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Abstract
Objective—Epidemiological studies strongly suggest that lipid factors independent of low-density lipoprotein cholesterol contribute significantly to cardiovascular disease risk. Because circulating lipoproteins comprise only a small fraction of total body cholesterol, the mobilization and excretion of cholesterol from plasma and tissue pools may be an important determinant of cardiovascular disease risk. Our hypothesis is that fecal excretion of endogenous cholesterol is protective against atherosclerosis.
Approach and Results—Cholesterol metabolism and carotid intima–media thickness were quantitated in 86 nondiabetic adults. Plasma cholesterol was labeled by intravenous infusion of cholesterol-d7 solubilized in a lipid emulsion and dietary cholesterol by cholesterol-d5 and the nonabsorbable stool marker sitostanol-d4. Plasma and stool samples were collected while subjects consumed a cholesterol- and phytosterol-controlled metabolic kitchen diet and were analyzed by mass spectrometry. Carotid intima–media thickness was negatively correlated with fecal excretion of endogenous cholesterol (r=−0.426; P<0.0001), total cholesterol (r=−0.472; P≤0.0001), and daily percent excretion of cholesterol from the rapidly mixing cholesterol pool (r=−0.343; P=0.0012) and was positively correlated with percent cholesterol absorption (r=+0.279; P=0.0092). In a linear regression model controlling for age, sex, systolic blood pressure, hemoglobin A1c, low-density lipoprotein, high-density lipoprotein cholesterol, and statin drug use, fecal excretion of endogenous cholesterol remained significant (P=0.0008).
Conclusions—Excretion of endogenous cholesterol is strongly, independently, and negatively associated with carotid intima–media thickness. The reverse cholesterol transport pathway comprising the intestine and the rapidly mixing plasma, and tissue cholesterol pool could be an unrecognized determinant of cardiovascular disease risk not reflected in circulating lipoproteins. Further work is needed to relate measures of reverse cholesterol transport to atherosclerotic disease.
Clinical Trial Registration—URL: http://www.clinicaltrials.gov. Unique identifier: NCT01603758.
Introduction
Guidelines for preventing and treating atherosclerotic disease are heavily focused on low-density lipoprotein (LDL) cholesterol because of strong clinical trial evidence and readily-available plasma measurements.1 Atherosclerosis originates from cholesterol deposition in the artery from β-lipoproteins, including LDL. However, circulating LDL is a small contributor to total body cholesterol, comprising ≈3 g in a person with circulating LDL cholesterol concentration of 100 mg/dL compared with ≈72 g of total body cholesterol.2–6 Moreover, LDL cholesterol is a static measurement that contains no kinetic information about rates of cholesterol production or excretion.
The reverse transport of cholesterol is a multistep pathway beginning with cholesterol extraction from peripheral tissues and macrophages, followed by transfer into a rapidly mixing body cholesterol pool, secretion into the intestine, and excretion in the stool.7 Reverse cholesterol transport (RCT) may mitigate the effect of LDL cholesterol by removing cholesterol deposited in the arterial wall. This work focuses on the distal portion of RCT, emphasizing the rapidly mixing body cholesterol pool and the intestine. Using multiple stable isotopic tracers to label both dietary and endogenous cholesterol and using a metabolic kitchen diet to provide a constant baseline for measurement, it is possible to quantify total fecal cholesterol excretion and to characterize it as originating from the endogenous pool, the diet, or unlabeled hepatobiliary components.8 The final common pathway for RCT is the fecal excretion of endogenous cholesterol (FEEC). That measurement, along with related cholesterol fluxes, forms the basis for this report. We define endogenous cholesterol as body cholesterol derived principally from biosynthesis and, to a lesser extent, from absorbed dietary cholesterol. The endogenous cholesterol was labeled by the intravenous administration of cholesterol-d7 tracer.
Although little evidence exists on the relationship between FEEC and cardiovascular disease (CVD) risk, epidemiological studies have examined plasma biomarkers for the related processes of cholesterol absorption and synthesis, but the results have been inconsistent.9,10 Case–control studies also have been controversial, in part because the proposed biomarkers are not fully understood and have not been validated.11–16 Actual measurements of whole-body cholesterol metabolism, rather than its biomarkers, are needed to establish a possible relationship between whole-body cholesterol metabolism and CVD.
Increased carotid intima–media thickness (CIMT) has been associated with increased risk of cardiovascular events, such as myocardial infarction and stroke.17–20 The Atherosclerosis Risk in Communities study showed a strong relationship between CIMT and cardiovascular events.20 More recently, a study of the Framingham Offspring Study cohort showed that both the maximum internal CIMT and mean common CIMT predict cardiovascular outcomes.21 Thus, CIMT is a well-accepted imaging biomarker of atherosclerosis and CVD risk. In the present observational study, we explored whether FEEC was related to CIMT in adults.
