Genome-wide association study and meta-analysis of phytosterols identifies a novel locus for serum levels of campesterol.

Sitosterolemia is a rare inherited disorder caused by mutations in the ABCG5/ABCG8 genes. These genes encode proteins involved in the transport of plant sterols. Mutations in these genes lead to decreased excretion of phytosterols, which can accumulate in the body and lead to a variety of health problems, including premature coronary artery disease. We conducted the first genome-wide association study (GWAS) in the Middle East/North Africa population to identify genetic determinants of plant sterol levels in Qatari people. GWAS was performed on serum levels of ß-sitosterol and campesterol using the Metabolon platform from Qatar Biobank (QBB) and genome sequence data provided by Qatar Genome Program. A trans-ancestry meta-analysis of data from our Qatari cohort with summary statistics from a previously published large cohort (9758 subjects) of European ancestry was conducted. Using conditional analysis, we identified two independent single nucleotide polymorphisms associated with ß-sitosterol (rs145164937 and rs4299376), and two others with campesterol (rs7598542 and rs75901165) in the Qatari population in addition to previously reported variants. All of them map to the ABCG5/8 locus except rs75901165 which is located within the Intraflagellar Transport 43 (IFT43) gene. The meta-analysis replicated most of the reported variants, and our study provided significant support for the association of variants in SCARB1 and ABO with sitosterolemia. Evaluation of a polygenic risk score devised from European GWAS data showed moderate performance when applied to QBB (adjusted-R2 = 0.082). These findings provide new insights into the genetic architecture of phytosterol metabolism while showing the importance including under-represented populations in future GWAS studies.

Dried blood spot-based free sterol signatures in sitosterolemia diagnostics.

BACKGROUND: Sitosterolemia is a rare inherited lipid metabolic disorder characterized by increased levels of plant sterols and accelerated atherosclerosis. Although early detection is beneficial for the prevention of disease progression, it is largely underdiagnosed by routine screening based on conventional lipid profiles. MATERIALS AND METHODS: A gas chromatography-mass spectrometry (GC-MS)-based profiling has been developed and validated to measure the levels of biologically active free sterols, including five endogenous sterols and three plant sterols (sitosterol, campesterol, and stigmasterol) in dried blood spot (DBS). RESULTS: Within- and between-run precisions were 1.4-11.1 % and 2.2-14.1 %, respectively, while the accuracies were all 86.3 âˆ¼ 121.9 % with the correlation coefficients (r2) > 0.988 for all the sterols. In the patients (four girls and two boys, 6.5 ± 2.8 years), sitosterol levels were significantly increased, with an optimal cut-off value of 2.5 µg/mL distinguishing them from ninety-three age-matched healthy children. A cut-off value of 31.9 µg/mL differentiated the patients from six ABCG5/ABCG8 heterozygous carriers. In addition, the molecular ratios of sitosterol to cholesterol, desmosterol, and 7-dehydrocholesterol provided excellent cut-off values of 26.3, 67.6, and 21.6, respectively, to distinguish patients from both healthy controls and heterozygous carriers. CONCLUSIONS: The novel DBS-based GC-MS profiling of free sterols accurately identified patients with sitosterolemia, with a performance comparable to that of a serum assay. The DBS profiling could be more feasible method in clinical practice as well as population screening programs, and it can provide diagnostic cut-off values for individual plant sterols.

Phytosterols in human serum as measured using a liquid chromatography tandem mass spectrometry.

