Catherine A. Vanstone

Dietary phytosterols as cholesterol-lowering agents in humans

Phytosterols (plant sterols), abundant in fat-soluble fractions of plants, are consumed at levels of 200-400 mg/day in Western diets. Chemically resembling cholesterol, phytosterols inhibit the absorption of cholesterol. Phytosterol consumption in human subjects under a wide range of study conditions has been shown to reduce plasma total and low density lipoprotein (LDL) cholesterol levels; however, the response varies widely. Greater cholesterol-lowering efficacy occurs with consumption of the saturated phytosterol sitostanol versus sitosterol or campesterol. Most studies report no effect of phytosterol administration in high density lipoprotein (HDL) cholesterol or triglyceride levels, although certain evidence exists for an HDL cholesterol raising effect of sitostanol. Phytosterol absorption is limited, although serum phytosterol levels have proven to be important indicators of both cholesterol absorption and synthesis. Serum phytosterols correlate with HDL cholesterol level. In addition, higher phytosterol/cholesterol ratios appear in HDL versus LDL particles, suggesting the existence of an intrinsic phytosterol action, in addition to the extrinsic effect on cholesterol absorption. In conclusion, addition to diet of the phytosterol sitostanol represents an effective means of improving circulating lipid profiles to reduce risk of coronary heart disease.

Effect of Different Dosages of Oral Vitamin D Supplementation on Vitamin D Status in Healthy, Breastfed Infants: A Randomized Trial

IMPORTANCE Vitamin D supplementation in infancy is required to support healthy bone mineral accretion. A supplement of 400 IU of vitamin D per day is thought to support plasma 25-hydroxyvitamin D (25[OH]D) concentrations between 40 and 50 nmol/L; some advocate 75 to 150 nmol/L for bone health. OBJECTIVE To investigate the efficacy of different dosages of vitamin D in supporting 25(OH)D concentrations in infants. DESIGN, SETTING, AND PARTICIPANTS Double-blind randomized clinical trial conducted among 132 one-month-old healthy, term, breastfed infants from Montréal, Québec, Canada, between March 2007 and August 2010. Infants were followed up for 11 months ending August 2011 (74% completed study). INTERVENTION Participants were randomly assigned to receive oral cholecalciferol (vitamin D3) supplements of 400 IU/d (n=39), 800 IU/d (n=39), 1200 IU/d (n=38), or 1600 IU/d (n=16). MAIN OUTCOMES AND MEASURES The primary outcome was a plasma 25(OH)D concentration of 75 nmol/L or greater in 97.5% of infants at 3 months. Secondary outcomes included 25(OH)D concentrations of 75 nmol/L or greater in 97.5% of infants at 6, 9, and 12 months; 25(OH)D concentrations of 50 nmol/L or greater across all times; growth; and whole body and regional bone mineral content. Data were analyzed by intention to treat using available data, logistic regression, and mixed-model analysis of variance. RESULTS By 3 months, 55% (95% CI, 38%-72%) of infants in the 400-IU/d group achieved a 25(OH)D concentration of 75 nmol/L or greater vs 81%(95% CI, 65%-91%) in the 800-IU/d group, 92% (95% CI, 77%-98%) in the 1200-IU/d group, and 100% in the 1600-IU/d group. This concentration was not sustained in 97.5% of infants at 12 months in any of the groups. The 1600-IU/d dosage was discontinued prematurely because of elevated plasma 25(OH)D concentrations. All dosages established 25(OH)D concentrations of 50 nmol/L or greater in 97% (95% CI, 94%-100%) of infants at 3 months and sustained this in 98% (95% CI, 94%-100%) to 12 months. Growth and bone mineral content did not differ by dosage. CONCLUSIONS AND RELEVANCE Among healthy, term, breastfed infants, only a vitamin D supplement dosage of 1600 IU/d (but not dosages of 400, 800, or 1200 IU/d) increased plasma 25(OH)D concentration to 75 nmol/L or greater in 97.5% of infants at 3 months. However, this dosage increased 25(OH)D concentrations to levels that have been associated with hypercalcemia. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00381914.

