66 { 4 5/1

54 { 2 35/6

56 { 4 10/3

29.4 { 0.7

27.1 { 0.7

27.2 { 1.0

8.8 25.5 66.8 5.7

{ { { {

2.4 7.8 20.2 1.7

14.2 34.5 106.3 8.2

{ { { {

1.5 2.9 12.2 0.8

19.5 47.0 135.1 11.0

{ { { {

3.5 7.1 24.3 1.7

123 { 11

131 { 7

124 { 15

82 { 4

87 { 3

90 { 2

NOTE. No significant differences (P ú 0.05) for any group comparisons. a The 6 patients with the E2 isoform had the E3/2 phenotype, those with the E3 isoform were all E3/3 homozygous, and the 13 patients with the E4 isoform were 11 E4/3 and 2 E4/4.

Discussion The main finding of this study was that patients with gallstones carried the e4 allele of apo E with a frequency 2.7 times that of gallstone-free subjects. Furthermore, the odds of carrying the apo E4 isoform of surgical patients with cholesterol gallstones was 3.6 times that of controls without gallstones. Differences in e4 allelic frequency between patients and controls were apparent in women but not in men, probably attributable to a lack of statistical power because of the small number of men relative to women as in any series of patients with gallstones. However, the prevalence of the e4 allele was low in the three study groups (14% in patients with cholesterol gallstones that underwent cholecystectomy, 10% in patients with gallstones, and 4.0% in controls). apo E allele frequencies are heterogeneous in various populations around the world.5,34 In keeping with the role of apo E in the predisposition to atherosclerosis, the prevalence of apo E4 has consistently been reported as lower in the regions of southern Europe and in Eastern countries (rates ranging from 8% to 12%) than in northern Europe and in the United States (rates ranging from 13% to 22%), where coronary heart disease mortality rates are higher.5,11,34,35 Moreover, E4 bearers may have reduced life expectancy, as suggested by the lower prevalence of the e4 allele in older than in younger people.36 Because we only studied subjects 40 years of age or older, the frequency of the e4 allele might be expected to be WBS-Gastro

1608 BERTOMEU ET AL.

GASTROENTEROLOGY Vol. 111, No. 6

lower than in the general adult population of Barcelona, where a random survey of 512 individuals found a prevalence of only 7.6%.37 Another recent study including 147 older subjects drawn from a nonrandom population in the same geographical area found an e4 allelic frequency of 6.1%.38 Underexpression of the e4 allele (7.5%) has also been reported in a population survey from a different geographical area in Spain.39 The presence of the e4 allele of apo E is strongly associated with the risk of atherosclerosis5 and Alzheimer’s disease.13,14 Our findings add another adverse effect of the apo e4 allele on health, indicating that it is a risk factor for the development of gallstones, albeit it would account for only a minority of disease cases within the populations studied. Nevertheless, these results provide evidence of a genetic predisposition to gallstone formation in humans that may explain, at least in part, the known increased familial frequency of gallstones.40,41 A family history of gallstone disease was not given significantly more often by carriers of the e4 allele than by e4negative subjects. However, because gallstones in the population are mostly asymptomatic,27 familial ultrasonography studies42 would have to be performed to reach a meaningful conclusion on this matter. As with many genetic traits, the gallstone risk derived from the presence of the e4 allele seems to increase with age, whereas its frequency in gallstone-free subjects decreases with age (Figure 2). The decrease in prevalence beyond 70 years of age could be explained by selection against age attributable to higher blood cholesterol levels and increased cardiovascular deaths of e4 subjects.8,9 Known derangements of hepatic cholesterol metabolism determining increased cholesterol content in liver cells with subsequent biliary cholesterol hypersecretion, found in conditions epidemiologically associated with gallstones, are enhanced cholesterol synthesis (obesity, hypertriglyceridemia), increased LDL uptake (estrogenic therapy), decreased cholesterol esterification (treatment with progestogens or fibric acid derivatives), and reduced degradation to bile acids (increasing age).15,16,22 Cholesterol absorbed from the intestine is delivered to the liver by chylomicron remnants4 and could be expected to behave similarly. Published studies on the effect of dietary cholesterol on biliary cholesterol are contradictory, showing either increases or no change of biliary cholesterol saturation (reviewed by Kern43), but apo E phenotypes were not considered in this context. The efficiency of hepatic cholesterol uptake from triglyceride-rich lipoproteins increases in the sequence E4 ú E3 ú E2.4,5 It has been shown recently44 that apo E influences intracellular cholesterol processing by enhancing cholesteryl ester hydrolysis, thus increasing cell free / 5e14$$0003

