Hypercholesterolemic Humans Research Articles

Effect of milk fat on LDL cholesterol and other cardiovascular risk markers in healthy humans: the INNOVALAIT project.

Milk has a specific saturated fatty acid profile and its calcium content may change the kinetics of fat absorption. The goal of this study was to compare the effect on LDL Cholesterol and other risk markers of four isolipidic diets differing by their fat food source, vegetable fat, spring milk fat, winter milk fat or winter milk fat supplemented with calcium, in healthy moderately hypercholesterolemic humans. This double-blind, randomized trial with four parallel arms included 172 healthy adults with plasma LDL cholesterol (LDL-C) from 130 to 220 mg/dL and triglycerides below 300 mg/dL. Individual meal plans ensured a stable energy intake. In the three diets containing milk fat, milk fat provided 38% of energy. Vegetable fat and spring milk fat diets provided the same amount of saturated fatty acids while the winter milk fat diets were slightly richer in saturated fatty acids. Vegetable fat diet and winter milk fat diets provided the same amount of palmitic acid (7.0% EI), while the spring milk fat diet was slightly poorer in this fatty acid (5.1% EI). Cardiovascular risk markers were analyzed after 8 weeks of dietary intervention. There was no significant difference in LDL-C and other markers, except total cholesterol (TC), apo C3 and CRP. TC was significantly higher with spring milk fat than with vegetable fat. In this trial, the chosen vegetable fat did not have a significant beneficial effect on LDL-C compared to dairy fat. However, sub-group analysis showed differences in TC, apo C3 and CRP. These results need confirmation and long-term studies aiming at cardiovascular endpoints are warranted.

Effect of daily intake of a low-alcohol orange beverage on cardiovascular risk factors in hypercholesterolemic humans

Oxidative stress, inflammation status, endothelial dysfunction, and imbalanced lipid metabolism play a major role in cardiovascular disease. Bioactive compounds and moderate alcohol consumption have been associated with decreased cardiovascular risk. This study aimed to evaluate the effect on cardiovascular risk factors of a low-alcohol beverage derived from the alcoholic fermentation of orange juice. Eighteen individuals with moderately high cholesterol levels were randomly assigned to an experimental group (n = 9) who drank 500 mL/day of the orange beverage for 2 weeks or a control group (n = 9) who drank no orange beverage. Blood samples were drawn at the beginning and end of the study period. Consumption of the beverage significantly decreased plasma levels of total cholesterol (−15.6%), LDL-cholesterol (−17.8%), LDL-cholesterol/HDL-cholesterol ratio (−21.4%), catalase (−25.5%), TBARS (−42.6%), and sVCAM-1 (−5.8%). This orange beverage would have a potential capacity to improve cardiovascular risk in hypercholesterolemic humans, supporting its future consideration as a functional beverage.

A Comparative Study of Some Functional Properties of Lactobacillus and Enterococcus Isolated from Feces of Normo and Hyper-Cholesterolemic Humans

There have been reports of an association between an imbalance of gastrointestinal microbiota and several kinds of diseases, mainly metabolic diseases. Lactobacillus and Enterococcus are two genera that form part of the native gastrointestinal microbiota. In this work, we isolated, identified and characterized 26 strains of Lactobacillus and 23 strains of Enterococcus from the feces of normo and hypercholesterolemic humans. We compared the behavior exhibited by all strains at acid pH and in presence of bile salts, their sensitivity to antibiotics, and their ability to hydrolyze bile salts and to reduce cholesterol in vitro. The highest percentage of all isolated and characterized Lactobacillus strains was from the normocolesterolemic group; in the hypercholesterolemic group, most strains belonged to the Enterococcus genus. The Lactobacillus strains showed greater capacity to reduce cholesterol levels; although this capacity has been related to bile salt hydrolase activity, four of the strains we isolated showed no such activity, but still reduced cholesterol. All strains of both genera isolated from normocholesterolemic participants showed a highest rate of cholesterol reduction than those isolated from hypercholesterolemic participants. Furthermore, the Lactobacillus strains showed greater resistance at pH 2.0, while strains of both genera showed similar survival rates at pH 3.0 and in the presence of bile salts after 24 h. These results support the evidence that a microbial imbalance involving the depletion of beneficial bacteria could be detrimental to the health of the host.

Effect of virgin olive oil and thyme phenolic compounds on blood lipid profile: implications of human gut microbiota.

