Ezetimibe Increases Endogenous Cholesterol Excretion in Humans.

OBJECTIVE: Ezetimibe improves cardiovascular outcomes when added to optimum statin treatment. It lowers low-density lipoprotein cholesterol and percent intestinal cholesterol absorption, but the exact cardioprotective mechanism is unknown. We tested the hypothesis that the dominant effect of ezetimibe is to increase the reverse transport of cholesterol from rapidly mixing endogenous cholesterol pool into the stool. APPROACH AND RESULTS: In a randomized, placebo-controlled, double-blind parallel trial in 24 healthy subjects with low-density lipoprotein cholesterol 100 to 200 mg/dL, we measured cholesterol metabolism before and after a 6-week treatment period with ezetimibe 10 mg/d or placebo. Plasma cholesterol was labeled by intravenous infusion of cholesterol-d7 in a lipid emulsion and dietary cholesterol with cholesterol-d5 and sitostanol-d4 solubilized in oil. Plasma and stool samples collected during a cholesterol- and phytosterol-controlled metabolic kitchen diet were analyzed by mass spectrometry. Ezetimibe reduced intestinal cholesterol absorption efficiency 30±4.3% (SE, P<0.0001) and low-density lipoprotein cholesterol 19.8±1.9% (P=0.0001). Body cholesterol pool size was unchanged, but fecal endogenous cholesterol excretion increased 66.6±12.2% (P<0.0001) and percent cholesterol excretion from body pools into the stool increased 74.7±14.3% (P<0.0001), whereas plasma cholesterol turnover rose 26.2±3.6% (P=0.0096). Fecal bile acids were unchanged. CONCLUSIONS: Ezetimibe increased the efficiency of reverse cholesterol transport from rapidly mixing plasma and tissue pools into the stool. Further work is needed to examine the potential relation of reverse cholesterol transport and whole body cholesterol metabolism to coronary events and the treatment of atherosclerosis. CLINICAL TRIALS REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01603758.

Endogenous Cholesterol Excretion Is Negatively Associated With Carotid Intima-Media Thickness in Humans.

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.

Multicenter performance evaluation of a second generation cortisol assay.

BACKGROUND: Untreated disorders of the adrenocortical system, such as Cushing's or Addison's disease, can be fatal, and accurate quantification of a patient's cortisol levels is vital for diagnosis. The objective of this study was to assess the analytical performance of a new fully-automated Elecsys® Cortisol II assay (second generation) to measure cortisol levels in serum and saliva. METHODS: Four European investigational sites assessed the intermediate precision and reproducibility of the Cortisol II assay (Roche Diagnostics) under routine conditions. Method comparisons of the Cortisol II assay vs. liquid chromatography-tandem mass spectrometry (LC-MS/MS), the gold standard for cortisol measurement, were performed. Cortisol reference ranges from three US sites were determined using samples from self-reported healthy individuals. RESULTS: The coefficients of variation (CVs) for repeatability, intermediate precision, and reproducibility for serum samples were ≤2.6%, ≤5.8%, and ≤9.5%, respectively, and for saliva were ≤4.4% and ≤10.9%, and ≤11.4%, respectively. Agreement between the Cortisol II assay and LC-MS/MS in serum samples was close, with a slope of 1.02 and an intercept of 4.473 nmol/L. Reference range samples were collected from healthy individuals (n=300) and serum morning cortisol concentrations (5-95th percentile) were 166.1-507 nmol/L and afternoon concentrations were 73.8-291 nmol/L. Morning, afternoon, and midnight saliva concentrations (95th percentile) were 20.3, 6.94, and 7.56 nmol/L, respectively. CONCLUSIONS: The Cortisol II assay had good precision over the entire measuring range and had excellent agreement with LC-MS/MS. This test was found suitable for routine diagnostic application and will be valuable for the diagnosis of adrenocortical diseases.