11-13-96 21:28:36

gasa

cholesterol. There is also preliminary evidence that apo E4 leads to more intracellular release of free cholesterol from internalized triglyceride-rich particle cholesteryl ester than does E3.45 It is tempting to hypothesize that different apo E isoforms will have specific effects on intracellular cholesterol metabolism, with increasing hepatic and biliary cholesterol concentration gradients from the E2 to the E3 to the E4 isoforms. We studied the composition of gallbladder biles of surgical patients with gallstones with different apo E phenotypes and showed a tendency to such a gradient for both biliary cholesterol and the cholesterol content of gallstones (Table 3) but, perhaps because of the small numbers of subjects with the two uncommon apo E alleles (e2 and e4), our study lacked statistical power to confirm this hypothesis. One must bear in mind, however, that bile composition at the time of cholecystectomy reflects a point years after stone nidation and may be far from representing conditions present in the gallbladder at that time. Previous studies46 – 48 have pointed to a genetic component in gallstone formation. Danziger et al.46 found higher biliary cholesterol saturation in the younger sisters of patients with cholesterol gallstones than in those of gallstone-free controls. In a study of monozygotic and dizygotic twin pairs, Kesaniemi et al.47 showed that biliary cholesterol metabolism is to some extent genetically controlled. In a recent study from the same group,48 patients with gallstones with cholesterol crystals in fresh gallbladder bile obtained at surgery showed a higher frequency of apo E4 phenotypes than those without crystals. The distribution of stone cholesterol content was also higher in apo E4 carriers than in the E3 or E2 groups.48 However, no distinction was made in that study48 between cholesterol and noncholesterol stones, and the results could be attributable to overrepresentation of apo E4 subjects among those with cholesterol stones (35% vs. 9% among noncholesterol stones) rather than to a specific effect of apo E4 on the crystallization of biliary cholesterol or its accumulation in stones. In a small subset of patients with cholesterol gallstones, these investigators48 found that those with the E4 phenotype had a shorter median nucleation time than subjects with E2 and E3. We could not detect any differences in median nucleation times among patients with different apo E subscripts in this study. apo E4 polymorphism seems to promote gallstone formation. Compositional studies of bile in gallstone-free subjects or in patients shortly after gallstone nidation in relation to apo E phenotypes should ascertain whether this effect is mediated by enhanced cholesterol secretion into bile, by increased susceptibility to the crystallization of biliary cholesterol, or by both mechanisms. WBS-Gastro

December 1996

APO E POLYMORPHISM AND GALLSTONES 1609

References 1. Zannis VI, Breslow JL. Human very low density lipoprotein apolipoprotein E isoprotein polymorphism is explained by genetic variation and post-translational modification. Biochemistry 1981;20: 1033–1041. 2. Mahley RW. Apolipoprotein E: cholesterol transport protein with expanding role in cell biology. Science 1988;240:622–630. 3. Kesa¨niemi YA, Ehnholm C, Miettinen TA. Intestinal cholesterol absorption efficiency in man is related to apoprotein E phenotype. J Clin Invest 1987;80:578–581. 4. Weintraub MS, Eisenberg S, Breslow JL. Dietary fat clearance in normal subjects is regulated by genetic variation in apolipoprotein E. J Clin Invest 1987;80:1571–1577. 5. Davignon J, Gregg RE, Sing CF. Apolipoprotein E polymorphism and atherosclerosis. Arteriosclerosis 1988;8:1–21. 6. Hixson JE, Pathobiological Determinants of Atherosclerosis in Youth (PDAY) Research Group. Apolipoprotein E polymorphisms affect atherosclerosis in young males. Arterioscler Thromb 1991; 11:1237–1244. 7. Katzel LI, Fleg JL, Paidi M, Ragoobarsingh N, Goldberg AP. ApoE4 polymorphism increases the risk for exercise-induced silent myocardial ischemia in older men. Arterioscler Thromb 1993;13: 1495–1500. 8. Eichner JE, Kuller LH, Orchard TJ, Grandits GA, McCallum L, Ferrell RE, Neaton JD. Relation of apolipoprotein E phenotype to myocardial infarction and mortality from coronary artery disease. Am J Cardiol 1993;71:160–165. 9. Stengard JH, Zerba KE, Pekkanen J, Enholm C, Nissinen A, Sing CF. Apolipoprotein E polymorphism predicts death from coronary heart disease in a longitudinal study of elderly Finnish men. Circulation 1995;91:265–269. 10. Pedro-Botet J, Senti M, Nogues X, Rubies-Prat J, Roquer J, D’Olaberriague L, Olive J. Lipoprotein and apolipoprotein profile in men with ischemic stroke: role of lipoprotein(a), triglyceride-rich lipoproteins, and apolipoprotein E polymorphism. Stroke 1992;23: 1556–1562. 11. Tiret L, de Knijff P, Menzel HJ, Ehnholm C, Nicaud V, Havekes L for the EARS group. ApoE polymorphism and predisposition to coronary heart disease in youths of different European populations. The EARS study. Arterioscler Thromb 1994;14:1617– 1624. 12. Strittmatter WJ, Saunders AM, Schmechel D, Pericak-Vance M, Enghild J, Salvesen GS, Roses AD. Apolipoprotein E: high-avidity binding to ß-amyloid and increased frequency of type 4 allele in late-onset familial Alzheimer’s disease. Proc Natl Acad Sci USA 1993;90:1977–1981. 13. Corder EH, Saunders AM, Strittmatter WJ, Schmechel DE, Gaskell PC, Small GW, Roses AD, Haines JL, Pericak-Vance MA. Gene dose of apolipoprotein-E type-4 allele and the risk of Alzheimer’s disease in late onset families. Science 1993;261:921–923. 14. Poirier J, Davignon J, Bouthillier D, Kogan S, Bertrand P, Gauthier S. Apolipoprotein E polymorphism and Alzheimer’s disease. Lancet 1993;342:697–699. 15. Cooper AD. Metabolic basis of cholesterol gallstone disease. Gastroenterol Clin North Am 1991;20:21–46. 16. Hay DW, Carey MC. Pathophysiology and pathogenesis of cholesterol gallstone formation. Semin Liver Dis 1990;10:159–170. 17. Dietschy JM, Turley SD, Spady DK. Role of the liver in the maintenance of cholesterol and low density lipoprotein homeostasis in different animal species, including humans. J Lipid Res 1993; 34:1637–1659. 18. Schwartz CC, Berman M, Vlahcevic ZR, Halloran LG, Gregory DH, Swell L. Multicompartmental analysis of cholesterol metabolism in man. Characterization of the hepatic bile acid and biliary cholesterol precursor sites. J Clin Invest 1978;61:408–423. 19. Turley SD, Dietschy JM. Regulation of biliary cholesterol output