To investigate the effect of virgin olive oil phenolic compounds (PC) alone or in combination with thyme PC on blood lipid profile from hypercholesterolemic humans, and whether the changes generated are related with changes in gut microbiota populations and activities. A randomized, controlled, double-blind, crossover human trial (n=12) was carried out. Participants ingested 25mL/day for 3weeks, preceded by 2-week washout periods, three raw virgin olive oils differing in the concentration and origin of PC: (1) a virgin olive oil(OO) naturally containing 80mgPC/kg, (VOO), (2) a PC-enriched virgin olive oil containing 500mgPC/kg, from OO (FVOO), and (3) a PC-enriched virgin olive oil containing a mixture of 500mgPC/kg from OO and thyme, 1:1 (FVOOT). Blood lipid values and faecal quantitative changes in microbial populations, short chain fatty acids, cholesterol microbial metabolites, bile acids, and phenolic metabolites were analysed. FVOOT decreased seric ox-LDL concentrations compared with pre-FVOOT, and increased numbers of bifidobacteria and the levels of the phenolic metabolite protocatechuic acid compared to VOO (P<0.05). FVOO did not lead to changes in blood lipid profile nor quantitative changes in the microbial populations analysed, but increased the coprostanone compared to FVOOT (P<0.05), and the levels of the faecal hydroxytyrosol and dihydroxyphenylacetic acids, compared with pre-intervention values and to VOO, respectively (P<0.05). The ingestion of a PC-enriched virgin olive oil, containing a mixture of olive oil and thyme PC for 3weeks, decreases blood ox-LDL in hypercholesterolemic humans. This cardio-protective effect could be mediated by the increases in populations of bifidobacteria together with increases in PC microbial metabolites with antioxidant activities.

Soy isoflavones in nutritionally relevant amounts have varied nutrigenomic effects on adipose tissue.

Soy consumption has been suggested to afford protection from cardiovascular disease (CVD). Indeed, accumulated albeit controversial evidence suggests that daily consumption of ≥25 g of soy protein with its associated phytochemicals intact can improve lipid profiles in hypercholesterolemic humans. However, the belief that soy foods and supplements positively impact human health has become increasingly controversial among the general public because of the reported estrogenic activities of soy isoflavones. In this study, we investigated the nutrigenomic actions of soy isoflavones (in nutritionally-relevant amounts) with a specific focus on the adipose tissue, due to its pivotal role in cardiometabolism. Young C57BL/6 mice were maintained for eight weeks under two different diet regimes: (1) purified control diet; or (2) purified control diet supplemented with 0.45 g% soybean dry purified extract (a genistein/daidzein mix). Soy isoflavones increased plasma total cholesterol concentrations and decreased triglyceride ones. Circulating leptin levels was also increased by soy consumption. Differentially expressed genes in adipose tissue were classified according to their role(s) in cellular or metabolic pathways. Our data show that soy isoflavones, administered in nutritionally-relevant amounts, have diverse nutrigenomic effects on adipose tissue. Taking into account the moderate average exposure to such molecules, their impact on cardiovascular health needs to be further investigated to resolve the issue of whether soy consumption does indeed increase or decrease cardiovascular risk.

Comparative Evaluation of NS-5 Mixture and its Components on Superoxide Production in HUVEC, and Inflammatory Biomarkers in Humans

Background: Inhibitory effects of NS-5 mixture of resveratrol, quercetin, δ-tocotrienol, nicotinic acid on several inflammatory and cardiovascular risk factors have been reported in normal cholesterolemic and hypercholesterolemic humans. The hypothesis was that combination of cholesterol-lowering and inflammatory-reducing properties of NS-5 mixture would be more effective than its individual components in reducing the serum levels of several biomarkers of cardiovascular disease in humans. However, effects of NS-5 mixture and its components did not report effects on cytokines, gene expression, and microRNAs were not reported in previous publication. As this area is gaining importance in the understanding of various transcriptional factors and signal pathways, which regulate several biomarkers in various diseases. Aims: Modulation of NS-5 mixture, and its components were evaluated on superoxide production in HUVEC in vitro, and on serum levels of total cholesterol, NO, CRP, TAS, plasma cytokines, gene expression, miRNAs in vivo in normal cholesterolemic and hypercholesterolemic humans. Study design: Study was carried out as double-blind randomized, trial of NS-5 mixture, resveratrol, quercetin, and δ-tocotrienol in free-living healthy and hypercholesterolemic humans. Results: The NS-5 mixture, resveratrol, quercetin, δ-tocotrienol, or nicotinic acid treatments caused reduction in superoxide production (11% to 24%; P<0.01) in HUVEC. These reductions were more pronounced with LPSstimulated HUVEC (26% to 40%; P<0.01) compared to predose values. These findings were further supported by decreases (P<0.01) in serum total cholesterol levels of NS-5 treated group (24%) versus resveratrol (18%), quercetin (20%), and δ-tocotrienol (22%) in hypercholesterolemic humans, followed by reduction of NO, CRP and increases in TAS in normal cholesterolemic and hypercholesterolemic humans. There was significant (P<0.001) down-regulation in pro-inflammatory cytokines and gene expression of resistin, IL-2α, IL-6, IL-12, IL-18, TNF-α, and others, that are normally involved in pathogenesis of atherosclerosis, diabetes, and aging processes. The plasma inflammatory miRNAs (miR-101a, miR-125a, miR-155, miR-223) were down-regulated as compared to predose values. The elevated levels of miRNA-146a during senescence were down-regulated after treatment with these compounds. Conclusions: This is the first report that describes the effects of NS-5 mixture, its components on proteomics, gene expression and levels of miRNAs in normal cholesterolemic and hypercholesterolemic humans. Results suggest that NS-5 mixture and its components are potent agents in the reduction of superoxide production, cardiovascular risk factors and inflammatory biomarkers, which are modulated by NF-κB. Maximum inhibition in superoxide production and other risk factors was observed with NS-5 mixture as compared with its individual components, thus supporting our hypothesis.