Phytosterols are lipophilic compounds found in plants with structural similarity to mammalian cholesterol. They cannot be endogenously produced by mammals and therefore always originate from diet. There has been increased interest in dietary phytosterols over the last few decades due to their association with a variety of beneficial health effects including low-density lipoprotein cholesterol lowering, anti-inflammatory and anti-cancerous effects. They are proposed as potential moderators for diseases associated with the central nervous system where cholesterol homeostasis is found to be imperative (multiple sclerosis, dementia, etc.) due to their ability to reach the brain. Here we utilised an enzyme-assisted derivatisation for sterol analysis (EADSA) in combination with a liquid chromatography tandem mass spectrometry (LC-MSn) to characterise phytosterol content in human serum. As little as 100 fg of plant sterol was injected on a reversed phase LC column. The method allows semi-quantitative measurements of phytosterols and their derivatives simultaneously with measurement of cholesterol metabolites. The identification of phytosterols in human serum was based on comparison of their LC retention times and MS2, MS3 spectra with a library of authentic standards. Free campesterol serum concentration was in the range from 0.30-4.10 µg/mL, ß-sitosterol 0.16-3.37 µg/mL and fucosterol was at lowest concentration range from 0.05-0.38 µg/mL in ten individuals. This analytical methodology could be applied to the analysis of other biological fluids and tissues.

Platelet proteomic profiling in sitosterolemia suggests thrombocytopenia is driven by lipid disorder and not platelet aberrations.

ABSTRACT: Sitosterolemia is a rare autosomal recessive genetic disorder in which patients develop hypercholesterolemia and may exhibit abnormal hematologic and/or liver test results. In this disease, dysfunction of either ABCG5 or ABCG8 results in the intestinal hyperabsorption of all sterols, including cholesterol and, more specifically, plant sterols or xenosterols, as well as in the impaired ability to excrete xenosterols into the bile. It remains unknown how and why some patients develop hematologic abnormalities. Only a few unrelated patients with hematologic abnormalities at the time of diagnosis have been reported. Here, we report on 2 unrelated pedigrees who were believed to have chronic immune thrombocytopenia as their most prominent feature. Both consanguineous families showed recessive gene variants in ABCG5, which were associated with the disease by in silico protein structure analysis and clinical segregation. Hepatosplenomegaly was absent. Thrombopoietin levels and megakaryocyte numbers in the bone marrow were normal. Metabolic analysis confirmed the presence of strongly elevated plasma levels of xenosterols. Potential platelet proteomic aberrations were longitudinally assessed following dietary restrictions combined with administration of the sterol absorption inhibitor ezetimibe. No significant effects on platelet protein content before and after the onset of treatment were demonstrated. Although we cannot exclude that lipotoxicity has a direct and platelet-specific impact in patients with sitosterolemia, our data suggest that thrombocytopenia is neither caused by a lack of megakaryocytes nor driven by proteomic aberrations in the platelets themselves.

Genome-wide meta-analysis of phytosterols reveals five novel loci and a detrimental effect on coronary atherosclerosis.

Phytosterol serum concentrations are under tight genetic control. The relationship between phytosterols and coronary artery disease (CAD) is controversially discussed. We perform a genome-wide meta-analysis of 32 phytosterol traits reflecting resorption, cholesterol synthesis and esterification in six studies with up to 9758 subjects and detect ten independent genome-wide significant SNPs at seven genomic loci. We confirm previously established associations at ABCG5/8 and ABO and demonstrate an extended locus heterogeneity at ABCG5/8 with different functional mechanisms. New loci comprise HMGCR, NPC1L1, PNLIPRP2, SCARB1 and APOE. Based on these results, we perform Mendelian Randomization analyses (MR) revealing a risk-increasing causal relationship of sitosterol serum concentrations and CAD, which is partly mediated by cholesterol. Here we report that phytosterols are polygenic traits. MR add evidence of both, direct and indirect causal effects of sitosterol on CAD.

Plasma oxyphytosterols most likely originate from hepatic oxidation and subsequent spill-over in the circulation.

