Off-Target Effect of Lovastatin Disrupts Dietary Lipid Uptake and Dissemination through Pro-Drug Inhibition of the Mesenteric Lymphatic Smooth Muscle Cell Contractile Apparatus.

Previously published, off-target effects of statins on skeletal smooth muscle function have linked structural characteristics within this drug class to myopathic effects. However, the effect of these drugs on lymphatic vascular smooth muscle cell function, and by proxy dietary cholesterol uptake, by the intestinal lymphatic network has not been investigated. Several of the most widely prescribed statins (Atorvastatin, Pravastatin, Lovastatin, and Simvastatin) were tested for their in-situ effects on smooth muscle contractility in rat mesenteric collecting lymphatic vessels. Lovastatin and Simvastatin had a concentration-dependent effect of initially increasing vessel contraction frequency before flatlining the vessel, a phenomenon which was found to be a lactone-ring dependent phenomenon and could be ameliorated through use of Lovastatin- or Simvastatin-hydroxyacid (HA). Simvastatin treatment further resulted in mitochondrial depolymerization within primary-isolated rat lymphatic smooth muscle cells (LMCs) while Lovastatin was found to be acting in a mitochondrial-independent manner, increasing the function of RhoKinase. Lovastatin's effect on RhoKinase was investigated through pharmacological testing and in vitro analysis of increased MLC and MYPT1 phosphorylation within primary isolated LMCs. Finally, acute in vivo treatment of rats with Lovastatin, but not Lovastatin-HA, resulted in a significantly decreased dietary lipid absorption in vivo through induced disfunction of mesenteric lymph uptake and trafficking.

Pravastatin Modulate Niemann-Pick C1-Like 1 and ATP-Binding Cassette G5 and G8 to Influence Intestinal Cholesterol Absorption.

PURPOSE: Niemann-Pick C1-like 1 (NPC1L1), ATP-binding cassette (ABC)G5, and ABCG8 mediate intestinal cholesterol absorption. It is unclear whether pravastatin (PR) or ezetimibe (EZ) affect expression of these transporters. We examined the effects of PR and EZ on NPC1L1, ABCG5, and ABCG8 expression in human hepatoma HepG2 cells and the murine small intestine. We also assessed expression of the transcription factors liver X receptor (LXR)a, LXRb and sterol regulatory element-binding protein. METHODS: Transporter mRNA levels were determined in murine small intestines 6 and 24 h after oral PR and EZ administration by real-time reverse-transcriptase polymerase chain reaction (RT-PCR). In PR- and EZ-treated HepG2 cells, transporter and transcription factor mRNA and protein levels were examined by RT-PCR and western blot, respectively. RESULTS: Significant decreases in NPC1L1, ABCG5, and ABCG8 mRNA expression were observed in the duodenum, but not jejunum and ileum, of mice 24 h after treatment with PR, but not EZ. In HepG2 cells, PR but not EZ treatment for 24 h also significantly decreased NPC1L1 protein and ABCG5, and ABCG8 mRNA expression, while increasing LXRa mRNA levels. CONCLUSION: PR but not EZ treatment reduced duodenal cholesterol transporter expression in mice. PR-induced increases in LXRa mRNA levels may be involved in attenuation of NPC1L1 expression, subsequently decreasing intestinal cholesterol absorption.

Rho-associated coiled-coil-containing kinase 2 deficiency in bone marrow-derived cells leads to increased cholesterol efflux and decreased atherosclerosis.