Hypotensive, hypoglycaemic and antioxidant effects of consuming a cocoa product in moderately hypercholesterolemic humans

The health benefits of dietary fiber and polyphenols in reducing cardiovascular risk have been evidenced. Cocoa husks are a good source of both components and a considerable by-product. A cocoa product rich in cocoa fiber (CP) has been produced from cocoa husks and this study assessed whether its regular consumption may be a strategy to improve lipid profile, serum glucose and antioxidant activity as well as blood pressure in moderately hypercholesterolemic subjects. In this free-living, non-controlled, non-randomized, two-month-long, open intervention trial. 21 volunteers consumed daily two servings of CP, which provided 12 g of dietary fiber and 283 mg of soluble polyphenols. Subjects were moderately hypercholesterolemic (>200 mg dl(-1)), non-vegetarian, non-smoker, women and men between 18 and 45 years old, with a body mass index under 30 kg m(-2), not suffering from any other chronic pathology. Blood samples were drawn and anthropometric measurements, systolic (SBP) and diastolic (DBP) blood pressures were evaluated at the baseline, and at weeks 2, 4, and 8. Serum lipids, creatinine, uric acid, glucose, C-reactive protein, ferric reducing/antioxidant power and malondialdehyde (MDA) levels were analyzed. Glucose (p = 0.019), SBP (p = 0.001), DBP (p = 0.001) and MDA (p = 0.036) decreased, HDL-cholesterol slightly increased, whereas the rest of the parameters remained similar. This preliminary study suggests that CP might be considered part of a dietary approach or a functional food or ingredient for the food industry to achieve hypotensive and hypoglycemic effects in moderately hypercholesterolemic subjects without inducing changes in body weight and waist circumference, although results should be confirmed in a longer, controlled human study.

Acute localized administration of tetrahydrobiopterin and chronic systemic atorvastatin treatment restore cutaneous microvascular function in hypercholesterolaemic humans

Elevated oxidized low-density lipoproteins (LDL) are associated with vascular dysfunction in the cutaneous microvasculature, induced in part by upregulated arginase activity and increased globalized oxidant stress. Since tetrahydrobiopterin (BH(4)) is an essential cofactor for endothelial nitric oxide synthase (NOS3), decreased bioavailability of the substrate l-arginine and/or BH(4) may contribute to decreased NO production with hypercholesterolaemia. We hypothesized that (1) localized administration of BH(4) would augment NO-dependent vasodilatation in hypercholesterolaemic human skin, which would be further increased when combined with arginase inhibition and (2) the improvement induced by localized BH(4) would be attenuated after a 3 month oral atorvastatin intervention (10 mg). Four microdialysis fibres were placed in the skin of nine normocholesterolaemic (NC: LDL = 95 ± 4 mg dl(-1)) and nine hypercholesterolaemic (HC: LDL = 177 ± 6 mg dl(-1)) men and women before and after 3 months of systemic atorvastatin. Sites served as control, NOS inhibited, BH(4), and arginase inhibited + BH(4) (combo). Skin blood flow was measured while local skin heating (42°C) induced NO-dependent vasodilatation. After the established plateau l-NAME was perfused in all sites to quantify NO-dependent vasodilatation (NO). Data were normalized to maximum cutaneous vascular conductance (CVC). Vasodilatation at the plateau and NO-dependent vasodilatation were reduced in HC subjects (plateau HC: 70 ± 5% CVC(max) vs. NC: 95 ± 2% CVC(max); NO HC: 45 ± 5% CVC(max) vs. NC: 64 ± 5% CVC(max); both P < 0.001). Localized BH(4) alone or combo augmented the plateau (BH(4): 93 ± 3% CVC(max); combo 89 ± 3% CVC(max), both P < 0.001) and NO-dependent vasodilatation in HC (BH(4): 74 ± 3% CVC(max); combo 76 ± 3% CVC(max), both P < 0.001), but there was no effect in NC subjects (plateau BH(4): 90 ± 2% CVC(max); combo 95 ± 3% CVC(max); NO-dependent vasodilatation BH(4): 68 ± 3% CVC(max); combo 58 ± 4% CVC(max), all P > 0.05 vs. control site). After the atorvastatin intervention (LDL = 98 ± mg * dl(-1)) there was an increase in the plateau in HC (96 ± 4% CVC(max), P < 0.001) and NO-dependent vasodilatation (68 ± 3% CVC(max), P < 0.001). Localized BH(4) alone or combo was less effective at increasing NO-dependent vasodilatation after the drug intervention (BH(4): 60 ± 5% CVC(max); combo 58 ± 2% CVC(max), both P < 0.001). These data suggest that decreased BH(4) bioavailability contributes in part to cutaneous microvascular dysfunction in hypercholesterolaemic humans and that atorvastatin is an effective systemic treatment for improving NOS coupling mechanisms in the microvasculature.