Effect of plant sterols and glucomannan on lipids in individuals with and without type II diabetes

Objective:The purpose of this study was to determine whether supplements of plant sterols and/or glucomannan improve lipid profile and cholesterol biosynthesis in mildly hypercholesterolemic type II diabetic and non-diabetic subjects and to compare the response of these two subject groups to the treatments.Design:A randomized, crossover study consisting of four phases of 21 days, with each phase separated by a 28-day washout.Setting:The Mary Emily Clinical Nutrition Research Unit of McGill University.Subjects:Eighteen non-diabetic individuals and 16 type II diabetic individuals aged 38–74 years.Interventions:Subjects were supplemented with plant sterols (1.8 g/day), glucomannan (10 g/day), a combination of glucomannan and plant sterols, and a placebo, provided in the form of bars.Results:Overall plasma cholesterol concentrations were lowered (P<0.05) after combination treatment (4.72±0.20 mmol/l) compared to control (5.47±0.18 mmol/l). Plasma low-density lipoprotein (LDL) cholesterol concentrations were decreased (P<0.05) after glucomannan (3.16±0.14 mmol/l) and combination treatments (2.95±0.16 mmol/l) compared to control (3.60±0.16 mmol/l). The results of lipid profiles did not differ between subject groups. Overall plasma lathosterol concentrations, an index of cholesterol biosynthesis, were lowered (P<0.05) after the combination treatment compared to the plant sterol treatment.Conclusions:The results suggest that glucomannan and a combination of glucomannan and plant sterols substantially improves plasma LDL cholesterol concentrations.Sponsorship:Forbes Medi-Tech Inc., Vancouver, British Columbia, Canada.

Plant sterol consumption frequency affects plasma lipid levels and cholesterol kinetics in humans

Background/Objectives:To compare the efficacy of single versus multiple doses of plant sterols on circulating lipid level and cholesterol trafficking.Subjects/Methods:A randomized, placebo-controlled, three-phase (6 days/phase) crossover, supervised feeding trial was conducted in 19 subjects. Subjects were provided (i) control margarine with each meal; (ii) 1.8 g/day plant sterols in margarine with breakfast (single-BF) and control margarine with lunch and supper or (iii) 1.8 g/day plant sterols in margarine divided equally at each of the three daily meals (three times per day).Results:Relative to control, end point plasma low-density lipoprotein (LDL) cholesterol concentrations were lower (P<0.05) after consuming plant sterols three times per day but were not different when consumed once per day (3.43±0.62, 3.22±0.58 and 3.30±0.65 mmol/l, control, three times per day and single-BF, respectively). Relative to the control, end point LDL level was 0.21±0.27 mmol/l (6%) lower (P<0.05) at the end of the three times per day phase. Cholesterol fractional synthesis rate was highest (P<0.05) after the three times per day phase (0.0827±0.0278, 0.0834±0.0245 and 0.0913±0.0221 pool/day, control, single-BF and three times per day, respectively). Cholesterol-absorption efficiency decreased (P<0.05) by 36 and 39% after the three times per day and single-BF phase, respectively, relative to control.Conclusions:Present data indicate that to obtain optimal cholesterol-lowering impact, plant sterols should be consumed as smaller doses given more often, rather than one large dose.

Injected phytosterols/stanols suppress plasma cholesterol levels in hamsters

Although plant sterols are known to suppress intestinal cholesterol absorption, whether plasma and hepatic lipid levels are influenced through non-gut related internal mechanisms has not been established. To examine this question 50 male hamsters were divided into 5 groups and fed semi-purified diets containing 20% energy as fat and 0.25% (w/w) cholesterol ad libitum for 60 days. The control group (i) received diet alone, while four additional groups consumed the diet plus one of four equivalent phytosterol mixtures (5 mg/kg/day) given either as (ii) tall oil phytosterols/stanols mixed with diet (oralSA), (iii) tall oil phytosterols/stanols subcutaneously injected (subSA), (iv) soybean oil phytosterols alone mixed with diet (oralSE), or (v) soybean oil subcutaneous injected phytosterols alone (subSE). The control group and both orally supplemented groups also received placebo subcutaneous sham injections. Neither food consumption, body weight, nor liver weight differed across treatment groups. Subcutaneous administration of SA and SE decreased plasma total cholesterol levels by 21% and 23% (p < 0.0001) and non-apolipoprotein-A cholesterol concentrations by 22% and 15% (p < 0.0002), respectively, compared to control. HDL cholesterol and TG concentrations remained unchanged across all groups, except for a decline of 25% (p < 0.0001) in HDL concentration in the subSE group versus control. Plasma campesterol levels were lower (p < 0.05) in the subSA group relative to all other groups. Plasma campesterol:cholesterol and campesterol:sitosterol ratios were, however, higher (p < 0.0001) for both the oral and subSE groups. Hepatic cholesterol levels were higher (p < 0.0001) in the oral and subSE phytosterol groups by 30% and 31%, respectively, relative to control. We conclude that low doses of subcutaneously administered plant sterols reduce circulating cholesterol levels through mechanisms other than inhibiting intestinal cholesterol absorption.