/ 5e14$$0003

11-13-96 21:28:36

gasa

20.

21.

22.

23.

24.

25.

26. 27. 28.

29.

30.

31. 32.

33. 34.

35.

36.

37.

38.

39.

in the rat: dissociation of the rate of cholesterol synthesis, the size of the cholesteryl ester pool, and the hepatic uptake of chylomicron cholesterol. J Lipid Res 1979;20:923–934. Robins SJ, Fasulo JM, Collins MA, Patton GM. Evidence for separate pathways of transport of newly synthesized and preformed cholesterol into bile. J Biol Chem 1985;260:6511–6513. Nervi F, Marinovic I, Rigotti A, Ulloa N. Regulation of biliary cholesterol secretion: functional relationship between the canalicular and sinusoidal cholesterol secretory pathways in the rat. J Clin Invest 1988;82:1818–1825. Robins SJ, Fasulo JM, LeDuc R, Patton GM. The transport of lipoprotein cholesterol into bile: a reassessment of kinetic studies in the experimental animal. Biochim Biophys Acta 1989; 1004:327–331. Stone BG, Evans CD. Evidence for a common biliary cholesterol and VLDL cholesterol precursor pool in rat liver. J Lipid Res 1992; 33:1665–1675. Scheibner J, Fuchs M, Schiemann M, Tauber G, Hormann E, Stange EF. Bile acid synthesis from newly synthesized vs. preformed cholesterol pools in the rat. Hepatology 1993;17:1095– 1102. Holan KR, Holzbach RT, Hermann RE, Cooperman AM, Claffey WJ. Nucleation time: a key factor in the pathogenesis of cholesterol gallstone disease. Gastroenterology 1979;77:611–617. Sedaghat A, Grundy SM. Cholesterol crystals and the formation of cholesterol gallstones. N Engl J Med 1980;302:1274–1277. Diehl AK. Epidemiology and natural history of gallstone disease. Gastroenterol Clin North Am 1991;20:1–19. Zambo´n D, Ros E, Casals E, Sanllehy C, Bertomeu A, Campero I. Effect of apolipoprotein E polymorphism on the serum lipid response to a hypolipidemic diet rich in monounsaturated fatty acids in patients with hypercholesterolemia and combined hyperlipidemia. Am J Clin Nutr 1995;61:141–148. Ros E, Navarro S, Ferna´ndez I, Reixach M, Ribo´ JM, Rode´s J. Utility of biliary microscopy for the prediction of the chemical composition of gallstones and the outcome of dissolution therapy with ursodeoxycholic acid. Gastroenterology 1986;91:703–712. Ros E, Navarro S, Bru C, GarcıB a-Puge´s A, Valderrama R. Occult microlithiasis in ’idiopathic’ acute pancreatitis: prevention of relapses by cholecystectomy or ursodeoxycholic acid therapy. Gastroenterology 1991;101:1701–1709. Carey MC. Critical tables for calculating the cholesterol saturation of native bile. J Lipid Res 1978;19:945–955. Trotman BW, Morris TA, Sanchez HM, Soloway RD, Ostrow DJ. Pigment versus cholesterol cholelithiasis: identification and quantification by infrared spectroscopy. Gastroenterology 1977; 72:495–498. Norussis MJ. SPSS/PC/. Chicago: SPSS Inc., 1990. Gerdes LU, Klausen IC, Sihm I, Faergeman O. Apolipoprotein E polymorphism in a Danish population compared to findings in 45 other study populations around the world. Genet Epidemiol 1992; 9:155–167. James RW, Boemi M, Giansanti R, Fumelli P, Pometta D. Underexpression of the apolipoprotein E4 isoform in an Italian population. Arterioscler Thromb 1993;13:1456–1459. Eggersten G, Tegelman R, Ericsson S, Angelin B, Berglund L. Apolipoprotein-E polymorphism in a healthy Swedish population: variation of allele frequency with age and relation to serum lipid concentrations. Clin Chem 1993;39:2125–2129. Fiol C, Argimo´n JM, Hurtado I, Machuca I, Pinto´ X, Castin ˜eiras MJ, Gracia V, Llargue`s E, Jime´nez J. Estudio poblacional de la distribucio´n del fenotipo de la apolipoproteina E. Clin Invest Arterioscler 1991;3:130–134. Adroer R, Santacruz P, Blesa R, Lo´pez-Pousa S, Ascaso C, Oliva R. Apolipoprotein E4 allele frequency in Spanish Alzheimer and control cases. Neurosci Lett 1995;189:1–5. Muros M, Rodriguez-Ferrer C. Apolipoprotein E polymorphism in-