Oral atorvastatin therapy increases nitric oxide-dependent cutaneous vasodilation in humans by decreasing ascorbate-sensitive oxidants

Elevated low-density lipoproteins (LDL) are associated with cutaneous microvascular dysfunction partially mediated by increased arginase activity, which is decreased following a systemic atorvastatin therapy. We hypothesized that increased ascorbate-sensitive oxidant stress, partially mediated through uncoupled nitric oxide synthase (NOS) induced by upregulated arginase, contributes to cutaneous microvascular dysfunction in hypercholesterolemic (HC) humans. Four microdialysis fibers were placed in the skin of nine HC (LDL = 177 ± 6 mg/dl) men and women before and after 3 mo of a systemic atorvastatin intervention and at baseline in nine normocholesterolemic (NC) (LDL = 95 ± 4 mg/dl) subjects. Sites served as control, NOS inhibited, L-ascorbate, and arginase-inhibited+L-ascorbate. Skin blood flow was measured while local skin heating (42°C) induced NO-dependent vasodilation. After the established plateau in all sites, 20 mM ≪ngname≫ was infused to quantify NO-dependent vasodilation. Data were normalized to maximum cutaneous vascular conductance (CVC) (sodium nitroprusside + 43°C). The plateau in vasodilation during local heating (HC: 78 ± 4 vs. NC: 96 ± 2% CVC(max), P < 0.01) and NO-dependent vasodilation (HC: 40 ± 4 vs. NC: 54 ± 4% CVC(max), P < 0.01) was reduced in the HC group. Acute L-ascorbate alone (91 ± 5% CVC(max), P < 0.001) or combined with arginase inhibition (96 ± 3% CVC(max), P < 0.001) augmented the plateau in vasodilation in the HC group but not the NC group (ascorbate: 96 ± 2; combo: 93 ± 4% CVC(max), both P > 0.05). After the atorvastatin intervention NO-dependent vasodilation was augmented in the HC group (HC postatorvastatin: 64 ± 4% CVC(max), P < 0.01), and there was no further effect of ascorbate alone (58 ± 4% CVC(max,) P > 0.05) or combined with arginase inhibition (67 ± 4% CVC(max,) P > 0.05). Increased ascorbate-sensitive oxidants contribute to hypercholesteromic associated cutaneous microvascular dysfunction which is partially reversed with atorvastatin therapy.

Oral atorvastatin therapy restores cutaneous microvascular function by decreasing arginase activity in hypercholesterolaemic humans