We evaluated oxyphytosterol (OPS) concentrations in plasma and various tissues of two genetically modified mouse models with either increased cholesterol (apoE KO mice) or increased cholesterol and plant sterol (PS) concentrations (apoExABCG8 dKO mice). Sixteen female apoE KO and 16 dKO mice followed the same standard, low OPS-chow diet. Animals were euthanized at 36 weeks to measure PS and OPS concentrations in plasma, brain, liver and aortic tissue. Cholesterol and oxysterol (OS) concentrations were analyzed as reference for sterol oxidation in general. Plasma campesterol (24.1 ± 4.3 vs. 11.8 ± 3.0 mg/dL) and sitosterol (67.4 ± 12.7 vs. 4.9 ± 1.1 mg/dL) concentrations were severely elevated in the dKO compared to the apoE KO mice (p < 0.001). Also, in aortic and brain tissue, PS levels were significantly elevated in dKO. However, plasma, aortic and brain OPS concentrations were comparable or even lower in the dKO mice. In contrast, in liver tissue, both PS and OPS concentrations were severely elevated in the dKO compared to apoE KO mice (sum OPS: 7.4 ± 1.6 vs. 4.1 ± 0.8 ng/mg, p < 0.001). OS concentrations followed cholesterol concentrations in plasma and all tissues suggesting ubiquitous oxidation. Despite severely elevated PS concentrations, OPS concentrations were only elevated in liver tissue, suggesting that OPS are primarily formed in the liver and plasma concentrations originate from hepatic spill-over into the circulation.

Plant Sterol-Poor Diet Is Associated with Pro-Inflammatory Lipid Mediators in the Murine Brain.

Plant sterols (PSs) cannot be synthesized in mammals and are exclusively diet-derived. PSs cross the blood-brain barrier and may have anti-neuroinflammatory effects. Obesity is linked to lower intestinal uptake and blood levels of PSs, but its effects in terms of neuroinflammation-if any-remain unknown. We investigated the effect of high-fat diet-induced obesity on PSs in the brain and the effects of the PSs campesterol and ß-sitosterol on in vitro microglia activation. Sterols (cholesterol, precursors, PSs) and polyunsaturated fatty acid-derived lipid mediators were measured in the food, blood, liver and brain of C57BL/6J mice. Under a PSs-poor high-fat diet, PSs levels decreased in the blood, liver and brain (>50%). This effect was reversible after 2 weeks upon changing back to a chow diet. Inflammatory thromboxane B2 and prostaglandin D2 were inversely correlated to campesterol and ß-sitosterol levels in all brain regions. PSs content was determined post mortem in human cortex samples as well. In vitro, PSs accumulate in lipid rafts isolated from SIM-A9 microglia cell membranes. In summary, PSs levels in the blood, liver and brain were associated directly with PSs food content and inversely with BMI. PSs dampen pro-inflammatory lipid mediators in the brain. The identification of PSs in the human cortex in comparable concentration ranges implies the relevance of our findings for humans.

Effects of an 8-week aerobic exercise program on plasma markers for cholesterol absorption and synthesis in older overweight and obese men.

BACKGROUND: Increased physical activity is inversely related to the risk to develop cardiovascular disease (CVD). In a recent systematic review, it was reported that CVD patients had an increased cholesterol absorption and a decreased synthesis as compared with control participants. As increased physical activity levels reduce CVD risk, we hypothesized that exercise training will reduce cholesterol absorption and increase endogenous cholesterol synthesis in older overweight and obese men. METHODS: A randomized, controlled, crossover trial was performed. Seventeen apparently healthy older overweight and obese men were randomized to start with an aerobic exercise or no-exercise control period for 8 weeks, separated by 12 weeks washout. Fasting serum total cholesterol (TC) and non-cholesterol sterol concentrations were measured at baseline, and after 4 and 8 weeks. RESULTS: The aerobic exercise program did not affect serum TC concentrations. In addition, exercise did not affect TC-standardized serum concentrations of sitosterol and cholestanol that are markers for cholesterol absorption. However, a trend for reduced TC-standardized campesterol concentrations, which is another validated marker for cholesterol absorption, was observed as compared with control. Lathosterol concentrations, reflecting cholesterol synthesis, did not differ between both periods. CONCLUSIONS: Aerobic exercise training for 8 weeks did not lower serum TC concentrations in older overweight and obese men, but a trend towards a decrease in the cholesterol absorption marker campesterol was found. The cholesterol synthesis marker lathosterol did not change. TRIAL REGISTRATION: posted on www.clinicaltrials.gov as NCT03272061 on 7 September 2017.

Genetic basis and hematologic manifestations of sitosterolemia in a group of Turkish patients.