WBS-Gastro

1610 BERTOMEU ET AL.

40.

41. 42.

43.

44.

45.

GASTROENTEROLOGY Vol. 111, No. 6

fluence on lipids, apolipoproteins and Lp(a) in a Spanish population underexpressing apo E4. Atherosclerosis 1996;121:13–21. Gilat T, Feldman C, Halpern Z, Dan M, Bar-Meir S. An increased familial frequency of gallstones. Gastroenterology 1983;84: 242–246. Jorgensen T. Gallstones in a Danish population: familial occurrence and social factors. J Biosoc Sci 1988;20:111–120. Sarin SK, Negi US, Dewan R, Sasan S, Saraya A. High familial prevalence of gallstones in the first-degree relatives of gallstone patients. Hepatology 1995;22:138–141. Kern F Jr. Effects of dietary cholesterol on cholesterol and bile acid homeostasis in patients with cholesterol gallstones. J Clin Invest 1994;93:1186–1194. Schwiegelshohn B, Presley JF, Gorecki M, Vogel T, Carpentier YA, Maxfield FR, Deckelbaum RJ. Effects of apoprotein E on intracellular metabolism of model triglyceride-rich particles are distinct from effects on cell particle uptake. J Biol Chem 1995;270: 1761–1769. Al-Haideri M, Presley J, Maxfield FR, Vogel T, Mahley RW, Pitas RE, Sturley SI, Deckelbaum RJ. Apoprotein E4 vs E3: differences

/ 5e14$$0003

11-13-96 21:28:36

gasa

in triglyceride-rich particle cell metabolism. Circulation 1995;92: I–688. 46. Danziger GR, Gordon H, Schoenfield LJ, Thistle JL. Lithogenic bile in siblings of young women with cholelithiasis. Mayo Clin Proc 1972;47:762–766. 47. Kesaniemi YA, Koskenvuo M, Vuoristo M, Miettinen TA. Biliary lipid composition in monozygotic and dizygotic pairs of twins. Gut 1989;30:1750–1756. 48. Juvonen T, Kervinen K, Kairaluoma MI, Lajunen LH, Kesa¨niemi YA. Gallstone cholesterol content is related to apolipoprotein E polymorphism. Gastroenterology 1993;104:1806–1813. Received April 9, 1996. Accepted September 5, 1996. Address requests for reprints to: Dr. Emilio Ros, Lipid Clinic, Nutrition and Dietetics Service, Hospital ClıB nic i Provincial, Villarroel 170, 08036 Barcelona, Spain. Fax: (34) 3-453-7829. Supported in part by grants from Fundacio´ ClıB nic per la Recerca Biome`dica and Fundacio´ Catalana de Nutricio´ i LıB pids. The authors thank Catherine Bouchet (lipoprotein analyses) and Eva Poca (biliary lipid determination) for excellent technical assistance.

WBS-Gastro