Altered Cholesterol Metabolism and Hypocholesterolemia in Patients with Single Ventricle following Fontan Palliation.

OBJECTIVE: To assess whether an abnormality in cholesterol absorption or synthesis may be associated with hypocholesterolemia in patients with single ventricle anatomy following Fontan palliation. STUDY DESIGN: This is a cross-sectional study of 21 patients with hypocholesterolemia following Fontan procedure and age/sex-matched healthy controls, with median age of 13.4 (IQR 10.6-16.1) years. Laboratory values of several biomarkers, including phytosterols and 5-α-cholestanol (for cholesterol absorption) and lathosterol (for cholesterol biosynthesis), as well as cholesterol levels, inflammatory markers, and indices of liver function were compared between patients following Fontan procedure and controls. RESULTS: The Fontan cohort had significantly lower total cholesterol (mean 117 ± SD 13.9, vs 128 ± 19.2 mg/dL, P = .03) and free cholesterol (35.5 ± 4.5 vs 39.2 ± 5.4 mg/dL, P = .02) compared with control patients. There was an increase in normalized 5-α-cholestanol (1.51 ± 0.6 vs 1.14 ± 0.37 µg/mL, P = .02), and a significantly lower lathosterol/5-α-cholestanol ratio (0.70 ± 0.38 vs 1.11 ± 0.76, P = .04). There was a strong correlation (r = 0.78, P < .0001) between lathosterol and cholesterol levels in the Fontan cohort, not seen in controls (r = 0.47, P = .04). The Fontan cohort also had significantly higher C-reactive protein, transaminases, total bilirubin, and gamma-glutamyl transferase levels. CONCLUSIONS: Patients with hypocholesterolemia following Fontan procedure have evidence of increased cholesterol absorption and decreased cholesterol synthesis. As cholesterol absorption efficiency is a regulated process, this finding suggests an upregulation of cholesterol absorption as a result of decreased cholesterol production. In the setting of elevated liver indices and possible inflammation, this finding supports a growing body of data suggesting development of liver disease in patients receiving Fontan.

Technical performance of a novel, fully automated electrochemiluminescence immunoassay for the quantitation of ß-amyloid (1-42) in human cerebrospinal fluid.

INTRODUCTION: Available assays for quantitation of the Alzheimer's disease (AD) biomarker amyloid-beta 1-42 (Aß [1-42]) in cerebrospinal fluid demonstrate significant variability and lack of standardization to reference measurement procedures (RMPs). We report analytical performance data for the novel Elecsys ß-amyloid (1-42) assay (Roche Diagnostics). METHODS: Lot-to-lot comparability was tested using method comparison. Performance parameters were measured according to Clinical & Laboratory Standards Institute (CLSI) guidelines. The assay was standardized to a Joint Committee for Traceability in Laboratory Medicine (JCTLM) approved RMP. RESULTS: Limit of quantitation was <11.28 pg/mL, and the assay was linear throughout the measuring range (200-1700 pg/mL). Excellent lot-to-lot comparability was observed (correlation coefficients [Pearson's r] >0.995; bias in medical decision area <2%). Repeatability coefficients of variation (CVs) were 1.0%-1.6%, intermediate CVs were 1.9%-4.0%, and intermodule CVs were 1.1%-3.9%. Estimated total reproducibility was 2.0%-5.1%. Correlation with the RMP was good (Pearson's r, 0.93). DISCUSSION: The Elecsys ß-amyloid (1-42) assay has high analytical performance that may improve biomarker-based AD diagnosis.

Preparation of intravenous cholesterol tracer using current good manufacturing practices.