BACKGROUND: Macrophages play a central role in the development of atherosclerosis. However, the signaling pathways that regulate their function are not well understood. The Rho-associated coiled-coil-containing kinases (ROCK1 and ROCK2) are serine-threonine protein kinases that are involved in the regulation of the actin cytoskeleton. Recent studies suggest that ROCK1 in macrophages and bone marrow-derived cells mediates atherogenesis. However, a similar role for ROCK2 in the pathogenesis of atherosclerosis has not been determined. METHODS AND RESULTS: The bone marrows from wild-type, ROCK2(+/-), and ROCK2(-/-) mice were transplanted into irradiated recipient low-density lipoprotein receptor(-/-) mice, and atherosclerosis was induced with a 16-week high-cholesterol diet. Compared with wild-type bone marrow-transplanted mice, ROCK2(+/-) bone marrow-transplanted and ROCK2(-/-) bone marrow-transplanted mice showed substantially less lipid accumulation in the aorta (8.46±1.42% and 9.80±2.34% versus 15.64±1.89%; P<0.01 for both) and decreased atherosclerotic lesions in the subaortic sinus (158.1±44.4 and 330.1±109.5×10(3)µm(2) versus 520.2±125.7×10(3)µm(2); P<0.01 for both). These findings correlated with decreased foam cell formation (2.27±0.57 versus 4.10±0.3; P<0.01) and increased cholesterol efflux (17.65±0.6 versus 9.75±0.8; P<0.05) in ROCK2-deficient mice that are mediated, in part, through the peroxisome proliferator-activated receptor-γ/liver X receptor/ATP-binding cassette transporter A1 pathway in macrophages. CONCLUSIONS: ROCK2 contributes to atherosclerosis, in part, by inhibiting peroxisome proliferator-activated receptor-γ-mediated reverse cholesterol transport in macrophages, which contributes to foam cell formation. These findings suggest that inhibition of ROCK2 in macrophages may have therapeutic benefits in preventing the development of atherosclerosis.

Molecular characterization of the NPC1L1 variants identified from cholesterol low absorbers.

Niemann-Pick C1-like 1 (NPC1L1) is an essential protein for dietary cholesterol absorption. Nonsynonymous (NS) variants of NPC1L1 in humans have been suggested to associate with cholesterol absorption variations. However, information concerning the characteristics and mechanism of these variants in cholesterol uptake is limited. In this study, we analyzed the cholesterol uptake ability of the 19 reported NS variants of NPC1L1 identified from cholesterol low absorbers. Among these variants, L110F, R306C, A395V, G402S, T413M, R693C, R1214H, and R1268H could partially mediate cellular cholesterol uptake and were categorized as partially dysfunctional variants. The other 11 variants including T61M, N132S, D398G, R417W, G434R, T499M, S620C, I647N, G672R, S881L, and R1108W could barely facilitate cholesterol uptake, and were classified into the severely dysfunctional group. The partially dysfunctional variants showed mild defects in one or multiple aspects of cholesterol-regulated recycling, subcellular localization, glycosylation, and protein stability. The severely dysfunctional ones displayed remarkable defects in all these aspects and were rapidly degraded through the ER-associated degradation (ERAD) pathway. In vivo analyses using adenovirus-mediated expression in mouse liver confirmed that the S881L variant failed to localize to liver canalicular membrane, and the mice showed defects in biliary cholesterol re-absorption, while the G402S variant appeared to be similar to wild-type NPC1L1 in mouse liver. This study suggests that the dysfunction of the 19 variants on cholesterol absorption is due to the impairment of recycling, subcellular localization, glycosylation, or stability of NPC1L1.

Combined effects of ezetimibe and phytosterols on cholesterol metabolism: a randomized, controlled feeding study in humans.

BACKGROUND: Both ezetimibe and phytosterols inhibit cholesterol absorption. We tested the hypothesis that the combination of ezetimibe and phytosterols is more effective than ezetimibe alone in altering cholesterol metabolism. METHODS AND RESULTS: Twenty-one mildly hypercholesterolemic subjects completed a randomized, double-blind, placebo-controlled, triple-crossover study. Each subject received a phytosterol-controlled diet plus (1) ezetimibe placebo+phytosterol placebo, (2) 10 mg/d ezetimibe+phytosterol placebo, and (3) 10 mg/d ezetimibe+2.5 g phytosterols for 3 weeks each. All meals were prepared in a metabolic kitchen. Primary outcomes were intestinal cholesterol absorption, fecal cholesterol excretion, and low-density lipoprotein cholesterol levels. The combined treatment resulted in significantly lower intestinal cholesterol absorption (598 mg/d; 95% confidence interval [CI], 368 to 828) relative to control (2161 mg/d; 95% CI, 1112 to 3209) and ezetimibe alone (1054 mg/d; 95% CI, 546 to 1561; both P<0.0001). Fecal cholesterol excretion was significantly greater (P<0.0001) with combined treatment (962 mg/d; 95% CI, 757 to 1168) relative to control (505 mg/d; 95% CI, 386 to 625) and ezetimibe alone (794 mg/d; 95% CI, 615 to 973). Plasma low-density lipoprotein cholesterol values during treatment with control, ezetimibe alone, and ezetimibe+phytosterols averaged 129 mg/dL (95% CI, 116 to 142), 108 mg/dL (95% CI, 97 to 119), and 101 mg/dL (95% CI, 90 to 112; (P<0.0001 relative to control). CONCLUSION: The addition of phytosterols to ezetimibe significantly enhanced the effects of ezetimibe on whole-body cholesterol metabolism and plasma low-density lipoprotein cholesterol. The large cumulative action of combined dietary and pharmacological treatment on cholesterol metabolism emphasizes the potential importance of dietary phytosterols as adjunctive therapy for the treatment of hypercholesterolemia. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT00863265.