Materials and Methods
Materials and Methods are available in the online-only Data Supplement.
Results
Subjects
Among the 100 subjects screened, 94 were eligible and entered the study, and 86 completed the protocol. Subjects included 52 women and 34 men with mean age of 53.7±13.0 years. Table 1 shows the subject characteristics on entry into the study. They comprised a wide range of nondiabetic individuals. Subjects over age 30 years were selected to increase the strength of the relationship of CVD risk factors to CIMT. Statin drug use was allowed because this is a common medication and does not seem to influence whole-body cholesterol metabolism significantly.22
Subject Characteristics at Baseline
CIMT and Traditional CVD Risk Factors
Pearson correlations between CIMT and several traditional CVD risk factors are shown in Table 2. Age, systolic blood pressure, hemoglobin A1c, and fasting plasma glucose were associated with increased CIMT, as expected. However, there was no significant relationship between circulating lipids or lipoproteins and CIMT in this sample.
Pearson Correlation Between CIMT and Traditional Risk Factors
Cholesterol Metabolism
Cholesterol metabolism results are shown in Table 3. Total daily fecal cholesterol excretion measured was 0.58 g/d. Of this amount, 68% was accounted for by recovery of the plasma tracer cholesterol-d7 and therefore had an endogenous origin, whereas 16% was because of unabsorbed dietary cholesterol measured with cholesterol-d5. An additional 16% of fecal cholesterol was not labeled and may represent newly synthesized or other nonequilibrating hepatobiliary cholesterol. Percent intestinal cholesterol absorption was 57.4%. The rapidly mixing cholesterol pool, which includes both circulating lipoproteins and a portion of tissue cholesterol, averaged 25.3 g. Because the calculated plasma pool of cholesterol was only ≈5.4 g, the rapidly mixing cholesterol pool was comprised 79% tissue cholesterol that equilibrated rapidly with plasma. Percent cholesterol excretion, the percent of the rapidly mixing plasma cholesterol pool excreted daily into the stool as cholesterol and bacterial cholesterol metabolites, was 1.64%. These measurements emphasize that endogenous tissue cholesterol dominates whole-body cholesterol metabolism.
Pearson Correlation Between CIMT and Cholesterol Metabolic Parameters
CIMT and Whole-Body Cholesterol Metabolism
Table 3 shows that CIMT was strongly and negatively correlated with several measures of fecal cholesterol elimination, including total cholesterol excretion (P<0.0001), endogenous cholesterol excretion (P<0.0001), unabsorbed dietary cholesterol excretion (P=0.019), and percent cholesterol excretion (P=0.0012). Figure 1 shows a scatterplot of the relationship between CIMT and FEEC. There was no relationship of CIMT to the small amount of unlabeled stool cholesterol, suggesting that unrecognized cholesterol pools do not contribute significantly to CIMT. Percent cholesterol absorption, an important determinant of fecal cholesterol excretion, was positively correlated with CIMT (P=0.0092). Secretion of endogenous cholesterol into the intestine, another determinant of fecal cholesterol excretion, was negatively correlated with CIMT (P=0.011). Although turnover of plasma cholesterol was not measured in the study, it was estimated by the decline in plasma cholesterol enrichment for 13 days after infusion of intravenous tracer. This measurement was positively correlated with CIMT (P=0.0083).
Relationship between carotid intima–media thickness (CIMT) and fecal excretion of endogenous cholesterol (FEEC). Simple regression of CIMT (mm) with FEEC (g/d) is presented for 86 subjects.
Potential Plasma Biomarkers for FEEC
Noncholesterol sterols were measured in plasma to assess their potential use as biomarkers for FEEC in larger studies. FEEC was positively correlated with the ratio of plasma lathosterol/cholesterol (r=0.283; P=0.0083) and marginally negatively correlated with the ratio of plasma 5α-cholestanol/cholesterol (r=−0.211; P=0.051). The ratio of 5α-cholestanol/lathosterol, reflecting the balance of cholesterol absorption to cholesterol biosynthesis, was significantly correlated with FEEC (r=−0.373; P=0.0004).
Regression Model
FEEC explained 18.1% of variation in CIMT (P<0.0001) in simple linear regression and 7.6% in a multiple regression model that controlled for age, sex, systolic blood pressure, hemoglobin A1c, LDL cholesterol, high-density lipoprotein (HDL) cholesterol, and statin drug use (model R2=0.52; P=0.0008 for FEEC).
