The association of bile acid excretion and atherosclerotic coronary artery disease

Abstract

Background: Excess cholesterol is usually eliminated from the body by conversion to bile acids excreted in feces as bile salts. The excretion of large amounts of bile protects against atherosclerosis, while diminished excretion may lead to coronary artery disease (CAD).

Objective: To investigate a relationship between CAD and bile acid excretion.

Methods: Bile acid excretion was compared between 36 patients with proven CAD and 37 CAD-free individuals (controls). The groups were comparable for demographics and selected risk factors. All subjects received a 4-day standard diet that included ∼500 mg of cholesterol. Fecal bile acids from 24-hour stool collections were measured by gas liquid chromatography.

Results: CAD patients excreted lower amounts of total bile acids (358 ± 156 mg) than controls (617 ± 293 mg; p < 0.01) and less deoxycholic acid (188.29 ± 98.12 mg versus 325.96 ± 198.57 mg; p < 0.0001) and less lithocholic acid (115.43 ± 71.89 mg versus 197.27 ± 126.87 mg; p < 0.01). Advanced age, male gender, left ventricular ejection fraction and total bile acid levels were significant independent factors that predicted CAD (p < 0.05). Mortality, CAD and cerebrovascular accident development rates were significantly lower for the controls at the 13-year follow up.

Conclusion: CAD patients have significantly decreased bile acid excretion levels than non-CAD patients. An impaired ability to excrete cholesterol may be an additional risk factor for CAD development.

Introduction

The role of cholesterol and different classes of lipoproteins in the development of coronary artery disease (CAD) has been investigated in much detail over the past five decades. The whole-body cholesterol metabolism is dependent on numerous factors, including dietary fat, fractional absorption of dietary cholesterol, tissue stores of cholesterol, endogenous cholesterol synthesis and fecal bile excretion [Lin and Connor, 1980]. Clinically, it became obvious that despite effective cholesterol-modulating treatment (e.g. statins), the development of atherosclerosis cannot be stopped in a significant number of patients. In addition to statins, low-density lipoprotein cholesterol (LDL-c) can be reduced by increasing the fecal bile acid waste and by compensatory hepatic upregulation of bile acid synthesis [Bhat et al. 2003; Izzat et al. 2000]. While a great deal of attention has been given to the factors that determine cholesterol homeostasis, cholesterol excretion via bile in patients with CAD has not been examined in depth.

It is known that cholesterol is mainly eliminated from the body via the liver in the form of bile acids [Batta et al. 1999]. It is reasonable to speculate that a reduced ability to convert cholesterol to bile acids would lead to body cholesterol overload, with the subsequent development of atherosclerosis [Assmann et al. 2006; Sudhop et al. 2002; Rajaratnam et al. 2000, 1999; Glucc et al.1991]. Studies in animals have revealed that mice and rats [Guorong et al. 2004; Bhat et al. 2003; Post et al. 2003; Lutton, 1990] do not develop experimental atherosclerosis, despite ingestion of a cholesterol-rich diet. They were able to react to the overload of cholesterol intake by excreting large amounts of bile acids. Squirrel monkeys and New Zealand white rabbits were also fed a cholesterol-rich diet in another study [Lofland et al. 1972]. Animals that were able to excrete large amounts of cholesterol did not develop hypercholesterolemia, whereas those with a less efficient excretion had increased plasma levels of cholesterol [Lofland et al. 1972]. Moreover, the degree of hypercholesterolemia was inversely correlated with the rate of bile acid elimination. These animal experiments suggest that the atherogenic effect of the cholesterol-rich diet is highly dependent on the animal’s ability to eliminate cholesterol in the form of bile acids [Guorong et al. 2004; Lutton, 1990]. Surprisingly, the possibility of a similar correlation between the elimination of cholesterol in bile and the development of atherosclerosis has not been adequately studied among humans. In an earlier investigation, our group had compared the elimination of bile acid in the feces of patients who had ischemic heart disease with that of healthy controls on the same diet and reported that the patients excreted much fewer bile acids than the controls [Charach et al. 1998]. Several other studies had shown an inverse relationship between CAD and bile acid excretion [Gylling et al. 2009; Assmann et al. 2006; Rajaratnam et al. 2001; Rajaratnam et al. 2000, 1999; Sudhop et al. 2000; Glucc et al. 1991). Recent human studies found disturbed metabolism of plant sterols (which reflects cholesterol) in postmenopausal CAD women who showed disturbed synthesis and disturbed secretion of bile acids [Gylling et al. 2009; Rajaratnam et al. 2001]. These data prompted us to extend our investigation of the relationship between CAD and bile acid excretion by comparing the bile acid excretion in two groups of patients who complained of chest pain: one group had confirmed CAD and the other was CAD-free. Patients were followed up for up to 13 years for cardiovascular events and mortality.