Validation of a single-isotope-labeled cholesterol tracer approach for measuring human cholesterol absorption

Cholesterol absorption is frequently determined using the plasma dual stable-isotope ratio method (PDSIRM). However, this method involves intravenous injection of stableisotope-labeled cholesterol with simultaneous oral administration of differently labeled cholesterol, which results in high study costs and involves additional ethical considerations. The objective of the present study was to validate a simpler singleisotope method for determining cholesterol absorption against PDSIRM by using data from two previous studies. Enrichments of carbon-13 (13C) and deuterium in red blood cells were analyzed by using differential isotope ratio MS. The area under the curve of 13C-enrichment in the plasma free-cholesterol pool was found to be significantly correlated with cholesterol absorption measured by using PDSIRM for study 1 (r=0.85, P<0.0001) and study 2 (r=0.81, P<0.0001). Average 13C-enrichment correlated with the area under the curve of 13C-enrichment in the plasma free cholesterol for both study 1 (r=0.98, P<0.0001) and study 2 (r=1.00, P<0.0001). Study 1 examined the efficacy and mechanisms of unesterified plant sterols and stanols on lipid profiles in hypercholesterolemic men and women, while study 2 investigated the effects of phytosterol vs. phytostanol esters on plasma lipid levels and cholesterol kinetics in hyperlipidemic men. Experimental approaches to determine cholesterol absorption were identical between the two studies. Consequently, in both studies, correlations (r=0.88, P<0.0001 for study 1, and r=0.82, P<0.0001 for study 2) were found between the average 13C-enrichment of plasma free cholesterol and cholesterol absorption measured by PDSIRM. These results suggest that a single-isotope-labeled cholesterol tracer approach can be used as a reliable noninvasive method to replace PDSIRM for examining changes in cholesterol absorption.

Vitamin D Status in Montréal Preschoolers Is Satisfactory Despite Low Vitamin D Intake

The 2007 to 2009 Canadian Health Measures Survey reported vitamin D status in a representative sample of Canadians (6-79 y); however, children <6 y were not assessed. Our objective was to measure vitamin D intake from food and supplements, sun exposure, and biological vitamin D status of children ages 2 through 5 y in Montréal (latitude 45°N). Preschoolers (n = 508) were recruited between June 2010 and 2011 in a random sample of licensed daycares in the regions of greater Montréal, Canada in a cross-sectional study. The total plasma 25-hydroxyvitamin D [25(OH)D] concentration was measured using a chemiluminescence assay (Liaison, Diasorin). Dietary intake was assessed during one 24-h period plus a 30-d FFQ. Socioeconomic, demographic, anthropometry, and sun exposure data were collected. Plasma 25(OH)D was ≥50 nmol/L in 88% of children, whereas 49.4% had concentrations ≥75 nmol/L during the 1-y study. Almost 95% of preschoolers had vitamin D intakes less than the Estimated Average Requirement (EAR), and 4.8% of preschoolers ≤3.9 y and 25.9% of preschoolers ≥4 y had calcium intakes less than the EAR. Plasma 25(OH)D was different across age, income, sun index, milk intake, and dietary and supplemental vitamin D intake tertiles. Despite vitamin D intakes less than the EAR, the vitamin D status of Montréal preschoolers attending daycare is mostly satisfactory even in winter, suggesting that the EAR value is too high in the context of typical exogenous intakes of vitamin D in North America.

Baseline plasma plant sterol concentrations do not predict changes in serum lipids, C-reactive protein (CRP) and plasma plant sterols following intake of a plant sterol-enriched food

Background/Objectives:Plant sterol (PS) consumption lowers serum cholesterol levels, while modestly increasing plasma PS concentrations. Plasma PS concentrations may reflect sterol absorption, thus individuals with high plasma plant sterol (HPS) concentrations may show greater changes in circulating cholesterol and PS than individuals with low plasma plant sterol (LPS) concentrations. The objective of this study was to examine whether HPS and LPS concentrations are related to subsequent changes in plasma PS, serum lipid and C-reactive protein (CRP) concentrations, following dietary PS intake in otherwise healthy hypercholesterolemic men.Subjects/Methods:This single-blinded, randomized, diet-controlled study consisted of two 4-week phases, separated by a 4-week washout, where a diet with a placebo or the 2.0 g per day PS-enriched spread was consumed during the phases.Results:At baseline, men with HPS possessed higher (P<0.01) mean serum cholesterol concentration, while those with LPS had higher (P<0.05) body mass index. Following PS intake, plasma sum of campesterol plus sitosterol concentrations were elevated from 34.6±4.2 to 46.2±3.3 μmol l−1 (mean±SE) and 16.5±0.9 to 20.8±1.2 μmol l−1 after PS intake in men with HPS and LPS, respectively. Changes in plasma PS concentrations, however, were not different between individuals with either HPS or LPS baseline concentrations. Total cholesterol and low-density lipoprotein cholesterol levels were decreased (P<0.0001) by 6.3 and 7.8%, respectively, with PS consumption for all individuals. Changes in lipid parameters were not different between individuals with HPS or LPS baseline concentrations. No changes in CRP were apparent subsequent to PS intervention.Conclusions:Baseline plasma PS concentrations are not associated or predictive of changes in serum cholesterol or plasma PS concentrations after PS intervention. Thus, individuals with HPS show similar increases in PS concentrations as individuals with LPS following PS supplementation. Plasma PS remained in the range of previously reported concentrations.