Do clinical and experimental investigations support an antiatherogenic role for dietary phytosterols/stanols?

The plasma cholesterol-reducing effect of phytosterols (PS) has been recognized in several studies, but the usefulness of PS in preventing coronary heart disease remains controversial, as some investigations claim that the high PS concentrations found in plasma and specific tissues are related to an increased risk of cardiovascular events. It has also been demonstrated that PS may induce inflammation and reduce cholesterol efflux from macrophages, conditions that are directly implicated in the development of atherosclerosis. As to arterial dysfunction and atherosclerosis, some studies have concluded that plasma PS concentrations are unrelated or only weakly related or that PS intake or plasma PS concentrations are harmful. Thus, in light of the National Cholesterol Education Program-ATPIII report, it is necessary to evaluate the relevance of their findings. To this end, we have evaluated the studies conducted on cells, animal models, and humans regarding the influence of PS on the development of atherosclerosis.

Neovascularization of coronary tunica intima (DIT) is the cause of coronary atherosclerosis. Lipoproteins invade coronary intima via neovascularization from adventitial vasa vasorum, but not from the arterial lumen: a hypothesis.

BACKGROUND: An accepted hypothesis states that coronary atherosclerosis (CA) is initiated by endothelial dysfunction due to inflammation and high levels of LDL-C, followed by deposition of lipids and macrophages from the luminal blood into the arterial intima, resulting in plaque formation. The success of statins in preventing CA promised much for extended protection and effective therapeutics. However, stalled progress in pharmaceutical treatment gives a good reason to review logical properties of the hypothesis underlining our efforts, and to reconsider whether our perception of CA is consistent with facts about the normal and diseased coronary artery. ANALYSIS: To begin with, it must be noted that the normal coronary intima is not a single-layer endothelium covering a thin acellular compartment, as claimed in most publications, but always appears as a multi-layer cellular compartment, or diffuse intimal thickening (DIT), in which cells are arranged in many layers. If low density lipoprotein cholesterol (LDL-C) invades the DIT from the coronary lumen, the initial depositions ought to be most proximal to blood, i.e. in the inner DIT. The facts show that the opposite is true, and lipids are initially deposited in the outer DIT. This contradiction is resolved by observing that the normal DIT is always avascular, receiving nutrients by diffusion from the lumen, whereas in CA the outer DIT is always neovascularized from adventitial vasa vasorum. The proteoglycan biglycan, confined to the outer DIT in both normal and diseased coronary arteries, has high binding capacity for LDL-C. However, the normal DIT is avascular and biglycan-LDL-C interactions are prevented by diffusion distance and LDL-C size (20 nm), whereas in CA, biglycan in the outer DIT can extract lipoproteins by direct contact with the blood. These facts lead to the single simplest explanation of all observations: (1) lipid deposition is initially localized in the outer DIT; (2) CA often develops at high blood LDL-C levels; (3) apparent CA can develop at lowered blood LDL-C levels. This mechanism is not unique to the coronary artery: for instance, the normally avascular cornea accumulates lipoproteins after neovascularization, resulting in lipid keratopathy. HYPOTHESIS: Neovascularization of the normally avascular coronary DIT by permeable vasculature from the adventitial vasa vasorum is the cause of LDL deposition and CA. DIT enlargement, seen in early CA and aging, causes hypoxia of the outer DIT and induces neovascularization. According to this alternative proposal, coronary atherosclerosis is not related to inflammation and can occur in individuals with normal circulating levels of LDL, consistent with research findings.

Cholesterol synthesis is increased and absorption decreased in non-alcoholic fatty liver disease independent of obesity.

BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD) is associated with impaired glucose and lipoprotein metabolism. However, the metabolism of cholesterol in NAFLD remains unexplored. We investigated how fatty liver influences cholesterol metabolism in 242 non-diabetic subjects. METHODS: Liver fat content was measured with proton magnetic resonance spectroscopy. Cholesterol metabolism was assayed with serum non-cholesterol sterols, surrogate markers of cholesterol synthesis and absorption. The analyses were performed with gas-liquid chromatography. RESULTS: A total of 114 subjects had NAFLD and 128 subjects had normal liver fat content. Non-cholesterol sterols reflecting cholesterol synthesis (cholestenol, desmosterol, and lathosterol) were higher, and those reflecting cholesterol absorption (cholestanol and plant sterols) were lower in subjects with NAFLD than in controls, independent of body mass index. Liver fat content was positively associated with markers of cholesterol synthesis (r = from 0.262 to 0.344, p < 0.001 for all) and inversely associated with markers of cholesterol absorption (r = from -0.299 to -0.336, p < 0.001 for all). In the entire study group, synthesis and absorption markers were interrelated, indicating that the homeostasis of cholesterol metabolism was maintained. LDL cholesterol was similar in the two groups. CONCLUSIONS: We demonstrated that although LDL cholesterol concentrations are unchanged, cholesterol metabolism in NAFLD is characterized by increased synthesis and diminished absorption of cholesterol. These changes are associated with liver fat content independent of body weight.

Targeting cholesterol at different levels in the mevalonate pathway protects fatty liver against ischemia-reperfusion injury.

BACKGROUND & AIMS: Liver steatosis enhances ischemia/reperfusion (I/R) injury and is considered a primary factor in graft failure after liver transplantation. Although previous reports have shown a role for qualitative steatosis (macrovesicular vs. microvesicular) in hepatic I/R injury, no studies have compared side by side the specific contribution of individual lipids accumulating in fatty liver to I/R damage. METHODS: We used nutritional and genetic models of micro and macrovesicular fatty livers exhibiting specific lipid profiles to assess their susceptibility to normothermic I/R injury. RESULTS: Unlike choline-deficient (CD) diet-fed mice, characterized by predominant liver triglycerides/free fatty acids (TG/FFA) accumulation, mice fed a cholesterol-enriched (HC) diet, which exhibited enhanced hepatic cholesterol loading in mitochondria, were highly sensitive to I/R-induced liver injury. In vivo two-photon confocal imaging revealed enhanced mitochondrial depolarization and generation of reactive oxygen species following hepatic I/R in HC-fed but not in CD-fed mice, consistent with decreased mitochondrial GSH (mGSH) observed in HC-fed mice. Moreover, ob/ob mice, characterized by increased hepatic TG, FFA, and cholesterol levels, were as sensitive to I/R-mediated liver injury as mice fed the HC diet. Livers from ob/ob mice displayed increased StAR expression and mitochondrial cholesterol accumulation, resulting in mGSH depletion. Interestingly, atorvastatin therapy or squalene synthase inhibition in vivo attenuated StAR overexpression, mitochondrial cholesterol loading, and mGSH depletion, protecting ob/ob mice from I/R-mediated liver injury. CONCLUSIONS: Cholesterol accumulation, particularly in mitochondria, sensitizes to hepatic I/R injury, and thus represents a novel target to prevent the enhanced damage of steatotic livers to I/R-mediated damage.

An NPC1L1 gene promoter variant is associated with autosomal dominant hypercholesterolemia.

BACKGROUND AND AIMS: A substantial number of subjects with autosomal dominant hypercholesterolemia (ADH) do not have LDL receptor (LDLR) or apolipoprotein B (APOB) mutations. Some ADH subjects appear to hyperabsorb sterols from the intestine, thus we hypothesized that they could have variants of the Niemann-Pick C1-Like 1 gene (NPC1L1). NPC1L1 encodes a crucial protein involved in intestinal sterol absorption. METHODS AND RESULTS: Four NPC1L1 variants (-133A>G, -18C>A, 1679C>G, 28650A>G) were analyzed in 271 (155 women and 116 men) ADH bearers without mutations in LDLR or APOB aged 30-70years and 274 (180 women and 94 men) control subjects aged 25-65years. The AC haplotype determined by the -133A>G and -18C>A variants was underrepresented in ADH subjects compared to controls (p=0.01). In the ADH group, cholesterol absorption/synthesis markers were significantly lower in AC homozygotes that in all others haplotypes. Electrophoretic mobility shift assay (EMSA) results revealed that the -133A-specific oligonucleotide produced a retarded band stronger than the -133G allele. Luciferase activity with NPC1L1 -133G variant was 2.5-fold higher than with the -133A variant. CONCLUSION: The -133A>G polymorphism exerts a significant effect on NPC1L1 promoter activity. NPC1L1 promoter variants might explain in part the hypercholesterolemic phenotype of some subjects with nonLDLR/nonAPOB ADH.