Methods

All hospitalized patients with chest pain suspected as being CAD (angina pectoris, unstable angina, or myocardial infarction) were eligible for participation in this study that was conducted between January 1995 and December 2008. Excluded from the study were patients younger than 40 years of age (because of a low risk for CAD), as well as those with psychiatric disease, malignancy, acute and chronic renal failure, hepatic disease, severe chronic obstructive pulmonary disease, or acute infectious disease, as well as patients receiving lipid-lowering drugs (statins, fibrates, bile acid sequestrants, antibiotics, antipsychotic drugs or any medicine that could cause cholestasis. A complete medical history was taken, and the medical charts of participants with previous visits to our clinic or admissions to our hospital were reviewed in order to retrieve data on a previous history of CAD and cardiac risk factors, i.e. diabetes mellitus (DM), hyperlipidemia, hypertension, smoking and a body mass index (BMI) >25. The study candidates underwent a careful physical examination, an electrocardiogram, and laboratory tests (complete blood count, glucose level, renal function tests, liver enzyme levels, creatinine kinase to exclude myocardial infarction, and lipid profile) to ensure that they were clinically stable. After undergoing coronary angiography, the patients were divided in two groups: those with confirmed CAD (Group 1) and those without CAD (Group 2). All Group 1 patients were entered into the current study if at least 3 months had elapsed since the acute coronary event in order to exclude any effects of most infarction-related metabolic changes. The study was approved by the local ethics committee and each entrant signed informed consent to participate.

Study protocol

All patients performed a 24-hour stool collection into special containers. A continuous nonabsorbable marker was used to evaluate the quantity of stool excretion. Each subject received 12 capsules of 75 mg copper isothiocyanate to be taken one per meal for the 4 days before stool collection to serve as an internal standard for the purposes of calculation. One week prior to the ingestion of this internal standard, the patients received a standard in-hospital diet containing 490 mg/day of cholesterol. They were asked to record the contents of each meal they consumed. Participants who did not conform to the strict diet were dropped from the study.

Measurement of the lipids content in the serum

Total cholesterol and triglyceride levels were measured enzymatically on a SIEMENS ADVIA 1650 using the manufacturer’s reagents.

Quantitative determination of the bile acids in feces

We used a modification of the method described by Grundy and colleagues [Grundy et al. 1965]. This method allows for complete recovery and separation of bile acids, together with a high level of accuracy, reliability, and simplicity in performance. The stools were collected throughout 24 hours. The amount of bile acids was determined per 225 mg of copper isothiocyanate that served as an internal standard [Gilat and Ronen, 1972]. One week prior to ingesting the capsules, participants were put on a standard diet containing 490 mg cholesterol daily. Fecal bile acids were obtained quantitatively after separation from neutral steroids and saponification of taurine and glycine conjugates. Free bile acids were extracted with chloroform–methanol. The extract was removed from fatty acids and pigments by preparative thin layer chromatography (TLC). TLC 5ά-cholestan was later added as internal standard for gas–liquid chromatography (GLC). The bile acids were converted to trimethyl silylalanine (TMS) ethers which were quantified by GLC. Standards bile acids and 5ά-cholestane were obtained from Sigma Chemical Co. (St Louis, MO, USA), pyridine super dried from E. Merck (Darmstadt, Germany), and hexamethyldisalazan and trimetylchlorosilane from Fluka AG (Buch SG, Switzerland). Thin-layer chromatography was carried out on 0.25 mm of Silica Gel layers (Merck). GLC of bile acid derivatives was performed by a gas–liquid chromatograph (Unigam, Cambridge, UK) equipped with a hydrogen flame ionization detector. A glass column 6 ft × 4 mm packed with 3% OV-1 on 100–120 mesh chromosorb W-HP (Alltech Associates, Inc., Deemfield, IL, USA) was used. Nitrogen with a flow between 6 and 30 ml/min was used as the carrier gas. The column temperature was 275°C, and that of the flash heater and hydrogen flame ionization detector was 280°C.