Studies of human reverse cholesterol transport require intravenous infusion of cholesterol tracers. Because insoluble lipids may pose risk and because it is desirable to have consistent doses of defined composition available over many months, we investigated the manufacture of cholesterol tracer under current good manufacturing practice (CGMP) conditions appropriate for phase 1 investigation. Cholesterol tracer was prepared by sterile admixture of unlabeled cholesterol or cholesterol-d7 in ethanol with 20% Intralipid(®). The resulting material was filtered through a 1.2 micron particulate filter, stored at 4°C, and tested at time 0, 1.5, 3, 6, and 9 months for sterility, pyrogenicity, autoxidation, and particle size and aggregation. The limiting factor for stability was a rise in thiobarbituric acid-reacting substances of 9.6-fold over 9 months (P < 0.01). The emulsion was stable with the Z-average intensity-weighted mean droplet diameter remaining at 60 nm over 23 months. The zeta potential (a measure of negative surface charge protecting from aggregation) was unchanged at -36.2. Rapid cholesterol pool size was 25.3 ± 1.3 g. Intravenous cholesterol tracer was stable at 4°C for 9 months postproduction. CGMP manufacturing methods can be achieved in the academic setting and need to be considered for critical components of future metabolic studies.

Natural Dietary Phytosterols.

Most clinical phytosterol studies are performed by adding purified supplements to smaller phytosterol amounts present in the natural diet. However, natural dietary phytosterols themselves may also have important effects on cholesterol metabolism. Epidemiological work using food frequency questionnaires to estimate dietary intake suggest that extremes of normal consumption may be associated with 3-14% changes in LDL cholesterol. Standardized food databases do not have enough phytosterol values to allow calculation of phytosterol intake for individuals outside of specialized studies. Natural diets contain phytosterol amounts ranging from less than 60 mg/2000 kcal to over 500 mg/2000 kcal. Physiological studies in which whole body cholesterol metabolism is investigated show large effects of natural dietary phytosterols on cholesterol absorption efficiency, cholesterol biosynthesis and cholesterol excretion which exceed the magnitude of changes in LDL cholesterol. The dual effects of natural phytosterols on both LDL-C and whole body cholesterol metabolism need to be considered in relating them to potential protection from coronary heart disease risk.

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.

Human sodium/inositol cotransporter 2 (SMIT2) transports inositols but not glucose in L6 cells.

To characterize the function of the sodium/inositol symporter SMIT2 in skeletal muscle, human SMIT2 cDNA was transfected into L6 myoblasts using pcDNA3.1 expression vector. Compared with the pcDNA3.1 vector only transfection, this overexpression increased the uptake of [(3)H]D-chiro-inositol (DCI) by 159-fold. [(3)H]myo-Inositol uptake increased by 37-fold. In contrast, [(14)C]D-glucose, [(14)C]2-deoxy-D-glucose, or [(14)C]3-O-methyl-D-glucose uptake remained unchanged in the presence of either 0, 5.5, or 25 mM unlabeled glucose. The K(m) of DCI and myo-inositol for DCI uptake was 111.0 and 158.0 microM, respectively, whereas glucose competed for DCI uptake with a K(i) of 6.1 mM. Insulin treatment of non-transfected L6 cells (2 microM for 24 h) increased [(3)H]DCI specific uptake 18-fold. DCI transport is up regulated by insulin and competitively inhibited by millimolar levels of glucose. Therefore, expression and/or function of SMIT2, a high affinity transporter specific for DCI and myo-inositol, may be reduced in diabetes mellitus, insulin resistance and polycystic ovary syndrome causing the abnormal DCI metabolism observed in these conditions.

d- chiro-Inositol is absorbed but not synthesised in rodents.