GATA4 is essential for jejunal function in mice.

BACKGROUND & AIMS: Although the zinc-finger transcription factor GATA4 has been implicated in regulating jejunal gene expression, the contribution of GATA4 in controlling jejunal physiology has not been addressed. METHODS: We generated mice in which the Gata4 gene was specifically deleted in the small intestinal epithelium. Measurements of plasma cholesterol and phospholipids, intestinal absorption of dietary fat and cholesterol, and gene expression were performed on these animals. RESULTS: Mice lacking GATA4 in the intestine displayed a dramatic block in their ability to absorb cholesterol and dietary fat. Comparison of the global gene expression profiles of control jejunum, control ileum, and GATA4 null jejunum by gene array analysis revealed that GATA4 null jejunum lost expression of 53% of the jejunal-specific gene set and gained expression of 47% of the set of genes unique to the ileum. These alterations in gene expression included a decrease in messenger RNAs (mRNAs) encoding lipid and cholesterol transporters as well as an increase in mRNAs encoding proteins involved in bile acid absorption. CONCLUSIONS: Our data demonstrate that GATA4 is essential for jejunal function including fat and cholesterol absorption and confirm that GATA4 plays a pivotal role in determining jejunal vs ileal identity.

Hypercholesterolemia and 3-hydroxy 3-methylglutaryl Coenzyme A reductase regulation during ageing.

We present here a brief description of the path that cholesterol covers from its intestinal absorption to its effect exerted on some enzyme regulation. Some mechanisms underlying hypercholesterolemia onset and, in particular, the role and the regulation of 3-hydroxy 3-methylglutaryl Coenzyme A reductase (HMGR) during adult life and during aging, have been described. In addition some pharmacological interventions to control proper HMGR regulation and, in turn, cholesterol homeostasis maintenance will be introduced.

Tofu and fish oil independently modulate serum lipid profiles in rats: Analyses of 10 class lipoprotein profiles and the global hepatic transcriptome.

Soy protein and fish oil are food components that decrease the risk of cardiovascular disease. Previous studies demonstrated that these food components reduced serum cholesterol levels and suppressed hepatic lipogenesis. However, the underlying mechanisms of action of these food components remain unclear. Ten classes of serum lipoprotein profiles showed that dietary tofu, a soybean curd, suppressed cholesterol absorption, while fish oil reduced most of the lipoprotein classes in rats. Tofu and fish oil both halved the level of the lipoprotein class LAC1 (LDL-anti-protease complex), a 15-nm LDL-anti-protease complex, which is speculated to be a cause of atherosclerosis. Moreover, a global transcriptome analysis revealed that tofu inhibited the mRNA expression of genes involved in hepatic lipogenesis, while fish oil stimulated that of genes related to fatty acid degradation. Therefore, tofu and fish oil independently regulate lipid metabolism. The decrease observed in LAC1 may have been due to reduced cholesterol absorption in the tofu diet group and the interference of lipogenesis via the activation of polyunsaturated fatty acid detoxification in the fish oil group.

Protective properties of milk sphingomyelin against dysfunctional lipid metabolism, gut dysbiosis, and inflammation.