Statistical analysis

Comparisons between CAD and non-CAD patients with regard to demographic and clinical parameters were by the chi-squared test for categorical variables and the two-sample t-test for continuous variables. The same tests were used for comparing between groups of patients defined according to various risk factors (hypercholesterolemia, smoking, hypertension, and DM). Pearson correlation coefficients were calculated to evaluate the association between triglycerides, LDL-c, high-density lipoprotein cholesterol (HDL-c), total cholesterol, non-HDL-c, and total bile acids. Correlations were computed for each group separately and for the whole sample in combination. Logistic regression model with the CAD group as the dependent variable was applied to locate the variables that best differentiated between CAD and non-CAD patients while adjusting for other variables. Three model building techniques were used: forced entry of all variables to the model, forward selection and backward elimination. All statistical analyses were performed using SAS for Windows, version 9.1.3.

Results

Of the 111 patients who were suitable for enrollment into the study, 38 were excluded for technical reasons, noncompliance, and inadequate compliance to the prescribed diet or lack of follow–up information. The remaining 73 patients underwent coronary catheterization and were subsequently divided into two groups. Group 1 consisted of 36 patients diagnosed as having CAD (mean age 62 years, 26 males [72%]), and Group 2 consisted of 37 patients (mean age 58 years, 18 males [60%]) in whom CAD was ruled out (controls). Table 1 lists the patients’ demographics and CAD risk factors. The Group 2 patients were slightly but significantly younger (p < 0.05, Table 1). There were no significant differences between the groups in the incidence of DM, hypertension, smoking, level of total cholesterol, LDL, triglycerides, BMI, and left ventricular ejection fraction (LVEF). Hemoglobin, glucose albumin, creatinine, and creatinine clearance test findings were similar for both groups.

Table 1.

Patient demographics and frequency of risk factors for coronary artery disease.

Parameter	Group 1 (CAD) n = 36	Group 2 (non-CAD) n = 37	p-value
Age (mean)	62.8 ± 7.2	56.8 ± 9.1	.003
Gender (male)	72	60	NS
BMI (mean)	28.27 ± 3.94	26.75 ± 3.58	NS
Hypercholesterolemia	78%	81%	NS
Smoking	39%	51%	NS
Hypertension	39%	30%	NS
Diabetes mellitus	48%	51%	NS
LVEF	46%	55%	NS
CVA/TIA	18%	3%	<0.0001
Laboratory data (mg/dl):
ALT	27	26	NS
AST	32	31	NS
Alkaline phosphatase	47	46	NS
Albumin	41	42	NS
Bilirubin	0.6	0.5	NS
Creatinine	1.0	0.9	NS

CAD, coronary artery disease; BMI, body mass index; LVEF, left ventricular ejection fraction; CVA, cerebrovascular accident; TIA, transient ischemic attack; ALT, alanine aminotransferase; AST, aspartate aminotransferase.

Table 2 lists the patients’ lipid profiles and 24-hour fecal bile acids. After adjustment to participants’ age the mean HDL-c level was significantly higher in Group 2 (factor of 1.2; p = 0.017). The mean 24-hour fecal bile acid was also higher in Group 2 (p < 0.0001). The CAD patients excreted markedly less total bile acids than the non-CAD patients (1.75-fold; p < 0.0001). The ratios of bile acids between Group 2 and Group 1 were as follows: cholic 1.78, chenodeoxycholic 1.6, deoxycholic 1.7, and lithocholic 1.5. Significant differences in the amounts of the excreted bile acids between the two groups were found in deoxycholic acid and lithocholic acid levels, which were higher for Group 2 than for Group 1. A large variability in the excretion of chenodeoxycholic and cholic acids did not allow the difference in their excretion between CAD and non-CAD patients to reach statistical significance. However, there was a trend in both acids towards decreased excretion in CAD patients.