Methodological issues in assessing plasma 25-hydroxyvitamin D concentration in newborn infants.

BACKGROUND Although no gold standard exists, liquid chromatography tandem mass spectrometry (LC-MS/MS) is a precise and accurate method for the analysis of plasma 25-hydroxyvitamin D (25(OH)D). Immunoassays are more readily available and require small volume sampling, ideal for infant testing. The objective was to compare two commercially available immunoassays for measuring circulating 25(OH)D concentration in infant plasma against LC-MS/MS. METHODS Capillary blood samples from 103 infants were analyzed for plasma 25(OH)D using an enzyme immunoassay (EIA, Octeia, IDS Ltd.) and radioimmunoassay (RIA, DiaSorin). Plasma 25(OH)D(3), C-3 epimer of 25(OH)D(3) (3-epi-25(OH)D(3)) and 24,25-dihydroxyvitamin D (24,25(OH)(2)D(3)) were measured on the same samples using LC-MS/MS. To establish whether plasma 24,25(OH)(2)D(3) or 3-epi-25(OH)D(3) interferes with these immunoassay results, the zero 25(OH)D calibrator from each assay kit was spiked with increasing amounts of 24,25(OH)(2)D(3) or 3-epi-25(OH)D(3). RESULTS Classifying infants below the common vitamin D status targets of 50 nmol/L and 75 nmol/L respectively, 58% and 99% fell below using the RIA, 19% and 56% with the EIA and 31% and 76% with LC-MS/MS. Compared to LC-MS/MS, both immunoassays showed poor Bland-Altman limits of agreement for 25(OH)D concentrations (RIA: limits of agreement -27 to +13%; EIA: -12 to +41%), and mountain plots (folded cumulative distribution) depicted significant skew and bias. Spiked 24,25(OH)2D3 concentrations, but not 3-epi-25(OH)D3, appeared as >100% of known values on the EIA but not on the RIA thus, suggesting that the EIA may cross-react with 24,25(OH)(2)D(3) to a greater extent than 3-epi-25(OH)D(3). CONCLUSION Two common immunoassays resulted in very different classifications of vitamin D status possibly related to the interference of other vitamin D metabolites. Based on these data, LC-MS/MS assessment of vitamin D status is recommended in young infants (4-6 weeks of age).

Efficacy of plant sterols is not influenced by dietary cholesterol intake in hypercholesterolemic individuals

Plant sterols (PSs) reduce plasma total and low-density lipoprotein cholesterol (LDL-C) levels by reducing cholesterol absorption; however, it is not known whether the level of dietary cholesterol intake has an impact on the efficacy of PSs on blood lipids. The purpose of this study was to determine the effect of high vs low dietary cholesterol levels on the lipid-lowering efficacy of free PSs. The study was a semirandomized, double-blind, crossover trial consisting of four 28-day feeding phases each separated by a 4-week washout period. Otherwise healthy hypercholesterolemic subjects (n = 22) consumed each of (a) low-cholesterol control (C(-)S(-)), (b) high-cholesterol control (C(+)S(-)), (c) 22 mg PSs per kilogram of body weight with a low-cholesterol diet (C(-)S(+)), and (d) 22 mg PSs per kilogram of body weight with a high-cholesterol diet (C(+)S(+)). Blood was drawn on the first and last 2 days of each phase to measure plasma total cholesterol, LDL-C, high-density lipoprotein cholesterol, and triacylglycerols as well as plasma campesterol and beta-sitosterol concentrations. Dietary cholesterol had no effect on PS efficacy as a cholesterol-lowering agent because no interaction was found between the 2 factors. However, dietary cholesterol and PS intake had significant independent effects on plasma total cholesterol, LDL-C, and high-density lipoprotein cholesterol levels. beta-Sitosterol levels in plasma increased (P < .0001) as a result of PS supplementation. Data from the present study indicate that, although PSs and dietary cholesterol exert independent effects on plasma cholesterol, PS efficacy is not affected by varying levels of cholesterol intake.