The identification of natural bioactive compounds aimed at promoting optimal gut health and improving lipid metabolism is paramount in the prevention of chronic disease. In this review, we summarize basic science and clinical research examining the protective properties of milk sphingomyelin (SM) against dysfunctional lipid metabolism, gut dysbiosis, and inflammation. Dietary SM dose-dependently reduces the intestinal absorption of cholesterol, triglycerides, and fatty acids in cell culture and rodent studies. Overall, rodent feeding studies show dietary milk SM, milk polar lipid mixtures, and milk fat globule membrane reduce serum and hepatic lipid concentrations. Furthermore, these hypolipidemic effects are observed in some supplementation studies in humans, although the extent of reductions in serum cholesterol is typically smaller and only one trial was conducted with purified SM. Dietary milk SM has been reported to affect the gut microbiota in rodent studies and its hydrolytic product, sphingosine, displays bactericidal activity in vitro. Milk SM may also improve gut barrier function to prevent the translocation of inflammatory gut bacteria-derived molecules. Current evidence from pre-clinical studies indicates that dietary milk SM has protective properties against dysfunctional lipid metabolism, gut dysbiosis, and inflammation. The hypolipidemic effects of milk SM observed in animal studies have been reported in some human studies, although the magnitude of such effects is typically smaller. More research is warranted to clearly define how dietary milk SM influences lipid metabolism, gut microbiota, and inflammation in humans.

Cholesterol homeostasis by the intestine: lessons from Niemann-Pick C1 Like 1 [NPC1L1).

Ezetimibe is a selective cholesterol absorption inhibitor, which potently inhibits the uptake and absorption of biliary and dietary cholesterol from the small intestine without affecting the absorption of fat-soluble vitamins, triglycerides or bile acids. Identification and characterization of Niemann-Pick C1 Like 1 (NPC1L1) has established NPC1L1 as an essential protein in the intestinal cholesterol absorption process. While otherwise phenotypically normal, Npc1l1 null mice exhibit a significant reduction in the intestinal uptake and absorption of cholesterol and phytosterols. Characterization of the NPC1L1 pathway revealed that ezetimibe specifically binds to NPC1L1 and inhibits its sterol transport function. Npc1l1 null mice were resistant to diet-induced hypercholesterolemia, and when crossed with apoE null mice, were completely resistant to the development of atherosclerosis. In Npc1l1/apoE null mice or apoE null mice treated with ezetimibe plasma cholesterol levels were reduced primarily in the apoB48 containing chylomicron remnant lipoproteins relative to untreated apoE null mice. SR-B1 has been proposed to play a role in intestinal cholesterol uptake, but in Npc1l1/SR-B1 double null mice intestinal cholesterol absorption was not different than Npc1l1 null alone mice. Therefore, NPC1L1 is the critical intestinal sterol transporter which influences whole body cholesterol homeostasis, and is the molecular target of ezetimibe.

Effects of dietary cholesterol and fat on serum non-cholesterol sterols according to different apolipoprotein E subgroups among healthy men.

The impact of apo E phenotypes on applicability of relative cholesterol synthesis (lathosterol:cholesterol) and absorption (ratios of cholestanol, campesterol and sitosterol to cholesterol) during diets of various cholesterol and fat content is unclear. We examined and compared with each other both relative and absolute synthesis and absorption among twenty-nine men, of whom eight, nine and twelve had apo E phenotypes 2 (2/2, 2/3, 2/4), 3 (3/3) and 4 (3/4, 4/4), respectively. Serum lipids, lipoproteins, sterols and cholesterol metabolism were examined on four subsequent diets: high-cholesterol high-fat (home diet; HD), low-cholesterol low-fat (LCLF), high-cholesterol low-fat (HCLF) and low-cholesterol high-fat (LCHF). LDL-cholesterol (LDL-C) level was about 40 % lower (P < 0.05) in apo E2 than apo E3 and E4 groups irrespective of dietary fat and cholesterol. Serum proportions of phytosterols were determined apo E-dependently on LCLF and HCLF, and those of lathosterol, cholestanol and campesterol were increased in apo E2 and E3 groups (P < 0.05 for each v. HD). Serum proportion of sitosterol reflected almost consistently apo E phenotype (r range+0.308 to+0.383; P range 0.214-0.011). Relative cholesterol synthesis and absorption reflected respective absolute values during each diet in the apo E4 group (r range+0.713 to+0.893; P < 0.05 for each), but only during HD (r+0.594; P = 0.015) in the apo E2+E3 group. The consumption of a high amount of fat did not interfere with cholesterol metabolism or serum levels of LDL-C differently in apo E phenotypes. Surrogate sterol markers of cholesterol metabolism reflected absolute ones (especially in the apo E4 group) and apo E phenotypes despite variable amounts of dietary cholesterol and fat.