Elevated low-density lipoproteins (LDLs) are associated with vascular dysfunction evident in the cutaneous microvasculature. We hypothesized that uncoupled endothelial nitric oxide synthase (NOS3) through upregulated arginase contributes to cutaneous microvascular dysfunction in hyperocholesterolaemic (HC) humans and that a statin intervention would decrease arginase activity. Five microdialysis fibres were placed in the skin of nine normocholesterolaemic (NC: LDL level 95±4 mg dl⁻¹) and nine hypercholesterolaemic (HC: LDL: 177±6 mg dl⁻¹) men and women before and after 3 months of systemic atrovastatin. Sites served as control, NOS inhibited, arginase inhibited, L-arginine supplemented and arginase inhibited plus L-arginine supplemented. Skin blood flow was measured while local skin heating (42°C) induced NO-dependent vasodilatation. L-NAME was infused after the established plateau in all sites to quantify NO-dependent vasodilatation. Data were normalized to maximum cutaneous vascular conductance (CVC(max)). Skin samples were obtained to measure total arginase activity and arginase I and arginase II protein. Vasodilatation was reduced in hyperocholesterolaemic subjects (HC: 76±2 vs. NC: 94±3%CVC(max), P < 0.001) as was NO-dependent vasodilatation (HC: 43±5 vs. NC: 62±4%CVC(max), P < 0.001). The plateau and NO-dependent vasodilatation were augmented in HC with arginase inhibition (92±2, 67±2%CVC(max), P < 0.001), L-arginine (93±2, 71±5%CVC(max), P < 0.001) and combined treatments (94±4, 65±5%CVC(max), P < 0.001) but not in NC. After statin intervention (LDL: 98±5 mg dl⁻¹) there was no longer a difference between control sites (88±4, 61±5%CVC(max)) and localized microdialysis treatment sites (all P > 0.05). Arginase activity and protein were increased in HC skin (P < 0.05 vs. NC) and activity decreased with atrovastatin treatment (P < 0.05). Reduced NOS3 substrate availability through upregulated arginase contributes to cutaneous microvascular dysfunction in hyperocholesterolaemic humans, which is corrected with atorvastatin therapy.

Men and not women showed cholesterol lowering after 12 weeks of supplementation with secoisolariciresinol diglucoside

We hypothesized that secoisolariciresinol diglucoside (SDG, Beneflax™) derived from flaxseed, would lower serum cholesterol in hypercholesterolemic humans and that the effect would be correlated with serum lignan metabolites. 90 subjects ingested 0 mg (18 male, 12 female), 150 mg (19 male, 11 female), or 300 mg SDG (, 20 male, 10 female) total daily dose for 12 w in a randomized, placebo‐controlled double‐blind trial. At 0 and 12 weeks, serum secoisolariciresinol (SECO), enterodiol (ED), and enterolactone (EL) were measured by LC/MS. Fasting serum cholesterol, glucose, and HbA1c were measured at −2, 0, 4, 8 and 12 wks. Decrease of total cholesterol (TC) and LDL‐cholesterol (LDL‐C) from baseline were significant in men ingesting either dose of SDG. Plasma glucose was not changed from baseline by any treatment. The decrease of TC and LDL‐C from baseline was significantly greater in men than in women ingesting 300 mg SDG/d. Serum SECO, EL and ED were significantly increased in subjects ingesting either dose of lignans. LDL‐C was negatively correlated with serum ED (r = − 0.50, P = 0.03); and LDL decrease from baseline was positively correlated with serum SECO (r = 0.59, P = 0.01) in subjects ingesting 150 mg SDG/d. In conclusion, SDG decreased serum TC and LDL‐C in men but not in women and this function was related to the bioavailability of lignan metabolites. Research funded by Archer Daniels Midland.Grant Funding Source: Archer Daniels Midland

Hepatocyte Nuclear Factor 1α Plays a Critical Role in PCSK9 Gene Transcription and Regulation by the Natural Hypocholesterolemic Compound Berberine

PCSK9 is a natural inhibitor of LDL receptor (LDLR) that binds the extracellular domain of LDLR and triggers its intracellular degradation. PCSK9 and LDLR are coordinately regulated at the transcriptional level by sterols through their promoter-imbedded sterol response elements (SRE) and co-induced by statins. Identification of regulatory networks modulating PCSK9 transcription is important for developing selective repressors of PCSK9 to improve statin efficacy by prolonging the up-regulation of LDLR. Interestingly, the plant-derived hypocholesterolemic compound berberine (BBR) up-regulates LDLR expression while down-regulating PCSK9. In our investigations to define mechanisms underlying the transcriptional suppression of PCSK9 by BBR in HepG2 cells, we have identified a highly conserved hepatocyte nuclear factor 1 (HNF1) binding site residing 28 bp upstream from SRE as a critical sequence motif for PCSK9 transcription and its regulation by BBR. Mutation of the HNF1 site reduced PCSK9 promoter activity >90%. A battery of functional assays identified HNF1α as the predominant trans-activator for PCSK9 gene working through this sequence motif. We further provide evidence suggesting that HNF1 site works cooperatively with SRE as HNF1 mutation significantly attenuated the activity of nuclear SREBP2 to transactivate PCSK9 promoter. Finally, we show that a coordinate modest reduction of HNF1α and nuclear SREBP2 by BBR led to a strong suppression of PCSK9 transcription through these two critical regulatory sequences. This is the first described example of SREBP pairing with HNF1 to control an important regulatory pathway in cholesterol homeostasis. This work also provides a mechanism for how BBR suppresses PCSK9 transcription.