Discussion
This work shows that FEEC, the principal pathway of cholesterol elimination in humans, is negatively correlated with CIMT. The data provide a link between whole-body cholesterol metabolism and CIMT, a measure of early atherosclerosis. The association is strong and similar to that of traditional risk factors, such as age, systolic blood pressure, and hemoglobin A1c in a study group consisting of 86 relatively healthy nondiabetic subjects between the ages of 30 and 80 years. Moreover, FEEC seems to be a stronger risk factor than HDL and LDL cholesterol, which were not related to CIMT in this small study.
The cholesterol production rate (1.0 g/d) in the present study was similar to previous reports of 1.1 g/day22 and 1.14 g/d.23 The rapidly mixing cholesterol pool of 25.3 g in the current study also was similar to the reported pool sizes of 23.422 and 25.9 g.23 The cholesterol flux from the rapidly mixing cholesterol pool out of the body (production rate over the size of the rapidly mixing pool; 4.0%) in the present study was comparable to previous reported values of 4.7%22 and 4.4%.23 Others have reported a higher production rate (2.1 g/d)24 and a lower rapid pool size (11.7 g),25 probably because of differences in tracer and methods used.
The primary objective in this work was to quantify fecal cholesterol excretion and precisely determine its origins. The analytical methods also allowed us to correlate several secondary end points with CIMT. The complete results form a coherent model for how body cholesterol metabolism might influence CIMT and CVD risk. Figure 2 depicts a working model of cholesterol compartments (circles) and cholesterol fluxes (arrows). The rapidly mixing body cholesterol pool includes all of the cholesterol in liver and blood, as well as part of the cholesterol in other tissues. The rapid cholesterol pool is particularly important because it is the pool from which cholesterol is excreted from the body and into which cholesterol is absorbed. The size of the rapidly mixing cholesterol pool was not directly related to CIMT, but cholesterol fluxes into and out of the pool were significantly correlated with CIMT, either negatively (solid arrows) or positively (dashed arrows). Cholesterol transport processes tending to increase the rapidly mixing pool, such as inflow from unlabeled endogenous cholesterol and reabsorption of intestinal cholesterol, were positively correlated with CIMT, whereas processes tending to reduce the pool size, such as secretion of endogenous cholesterol into the intestine and elimination of cholesterol in the stool, were negatively correlated. Fecal bile acids were not correlated to CIMT and were not included in the model. The results suggest that cholesterol homeostasis is maintained by competing cholesterol transport processes and that individuals who have greater cholesterol excretion have more favorable CIMT values.
Partial model of body and intestinal endogenous cholesterol metabolism. Solid arrows depict cholesterol pathways with statistically significant negative correlations to carotid intima–media thickness (CIMT) while dotted lines show pathways with significant positive correlations to CIMT. Dietary cholesterol was controlled at a low level during measurements and is not included in the model. Cholesterol pathways that tend to reduce the rapidly mixing body cholesterol pool are associated with reduced CIMT. Primary data are found in Table 3. Endogenous cholesterol input into the rapidly mixing pool is estimated as the percent fall in plasma enrichment between days 2 and 15.
LDL and HDL cholesterol have great use in assessing CVD risk, but the information obtained is limited to a static measurement, obtained at a single point in time. HDL cholesterol concentration does not provide information about the quantity of cholesterol flowing into and out of the HDL particles. Previous investigators also found that whole-body cholesterol turnover studied with an intravenous cholesterol tracer was not related to circulating HDL or LDL cholesterol,26 which may be because of the fact that RCT is the combined actions of various lipoproteins, such as HDL and LDL. More attention has been given to functional assays for HDL. For example, cholesterol efflux capacity of HDL, a functional measure of cholesterol efflux from macrophages, has been shown to relate to atherosclerosis better than HDL cholesterol alone.27 What distinguishes many cholesterol metabolic parameters in the current study is that they are kinetic and represent cholesterol flows over time. It is possible that kinetic measurements may supplement lipoprotein concentration measurements to provide a stronger prediction of CVD risk.