Table 2.

Main laboratory and clinical data.

Parameter	Group 1 (CAD)	Group 2 (non-CAD)	p-value
Total cholesterol (mg/dl)	254.79 ± 64.81	246.77 ± 41.82	NS
HDL-c (mg/dl)	43.23 ± 10.93	50.56 ± 13.62	0.017
LDL-c (mg/dl)	165.39 ± 55.80	151.31 ± 36.43	NS
Triglycerides(mg/dl)	228.26 ± 163.50	227.51 ± 84.31	NS
Non-HDL cholesterol (mg/dl)	214.26 ± 54.41	197.57 ± 41.5	NS
Total bile acids (mg/24 h)	358.58 ± 156.6	616.91 ± 293.3	<0.0001
Cholic acid (mg/24 h)	36.76 ± 76.72	63.35 ± 121.21	NS
Chenodeoxycholic (mg/24 h)	19.07 ± 29.58	31.42 ± 70.36	NS
Deoxycholic acid (mg/24 h)	188.29 ± 98.12	325.96 ± 198.57	0.0115
Lithocholic acid (mg/24 h)	115.43 ± 71.89	197.27 ± 126.87	0.011

CAD, coronary artery disease; HDL-c, high-density lipoprotein cholesterol; LDL-c, low-density lipoprotein cholesterol.

Table 3 shows the Pearson correlations between total bile acids and various lipid parameters. There was a correlation, albeit weak, between the levels of triglycerides and total bile acids in the non-CAD group (r = 0.34, p = 0.04). There was no correlation between fecal bile acids and other lipid profile components in either group. Multivariate regression analysis was used in order to define the clinical relevance of total bile acids in the CAD group. Table 4 displays the adjusted odds ratios (ORs) of the main clinical and laboratory parameters and the major risk factors that were examined as predictors for CAD: advanced age, male gender, LVEF, and levels of total bile acids emerged as being significant (p < 0.05) independent factors that predicted CAD.

Table 3.

Pearson correlation coefficient (R) of bile acid amount and lipid profile variables.

Variables	Group 1		Group 2
	CAD R	p-value	Non-CAD R	p-value
LDL-c	−0.01	0.95	0.05	0.74
HDL-c	0.13	0.49	0.20	0.25
Non-HDL-c	−0.15	0.78	0.15	0.37
Triglycerides	0.02	0.91	0.34	0.04
Total cholesterol	−0.03	0.86	0.24	0.15

CAD, coronary artery disease; HDL-c, high-density lipoprotein cholesterol; LDL-c, low-density lipoprotein cholesterol.

Table 4.

Multi-adjusted significant odds ratio of clinical and laboratory variables for coronary heart disease.

	Odds ratio	Lower 95% CI	Upper 95% CI
Ejection fraction	1.247	1.094	1.421
Total bile acids	1.009	1.003	1.015
Gender	7.415	0.953	57.701
Age	0.868	0.769	0.979

During 13-year follow up, 16% of participants in Group 2 developed CAD events. Moreover, 19% of patients in Group 1 developed ischemic stroke compared with one patient (2.7%) in Group 2. A total of nine patients (25%) in Group 1 and three patients (8%) in Group 2 died (cardiovascular cause) during the long-term follow up (p < 0.001).