The in vivo role of arginase in cutaneous microvascular dysfunction in hypercholesterolemic humans before and after statin therapy

Elevated low‐density lipoproteins (LDL) are associated with systemic vascular dysfunction evident in the cutaneous microvasculature. Our aim was to determine the role of arginase and freely exchangeable L‐arginine (L‐arg) in vivo in reduced NO‐dependent vasodilation (VD) in hypercholesteroleΔic (HC) skin before and after statin therapy. Three intradermal microdialysis (MD) fibers were placed in 5 normocholesterolemic (NC) (LDL=92 ±8 mg/dl) and 6 HC subjects before (LDL=179±6 mg/dl) and after 3 months of atorvastatin (10 mg) (LDL=100±7 mg/dl): control, arginase‐inhibited (A‐I: 5 mM BEC), and L‐arg (10 mM). Skin blood flow (SkBF) was measured while local skin heating (42°C) induced NO‐dependent VD [quantified (ΔNO) by infusing 10 mM L‐NAME after the plateau (P) in SkBF]. Cutaneous vascular conductance (CVC= flux/MAP) was normalized to max (50 mM SNP). VD was reduced in HC (HC: 77±5 vs. NC: 94±5 % max, p&lt;0.05) as was ΔNO (HC: 37±5 vs. NC: 61±6 % max, p&lt;0.05). P and ΔNO were augmented in HC with A‐I (P: 92±3, ΔNO: 65±3% max, p&lt;0.05) and L‐arg (P: 95±3, ΔNO: 75±4% max, p&lt;0.05) but not in NC. After statin therapy there was no difference between control (P: 92±2, ΔNO: 51±4% max) and A‐I (P: 97±1, ΔNO: 62±6% max, p&gt;0.05) or L‐arg (P: 94±3, ΔNO: 56±5% max, p&gt;0.05) sites. Reduced NOS substrate availability through upregulated arginase contributes to cutaneous microvascular dysfunction with HC, which is corrected with statin therapy.

Whole and fractionated yellow pea flour consumption alters post‐ prandial glucose response and insulin parameters, but not lipids or glucose levels, in overweight or obese, hypercholesterolemic humans

Yellow pea flour is a low fat, high fiber food; however, underlying mechanisms regarding health benefits of whole (WPF) and fractionated (FPF) yellow pea flours remain understudied. In a controlled feeding crossover study, 23 hypercholesterolemic, overweight and obese men and women received for 28 d two muffins/d containing WPF, FPF or white wheat flour (WF) as control. Treatments were separated by a 28 d washout. Two WPF muffins contained 50 g of whole yellow peas, with the amount of FPF in FPF treatments equaling the amount of pea‐derived fiber in WPF treatments. WPF and FPF consumption failed to affect fasting total cholesterol, LDL‐C, HDL‐C, and glucose. Subjects with a BMI between 25 and 34.9 receiving FPF tended (p=0.075) to have reduced post‐prandial glucose responses compared to WF. Insulin levels for WPF (31.8 ±6.5 pmol/ml, p=0.02) and FPF (32.0±6.5 pmol/ml, p=0.026) were lower compared to WF (37.9±6.5 pmol/ml). Insulin homeostasis model‐assessment showed that consumption of WPF (p=0.019) and FPF (p=0.012) increased insulin sensitivity 20% compared to WF. Results suggest that at energy balance, WPF and FPF do not alter fasting lipid and glucose levels and exert minimal effects on post‐prandial glucose response in hypercholesterolemic men and woman with elevated BMIs. Conversely, 50 g/d WPF and FPF consumption improves fasting insulin levels and sensitivity and thus provide dietary health benefits. Supported by Pulse Innovation Project, Canada

Evaluation of cholesterol-lowering and antioxidant properties of sugar cane policosanols in hamsters and humans