d-chiro-inositol (DCI) and pinitol (1d-3-O-methyl-chiro-inositol) are distinctive inositols reported to possess insulin-mimetic properties. DCI-containing compounds are abundant in common laboratory animal feed. By GC-MS of 6 m-HCl hydrolysates, Purina Laboratory Rodent Diet 5001 (diet 5001) contained 0.23 % total DCI by weight with most found in the lucerne and soya meal components. In contrast, only traces of l-chiro-inositol were observed. The DCI moiety was present in a water-soluble non-ionic form of which most was shown to be pinitol. To measure the absorption of dietary inositols, rats were fed diet 5001 in a balance study or given purified pinitol or [2H6]DCI. More than 98 % of the total DCI fed to rats as diet 5001, purified pinitol or [2H6]DCI was absorbed from the gastrointestinal tract. Rats chronically on diet 5001 consumed 921 mumol total DCI/kg body weight per d but excreted less than 5.3 % in the stools and urine, suggesting that the bulk was metabolised. The levels of pinitol or DCI in plasma, stools or urine remained relatively stable in mice fed Purina PicoLab Rodent Diet 20 5053 over a 5-week period, whereas these values declined to very low levels in mice fed a pinitol/DCI-deficient chemically defined diet. To test whether DCI was synthesised or converted from myo-inositol, mice were treated with heavy water or [2H6]myo-inositol. DCI was neither synthesised endogenously from 2H-labelled water nor converted from [2H6]myo-inositol. DCI and pinitol in rodents appear to be derived solely from the diet.

Low dose chloroquine decreases insulin resistance in human metabolic syndrome but does not reduce carotid intima-media thickness.

BACKGROUND: Metabolic syndrome, an obesity-related condition associated with insulin resistance and low-grade inflammation, leads to diabetes, cardiovascular diseases, cancer, osteoarthritis, and other disorders. Optimal therapy is unknown. The antimalarial drug chloroquine activates the kinase ataxia telangiectasia mutated (ATM), improves metabolic syndrome and reduces atherosclerosis in mice. To translate this observation to humans, we conducted two clinical trials of chloroquine in people with the metabolic syndrome. METHODS: Eligibility included adults with at least 3 criteria of metabolic syndrome but who did not have diabetes. Subjects were studied in the setting of a single academic health center. The specific hypothesis: chloroquine improves insulin sensitivity and decreases atherosclerosis. In Trial 1, the intervention was chloroquine dose escalations in 3-week intervals followed by hyperinsulinemic euglycemic clamps. Trial 2 was a parallel design randomized clinical trial, and the intervention was chloroquine, 80 mg/day, or placebo for 1 year. The primary outcomes were clamp determined-insulin sensitivity for Trial 1, and carotid intima-media thickness (CIMT) for Trial 2. For Trial 2, subjects were allocated based on a randomization sequence using a protocol in blocks of 8. Participants, care givers, and those assessing outcomes were blinded to group assignment. RESULTS: For Trial 1, 25 patients were studied. Chloroquine increased hepatic insulin sensitivity without affecting glucose disposal, and improved serum lipids. For Trial 2, 116 patients were randomized, 59 to chloroquine (56 analyzed) and 57 to placebo (51 analyzed). Chloroquine had no effect on CIMT or carotid contrast enhancement by MRI, a pre-specified secondary outcome. The pre-specified secondary outcomes of blood pressure, lipids, and activation of JNK (a stress kinase implicated in diabetes and atherosclerosis) were decreased by chloroquine. Adverse events were similar between groups. CONCLUSIONS: These findings suggest that low dose chloroquine, which improves the metabolic syndrome through ATM-dependent mechanisms in mice, modestly improves components of the metabolic syndrome in humans but is unlikely to be clinically useful in this setting.Trial registration ClinicalTrials.gov (NCT00455325, NCT00455403), both posted 03 April 2007.

First international descriptive and interventional survey for cholesterol and non-cholesterol sterol determination by gas- and liquid-chromatography-Urgent need for harmonisation of analytical methods.