RCT is an important emerging pathway in which cholesterol efflux from macrophages and peripheral tissues is traced into HDL, through the plasma, and into the intestine, where it is excreted as neutral sterols and bile acids.7,28 Although attention has been focused on the arterial macrophage as a first step, the current work shows that cholesterol flowing from all tissues into and out of the rapidly mixing cholesterol pool and intestine may contribute importantly to RCT. In particular, the intestine may have a central role in regulating body cholesterol metabolism and possibly in protecting against the development of atherosclerotic disease.29 The data presented in Figure 1 show a large interindividual variability in FEEC and support previous work demonstrating similar variability in percent cholesterol absorption.30 This is likely because of interindividual differences in metabolic regulation rather than analytic error because our previous work shows that repeated FEEC measurements in the same individual have a coefficient of variation of ≈10%.8
Because of the difficulty of performing metabolic tracer studies such as this one, few data exist that address the relationship of whole-body cholesterol metabolism to CVD. It would be highly desirable to have a plasma biomarker for cholesterol excretion. Because cholesterol absorption from the intestine is not complete (averaging 57% in this study), cholesterol homeostasis must be maintained by increased cholesterol synthesis, increased cholesterol absorption, or a combination of these processes to avoid cholesterol deficiency. We used a composite plasma biomarker, the ratio of 5α-cholestanol/lathosterol, to reflect the balance between cholesterol absorption, represented by 5α-cholestanol, and cholesterol biosynthesis, represented by lathosterol, within each individual. The ratio was negatively correlated with FEEC (r=−0.373; P=0.0004) and positively correlated with CIMT (r=0.415; P<0.0001). Previous investigators have shown that similar plasma absorption/synthesis ratios are associated with CVD.16 It is possible that links to CVD in the absorption/synthesis biomarker literature might be explained, in part, by reduced FEEC as the final common pathway most closely linked to increased CIMT. Conversely, lower intestinal cholesterol absorption would result in cholesterol deficiency and increased compensatory biosynthesis, resulting in a lower absorption/synthesis ratio, higher fecal cholesterol excretion, and lower CVD risk. More work is needed to connect biomarkers with measurements of cholesterol metabolism, transport, CIMT, and CVD outcome.
Although CIMT is not a direct measure of clinical outcomes or events, it does reflect current information on the development of early atherosclerosis, and there is abundant evidence that it is strongly related to well-validated CVD risk factors. The present study demonstrates that FEEC is negatively related to CIMT independently of established CVD risk factors, pointing to FEEC and whole-body cholesterol metabolism as a novel potential target for modulating CVD risk.
It has been demonstrated that bile acid sequestrants effectively reduce coronary artery disease outcomes, probably by reducing LDL cholesterol and increasing sterol excretion or RCT.31,32 Failure of CETP (cholesteryl ester transfer protein) inhibitors to reduce cardiovascular events has indicated that it is not sufficient to simply raise HDL cholesterol.33 The functional capacity of HDL in RCT might be more important mechanistically. Increased fecal cholesterol excretion is a prominent mechanism of action of phytosterols in foods and supplements,34 but there have been no interventional studies with CVD outcomes to firmly establish the treatment effectiveness. A recent clinical trial of the drug ezetimibe, an inhibitor of cholesterol absorption, resulted in fewer CVD events even in the face of optimum statin drug treatment.35 Although the result has been attributed to LDL cholesterol lowering, the actual mechanism is unknown, and recent work shows that FEEC is increased ≈70% by ezetimibe.8 Further work is needed to establish the use of treatments directed toward whole-body cholesterol metabolism.
Acknowledgments
We thank the Alvin J. Siteman, Cancer Center at Washington University School of Medicine, for use of the Biologic Therapy Core Facility. We are grateful for the skilled assistance from the Center for Clinical Studies, the Clinical Research Unit nursing staff, and the metabolic kitchen staff at Washington University Institute of Clinical and Translational Sciences. We appreciate the dedication of the study participants.
Sources of Funding
This work was supported by National Institutes of Health grants R01-108160 to R.E. Ostlund, Washington University Mass Spectrometry Resource RR-00954, Washington University Diabetes Research Center P30DK020579, Washington University Nutrition Obesity Research Center P30 DK056341, and Washington University Institute of Clinical and Translational Sciences UL1 TR000448.
Disclosures
None.
Footnotes
The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.117.310081/-/DC1.
- Nonstandard Abbreviations and Acronyms
- CIMT
- carotid intima–media thickness
- FEEC
- fecal excretion of endogenous cholesterol
- HDL
- high-density lipoprotein
- LDL
- low-density lipoprotein
- RCT
- reverse cholesterol transport
- Received August 7, 2017.
- Accepted September 21, 2017.
- © 2017 American Heart Association, Inc.
References
Highlights
The quantity and efficiency of cholesterol excretion were strongly and negatively correlated with carotid intima–media thickness in nondiabetic adults aged 30 to 80 years old.
The association of fecal cholesterol excretion with carotid intima–media thickness was independent of age, sex, systolic blood pressure, hemoglobin A1c, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol.
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