Atherosclerosis prevention is now a main focus of the scientific community and pharmaceutical industry. Sugar cane policosanols (SCPs) have gained increasing popularity over the last decade as a result of numerous studies conducted in Cuba that consider SCPs as a natural alternative to statin drugs. However, independent research on policosanols was not able to replicate cholesterol reductions reported by Cuban laboratories. No independent study to date has examined the cholesterol-lowering effect and antioxidant capacity of original SCPs in humans. In addition, since independent research was criticized because of the use of alternative policosanol formulations, the source and composition of policosanol mixtures are now at the core of the policosanol controversy. The aim of this thesis project was first to compare the composition and cholesterol-lowering effects of different SCP preparations in hamsters, and second to test the cholesterol-lowering efficacy, mechanism of action, and antioxidant capacity of the original Cuban SCP in hypercholesterolemic humans. In study 1, 48 male hamsters were randomly assigned to 4 groups for a period of 4 weeks: (i) non-cholesterol control, (ii) cholesterol control, (iii) original SCPs, and (iv) alternative SCPs. Hamsters were sacrificed and blood was collected at the end of the feeding period for lipid measurements. In study 2, 21 hypercholesterolemic volunteers consumed 10 mg·d–1 of SCPs or a placebo for a period of 28 days in a crossover trial. Plasma lipid levels and low-density lipoprotein (LDL) oxidation were measured at the start and end of supplementation phases. Cholesterol absorption and synthesis were assessed using a single-isotope, single-tracer technique and deuterium incorporation, respectively. There was no significant difference in cholesterol-lowering efficacy between hamsters in the original and alternative SCP groups relative to the control. Similarly, in hypercholesterolemic humans, supplementation with original SCPs did not significantly improve lipid parameters and no change in cholesterol absorption or synthesis was observed relative to the control. In vivo assessment of LDL oxidation showed no significant changes in oxidized LDL concentration relative to baseline and control. The present thesis disagrees with previous Cuban data on the cardio-protective role of SCPs in animals and humans and supports independent studies showing no cholesterol-lowering effect and no antioxidant capacity.

The Influence of Pravastatin and Atorvastatin on Markers of Oxidative Stress in Hypercholesterolemic Humans

The aim of this study was to determine the effects of pravastatin and atorvastatin on markers of oxidative stress in plasma. Hydroxymethylglutaryl coenzyme A reductase inhibitors reduce low-density lipoprotein cholesterol (LDL-C) and cardiovascular risk, but their effects on circulating biomarkers of oxidative stress are not well-defined. Hypercholesterolemic subjects (n = 120, ages 21 to 80 years with LDL-C 130 to 220 mg/dl) were randomized in a double-blind, parallel design to pravastatin 40 mg/day (prava40), atorvastatin 10 mg/day (atorva10), atorvastatin 80 mg/day (atorva80), or placebo. At baseline and 16 weeks, urinary isoprostanes (8, 12-iso-iPF(2 alpha)-VI isoform), plasma lipoprotein-associated phospholipase A2 (Lp-PLA2), Mercodia oxidized LDL (OxLDL) with antibody 4E6, oxidized phospholipids/apolipoprotein B-100 particle (OxPL/apoB) with antibody E06, immunoglobulin (Ig)G/IgM autoantibodies to malondialdehyde (MDA)-LDL, and apolipoprotein B (apoB)-immune complexes (IC) were measured. After 16 weeks, there were no significant changes in urinary 8, 12-iso-iPF(2 alpha)-VI. The Lp-PLA2 and OxLDL were reduced in statin-treated groups, but after adjusting for apoB, only prava40 led to a reduction in Lp-PLA2 (-15%, p = 0.008) and atorva10 to a decrease in OxLDL (-12.9%, p = 0.01). The OxPL/apoB increased 25.8% (p < 0.01) with prava40 and 20.2% (p < 0.05) with atorva80. There were no changes in MDA-LDL autoantibodies, but significant decreases in IC were noted. This study suggests that statin therapy results in variable effects on oxidative stress markers in hypercholesterolemic subjects. Future outcome studies should collectively assess various oxidative markers to define clinical utility.

Tuning of the Outer Hair Cell Motor by Membrane Cholesterol

Cholesterol affects diverse biological processes, in many cases by modulating the function of integral membrane proteins. We observed that alterations of cochlear cholesterol modulate hearing in mice. Mammalian hearing is powered by outer hair cell (OHC) electromotility, a membrane-based motor mechanism that resides in the OHC lateral wall. We show that membrane cholesterol decreases during maturation of OHCs. To study the effects of cholesterol on hearing at the molecular level, we altered cholesterol levels in the OHC wall, which contains the membrane protein prestin. We show a dynamic and reversible relationship between membrane cholesterol levels and voltage dependence of prestin-associated charge movement in both OHCs and prestin-transfected HEK 293 cells. Cholesterol levels also modulate the distribution of prestin within plasma membrane microdomains and affect prestin self-association in HEK 293 cells. These findings indicate that alterations in membrane cholesterol affect prestin function and functionally tune the outer hair cell.