Serum concentrations of lathosterol, the plant sterols campesterol and sitosterol and the cholesterol metabolite 5α-cholestanol are widely used as surrogate markers of cholesterol synthesis and absorption, respectively. Increasing numbers of laboratories utilize a broad spectrum of well-established and recently developed methods for the determination of cholesterol and non-cholesterol sterols (NCS). In order to evaluate the quality of these measurements and to identify possible sources of analytical errors our group initiated the first international survey for cholesterol and NCS. The cholesterol and NCS survey was structured as a two-part survey which took place in the years 2013 and 2014. The first survey part was designed as descriptive, providing information about the variation of reported results from different laboratories. A set of two lyophilized pooled sera (A and B) was sent to twenty laboratories specialized in chromatographic lipid analysis. The different sterols were quantified either by gas chromatography-flame ionization detection, gas chromatography- or liquid chromatography-mass selective detection. The participants were requested to determine cholesterol and NCS concentrations in the provided samples as part of their normal laboratory routine. The second part was designed as interventional survey. Twenty-two laboratories agreed to participate and received again two different lyophilized pooled sera (C and D). In contrast to the first international survey, each participant received standard stock solutions with defined concentrations of cholesterol and NCS. The participants were requested to use diluted calibration solutions from the provided standard stock solutions for quantification of cholesterol and NCS. In both surveys, each laboratory used its own internal standard (5α-cholestane, epicoprostanol or deuterium labelled sterols). Main outcome of the survey was, that unacceptably high interlaboratory variations for cholesterol and NCS concentrations are reported, even when the individual laboratories used the same calibration material. We discuss different sources of errors and recommend all laboratories analysing cholesterol and NCS to participate in regular quality control programs.

Greek hyperinsulinemic women, with or without polycystic ovary syndrome, display altered inositols metabolism.

UNLABELLED: BACKGROUND We have shown that American women with polycystic ovary syndrome (PCOS) have decreased glucose-stimulated release of a putative mediator of insulin action, D-chiro-inositol (DCI)-containing inositolphosphoglycan (DCI-IPG), and increased urinary clearance of DCI (uCl(DCI)), which was associated with hyperinsulinemia. METHODS: DCI levels and the release of insulin and DCI-IPG during an oral glucose tolerance test (AUCs) were assessed in 27 Greek PCOS and 10 normal Greek women. RESULTS: PCOS women were heavier than controls (BMI = 28.4 versus 23.7 kg/m(2), P = 0.05) with higher waist-to-hip ratios (WHR = 0.78 versus 0.71, P = 0.009) and increased free testosterone (P = 0.048) and AUC(insulin) (P = 0.04). In PCOS women, incremental AUC(DCI-IPG) was significantly decreased by 59% (2158 versus 5276%.min, P = 0.01), even after correction for BMI and WHR. Finally, increased uCl(DCI) (r = 0.35, P = 0.04) and decreased AUC(DCI-IPG) (r = 0.46, P = 0.004) were significantly associated with hyperinsulinemia in all women together, even after correction for BMI and WHR (Ps = 0.02 and 0.007), and regardless of PCOS status. CONCLUSIONS: Greek women, with or without PCOS, display increased uCl(DCI) and decreased AUC(DCI-IPG) in association with higher insulin levels but independent of adiposity. Increased clearance of inositols might reduce tissue availability of DCI and decrease the release of DCI-IPG mediator, which could contribute to insulin resistance and compensatory hyperinsulinemia in Greek women, as previously described in American women.

Vitamin D3 supplementation decreases a unique circulating monocyte cholesterol pool in patients with type 2 diabetes.