Discussion

The findings of the current study showed a marked decrease of bile acid excretion in patients with CAD compared with patients in whom CAD was ruled out. This supports the possibility that CAD patients produce fewer bile acids than individuals without CAD, and that reduced production of bile acids could lead to advanced atherosclerosis. Our findings are in line with those of the few published human studies that showed increased fecal excretion of bile acids to have atheroprotective effect. Most of those investigations were performed on selected populations. For example, Simonen and Miettinen showed that males with heterozygous familial hypercholesterolemia (FH) and CAD excreted less bile acid than control males with FH and normal coronaries [Simonen and Miettinen, 1987]. Rajaratnam and colleagues showed that postmenopausal women with CAD had inefficient fecal elimination of cholesterol [Rajaratnam et al. 2001].

Our group had studied a general adult population with and without CAD and found that CAD patients eliminated subnormal fecal bile acids [Charach et al. 1998]. We now report 36 CAD patients and 37 non-CAD patients with a follow-up period of up to 13 years. Our current findings support our earlier results and allow us to reach more firm conclusions on the role of the elimination of fecal bile acids in CAD development. The differences in the amounts of the excreted bile acids between the two groups were found only in deoxycholic acid and lithocholic acid levels, which were higher for the healthy as it was shown in other studies [Poorman et al. 1993]. No differences were found for cholic and chenodeoxycholic acids. However, there was a trend in both acids towards decreased excretion in CAD patients.

We did not find significant differences in amounts of excreted total bile acids and their compositions in, diabetic patients (metabolic disorder), in every group separately p > 0.05 (not shown in tables).

Given the fact that 7-α-hydroxylase clears cholesterol from the plasma and the intracellular compartment, the possible mechanism of increased excretion of bile acids in the non-CAD patients can be explained mainly by increased activity of 7-α-hydroxylase [Princen et al. 1997; Poorman et al. 1993; Lutton, 1990]. In contrast, CAD patients are unable to effectively increase the activity and concentration of 7-α-hydroxylase [Princen et al. 1997]. Moreover, we had previously shown [Rabinovich and Guzachev, 1987] that cholesterol overload causes a decrease in bile acid excretion in CAD patients in contrast to controls whose bile acid excretion increases. There was no correlation between the amount of bile acid excretion and total cholesterol, LDL-c, and HDL-c, but the plasma HDL level was significantly lower in the CAD patients [Rabinovich and Guzachev, 1987].

Effective reverse transport of cholesterol by HDL from the tissues to the liver increases utility of cholesterol and its excretion [Princen et al. 1997]. The absence of correlation between HDL-c levels and fecal bile acid elimination (a process that reflects bile acid synthesis) led us to consider that the regulation of bile acid synthesis (described above) and the regulation of HDL are different, as had been shown in rabbits where no association was found between increased concentrations and activity of 7-α-hydroxylase and HDL levels [Machleder et al. 1999; Princen et al. 1997]. It follows, therefore, that both mechanisms were involved in the same process of eliminating cholesterol from the organism.

In contrast to total cholesterol, LDL-c, and HDL-c, there was a correlation between plasma triglycerides and bile acid excretion, but only in the non-CAD group. This can be explained by rapid and more complete intestinal absorption of triglycerides due to an excess of bile acids which are necessary for the emulsification of fats. CAD patients did not show this effect because the amount of excreted bile acids was significantly lower. It is important to emphasize that the ability to excrete large amounts of bile acids prevents more than CAD development. According to our data, it may prevent atherosclerosis in the cerebral arteries as well. Importantly, after a long follow up, the results of this study showed a sixfold higher incidence of ischemic stroke among the CAD patients compared with the non-CAD patients, with a threefold greater mortality in the CAD group.

A significant percentage of patients develop atherosclerosis despite statin treatment and suppressed levels of cholesterol. We reason that by decreasing cholesterol synthesis and increasing the utility of cholesterol, an additive anti-atherosclerosis effect might be achieved. This is particularly true in the case of patients at high risk for CAD requiring aggressive lipid-lowering therapy combining a statin with drugs affecting bile acid and cholesterol absorption in order to ensure the optimal management of dyslipidemia. Additional studies are necessary to validate our contention.

Conclusion

The findings of the present study showed significantly lower amounts of bile acid excretion in adult patients with CAD compared with non-CAD patients. The inability to efficiently excrete bile acids might be an independent risk factor for CAD.