Hydroxypropylmethylcellulose significantly lowers blood cholesterol in mildly hypercholesterolemic human subjects

To determine the cholesterol-lowering efficacy of hydroxypropylmethylcellulose (HPMC) in mildly hypercholesterolemic humans. Trial one: entry mean (range) total serum cholesterol values of eight female and four male subjects were 6.48 (5.57-7.51) mmol l(-1) (250 (215-290) mg dl(-1)) and 6.60 (5.57-7.64) mmol l(-1) (255 (215-295) mg dl(-1)), respectively. Trial two: corresponding values for 20 women and 20 men were 5.96 (5.43-6.48) mmol l(-1) 230 (210-250) mg dl(-1)) and 6.05 (5.46-6.63) mmol l(-1) 233 (211-256) mg dl(-1)), respectively. Trial one: HPMC decreased (P< or =0.05) total and LDL-cholesterol 9.3 and 15.3% (medium), 16.9 and 23.5% (high) and 13.8 and 19.4% (ultra-high), respectively, over placebo. Trial two: total and LDL-cholesterol decreased (P< or =0.05) throughout the 8 weeks, with mean (weeks 4-8) reductions of 7 and 8% at 5 g day(-1), and 12 and 15% at 15 g day(-1), respectively, over placebo. Adverse effects were minimal. Trial one: medium, high and ultra-high viscosity HPMC at 15 g day(-1) for 1 week each;1-week wash-out between treatments. Trial two: ultra-high viscosity HPMC at 5 or 15 g day(-1) for 8 weeks. HPMC soluble fiber, especially high-viscosity grades, significantly lowers cholesterol at well-tolerated doses, showing promise as a treatment of hypercholesterolemia.

Plasma LDL and HDL Cholesterol and Oxidized LDL Concentrations Are Altered in Normo- and Hypercholesterolemic Humans after Intake of Different Levels of Cocoa Powder

Cocoa powder is rich in polyphenols, such as catechins and procyanidins, and has been shown in a variety of subject models to inhibit oxidized LDL and atherogenesis. Our study evaluated plasma LDL cholesterol and oxidized LDL concentrations following the intake of different levels of cocoa powder (13, 19.5, and 26 g/d) in normocholesterolemic and mildly hypercholesterolemic humans. In this comparative, double-blind study, we examined 160 subjects who ingested either cocoa powder containing low-polyphenolic compounds (placebo-cocoa group) or 3 levels of cocoa powder containing high-polyphenolic compounds (13, 19.5, and 26 g/d for low-, middle-, and high-cocoa groups, respectively) for 4 wk. The test powders were consumed as a beverage after the addition of hot water, twice each day. Blood samples were collected at baseline and 4 wk after intake of the test beverages for the measurement of plasma lipids. Plasma oxidized LDL concentrations decreased in the low-, middle-, and high-cocoa groups compared with baseline. A stratified analysis was performed on 131 subjects who had a LDL cholesterol concentrations of ≥3.23 mmol/L at baseline. In these subjects, plasma LDL cholesterol, oxidized LDL, and apo B concentrations decreased, and the plasma HDL cholesterol concentration increased, relative to baseline in the low-, middle-, and high-cocoa groups. The results suggest that polyphenolic substances derived from cocoa powder may contribute to a reduction in LDL cholesterol, an elevation in HDL cholesterol, and the suppression of oxidized LDL.

Systemic Acyl-CoA:Cholesterol Acyltransferase Inhibition Reduces Inflammation and Improves Vascular Function in Hypercholesterolemia

Circulating lipids may initiate and progress atherosclerosis by causing vascular inflammation. Monocytes and tissue macrophages are involved and regulate lipid metabolism in the vascular wall through acetylation of cholesterol by acyl-CoA:cholesterol acyltransferase (ACAT). ACAT inhibition reduces atherosclerosis in animal models by mechanisms that may be independent of their effects on circulating lipids. Because endothelial dysfunction is an important factor in atherosclerosis, we tested the hypothesis that systemic ACAT inhibition would improve endothelial function in hypercholesterolemic humans and assessed its effects on circulating lipids and markers of systemic inflammation. We studied 21 hypercholesterolemic subjects in a double-blind, randomized-crossover, placebo-controlled trial with assessments of circulating lipids, markers of inflammation, resistance-vessel endothelial function (with venous occlusion plethysmography), and conduit-vessel vasoreactivity (brachial artery flow-mediated dilation at baseline and after placebo or treatment with avasimibe 750 mg QDS for 8 weeks. There was a small change in total cholesterol with treatment (326+/-25 to 311+/-22 mg/dL, P=0.04). Circulating tumor necrosis factor-alpha was significantly reduced (4.0+/-0.3 to 3.6+/-0.2 pg/mL, P=0.02); resistance vessel responses to acetylcholine, bradykinin, and verapamil were significantly enhanced; and responses to nitroglycerin and conduit-vessel vasoreactivity were unchanged after ACAT inhibition. Systemic ACAT inhibition reduces circulating tumor necrosis factor-alpha levels in hypercholesterolemic subjects and improves resistance-vessel endothelial function, with small effects on circulating cholesterol. This may be a novel therapeutic strategy to target vascular inflammation and endothelial dysfunction in atherosclerosis.