Cross-sectional studies indicate consistent associations between low 25(OH)D concentration and increased risk of cardiovascular disease (CVD), but results of randomized control trials (RCTs) are mixed. However, the majority of the RCTs do not focus on type 2 diabetics, potentially obscuring the effects of vitamin D in this population. In vitro 1,25(OH)2D3 downregulates macrophage cholesterol deposition, but the in vivo effects are unknown. To explore potential mechanisms of the effects of vitamin D on CVD risk in patients with type 2 diabetes, we isolated monocytes in a subset of 26 patients from our RCT of diabetics with baseline serum 25(OH)D <25ng/mL randomized to vitamin D3 4000 IU/day or placebo for 4 months. Upon enrollment, the mean 25(OH)D level was 17ng/mL, which increased to 36ng/mL after vitamin D and remained unchanged in the placebo group. Before randomization, groups demonstrated similar mean hemoglobin A1c and plasma lipids levels, none of which was significantly altered by vitamin D supplementation. Moreover, assessment of oxidized LDL uptake in monocytes cultured in the patient's own serum before vs. after treatment resulted in >50% reduction in the vitamin D group with no change in the placebo group. This was mediated through suppression of endoplasmic reticulum stress and scavenger receptor CD36 protein expression. The reduction in monocyte cholesterol uptake was reflected in a 19% decrease in total monocyte cholesterol content. Interestingly, cross-sectional analysis of circulating monocytes from vitamin D-deficient vs. sufficient diabetic patients revealed 8-fold higher cholesteryl ester content, confirming the capacity of these monocytes to uptake and carry cholesterol in the circulation. This study identifies a unique circulating cholesterol pool within monocytes that is modulated by vitamin D and has the potential to contribute to CVD in type 2 diabetes.

Progress and perspectives in plant sterol and plant stanol research.

Current evidence indicates that foods with added plant sterols or stanols can lower serum levels of low-density lipoprotein cholesterol. This review summarizes the recent findings and deliberations of 31 experts in the field who participated in a scientific meeting in Winnipeg, Canada, on the health effects of plant sterols and stanols. Participants discussed issues including, but not limited to, the health benefits of plant sterols and stanols beyond cholesterol lowering, the role of plant sterols and stanols as adjuncts to diet and drugs, and the challenges involved in measuring plant sterols and stanols in biological samples. Variations in interindividual responses to plant sterols and stanols, as well as the personalization of lipid-lowering therapies, were addressed. Finally, the clinical aspects and treatment of sitosterolemia were reviewed. Although plant sterols and stanols continue to offer an efficacious and convenient dietary approach to cholesterol management, long-term clinical trials investigating the endpoints of cardiovascular disease are still lacking.

Phytosterols, cholesterol absorption and healthy diets.

The purpose of this review is to outline the emerging role of dietary phytosterols in human health. Dietary saturated fat, cholesterol and fiber are currently emphasized in the reduction of low-density lipoprotein cholesterol levels. However, other dietary components such as phytosterols may have equivalent or even larger effects on circulating cholesterol and need further study with respect to the potential for coronary heart disease risk reduction. Phytosterol effects were not considered in classic fat-exchange clinical trials and may account for some of the differences attributed to the food fats studied. Phytosterols reduce cholesterol absorption while being poorly absorbed themselves and the effects can be studied in human subjects in single-meal tests using stable isotopic tracers. Because phytosterols are insoluble and biologically inactive when purified, careful attention needs to be given to ensuring that commercial supplement products are rendered bioavailable by dissolution in fat or by emulsification. Recent work shows that phytosterols in natural food matrices are also bioactive. The retention of phytosterols during food manufacturing and the use of foods with high phytosterol content may constitute an alternative to the use of supplements.

Altered D-chiro-inositol urinary clearance in women with polycystic ovary syndrome.

OBJECTIVE: Evidence suggests that some actions of insulin are effected by inositolphosphoglycan (IPG) mediators. We hypothesize that a deficiency in D-chiro-inositol (DCI) and/or a DCI-containing IPG (DCI-IPG) may contribute to insulin resistance in humans. RESEARCH DESIGN AND METHODS: To assess this possibility in polycystic ovary syndrome (PCOS), we determined insulin sensitivity (Si by frequently sampled intravenous glucose tolerance test), plasma and urinary DCI and myo-inositol (MYO) levels (by gas chromatography/mass spectrometry), and the release of insulin and DCI-IPG during the oral glucose tolerance test (area under the curve [AUC]) in 23 women with PCOS and 26 normal women. RESULTS: Women with PCOS were heavier than control subjects (P = 0.002 for BMI), but also had decreased Si (P < 0.001) and increased AUC(insulin) (P < 0.001) compared with normal women, even when corrected for BMI. The urinary clearance of DCI (uCl(DCI)) was increased almost sixfold in PCOS compared with normal women (P = 0.001), but not MYO clearance (P = 0.10). uCl(DCI) correlated inversely with Si when all women were analyzed together (n = 49, r = -0.50, P < 0.001) and was one of the three best independent parameters predicting Si. Finally, the ratio of AUC(DCI-IPG) to AUC(insulin) was decreased threefold in women with PCOS (P < 0.001). CONCLUSIONS: uCl(DCI) is inversely correlated with insulin sensitivity in women and is a strong independent predictor of insulin resistance in multivariate models. PCOS, which is characterized by insulin resistance, is associated with a selective increase in uCl(DCI) and impaired DCI-IPG release in response to insulin. These findings are consistent with a defect in tissue availability or utilization of DCI in PCOS that may contribute to the insulin resistance of the syndrome.

Multicenter evaluation of analytical characteristics of the Elecsys® Periostin immunoassay.

OBJECTIVE: The multifunctional cytokine IL-13 is thought to play a central role in Type 2 inflammation in asthma. Serum periostin has been explored as a candidate biomarker for evaluating IL-13 activity in the airway. We describe the technical performance characteristics of a novel, fully automated immunoassay for the determination of periostin in serum. DESIGN AND METHODS: Limit of blank [LoB], limit of detection [LoD] and limit of quantitation [LoQ], linearity, precision and reproducibility across sites and lots were evaluated according to Clinical and Laboratory Standards Institute guidelines. Interferences and sample stability were also investigated. RESULTS: The pre-specified values for LoB (2ng/mL), LoD (4ng/mL) and LoQ (10ng/mL) were met. The assay was linear throughout the measuring range (10-160ng/mL) with recoveries within ±10% of target at concentrations >30ng/mL and within ±3ng/mL at concentrations ≤30ng/mL. Recovered periostin concentrations were also within ±10% of target in presence of 43 potentially interfering substances and drugs. Samples were stable across various storage conditions and durations (24h at room temperature, 7days at 4°C, 12weeks at -20°C, and 3 freeze/thaw cycles). Repeatability experiments resulted in CVs across samples and controls ranging from 0.9-1.5%. Intermediate precision was 1.2-1.7% and reproducibility including 3 testing sites and 3 reagent lots was 1.7-3.1%. The final assay correlates to the assay version used in previous clinical trials (Pearson's r=0.998, bias at 50ng/mL=1.2%). CONCLUSION: The performance evaluation of the Elecsys® Periostin immunoassay including a multicenter precision analysis demonstrated that the assay is suitable for measuring serum periostin at clinically important concentrations around 50ng/mL.

Effect of plant stanol tablets on low-density lipoprotein cholesterol lowering in patients on statin drugs.

The objective of this study was to show that plant sterols in tablet form provide additional low-density lipoprotein (LDL) cholesterol lowering for patients on statin therapy. Dispersible phytosterol tablets were tested in a double-blind, placebo-controlled, parallel clinical trial. Twenty-six patients who were following the American Heart Association Heart Healthy Diet and on long-term statin therapy were studied for 9 weeks. After 3 weeks of placebo treatment, the subjects were randomized to receive either 1.8 g of soy stanols or placebo for 6 weeks in addition to their usual statin regimen. Stanol tablets reduced LDL cholesterol 9.1% (p = 0.007) or 12.2 mg/dl. Total cholesterol was reduced by 12.9 mg/dl (p = 0.03). A strong inverse correlation (r(s) = -0.82, p = 0.0007) was found between the baseline LDL cholesterol and the percent change in LDL cholesterol observed after stanol treatment. The additional LDL cholesterol lowering with stanol/lecithin tablets provided a potential adjunctive therapy for patients who have not reached their target LDL cholesterol goal during statin therapy.