BACKGROUND: Sitosterolemia is a rare lipid disorder caused by mutations in adenosine triphosphate-binding cassette genes (ABCG) 5 and 8. OBJECTIVE: To evaluate the phenotypic/genotypic features of sitosterolemia in a group of Turkish patients. METHODS: Seven probands with unexplained hematologic abnormalities and their 13 relatives were enrolled. Sterol levels were measured by gas chromatography and genetic studies were performed using Sanger sequencing. Individuals were diagnosed with sitosterolemia if they were found to have frankly elevated sitosterol level >15 µg/mL and/or pathogenic variants of the ABCG5/ABCG8. RESULTS: The seven probands and their six relatives  were diagnosed with frank sitosterolemia, and all these patients had hematologic abnormalities. The remaining seven relatives were asymptomatic heterozygous carriers. Three novel variants in the ABCG5 gene (c.161G>A, c.1375C>T, IVS10-1G>T), one novel variant in the ABCG8 gene (c.1762G>C) and one known variant in the ABCG5 gene (c.1336 C>T) were identified. No variant was identified in one case. The mean sitosterol level was significantly higher and mean platelet count was significantly lower in patients with homozygous variants compared to heterozygous variants (p<0.05, for all). Diets low in plant sterols were recommended for 13 symptomatic cases. Four homozygotes received ezetimibe, and their splenomegaly, anemia, and thrombocytopenia completely resolved except one. CONCLUSION: The five pathogenic variants identified in this study indicate the genetic heterogeneity of sitosterolemia in Turkish population. Patients with unexplained hematologic abnormalities (specifically macrothrombocytopenia) should have their sterol level measured as initial testing. Ezetimibe can be a good choice for sitosterolemia.

Severe Genotype, Pancreatic Insufficiency and Low Dose of Pancreatic Enzymes Associate with Abnormal Serum Sterol Profile in Cystic Fibrosis.

BACKGROUND: Several factors could lead to lipid disturbances observed in cystic fibrosis (CF). This study aimed to assess sterol homeostasis in CF and define potential exogenous and endogenous determinants of lipid dysregulation. METHODS: The study involved 55 CF patients and 45 healthy subjects (HS). Sterol concentrations (µg/dL) were measured by gas chromatography/mass spectrometry. CF was characterised by lung function, pancreatic status, liver disease and diabetes coexistence, Pseudomonas aeruginosa colonisation and BMI. CFTR genotypes were classified as severe or other. RESULTS: Campesterol and ß-sitosterol concentrations were lower (p = 0.0028 and p < 0.0001, respectively) and lathosterol levels (reflecting endogenous cholesterol biosynthesis) were higher (p = 0.0016) in CF patients than in HS. Campesterol and ß-sitosterol concentrations were lower in patients with a severe CFTR genotype, pancreatic insufficiency and lower pancreatic enzyme dose (lipase units/gram of fat). In multiple regression analyses, ß-sitosterol and campesterol concentrations were predicted by genotype and pancreatic insufficiency, whereas cholesterol and its fractions were predicted by phytosterol concentrations, age, dose of pancreatic enzymes, nutritional status and genotype. CONCLUSIONS: Independent determinants of lipid status suggest that malabsorption and pancreatic enzyme supplementation play a significant role in sterol abnormalities. The measurement of campesterol and ß-sitosterol concentrations in CF patients may serve for the assessment of the effectiveness of pancreatic enzyme replacement therapy and/or compliance, but further research is required.

Markers of cholesterol synthesis are elevated in adolescents and young adults with type 2 diabetes.

BACKGROUND: Changes in cholesterol absorption and cholesterol synthesis may promote dyslipidemia and cardiovascular disease in individuals with type 2 diabetes mellitus (T2DM). OBJECTIVE: To assess cholesterol synthesis and absorption in lean individuals, obese individuals, and individuals with T2DM. METHODS: We measured lathosterol and lanosterol (markers of cholesterol synthesis) as well as campesterol and ß-sitosterol (markers of cholesterol absorption) in the serum of 15 to 26 years old individuals with T2DM (n = 95), as well as their lean (n = 98) and obese (n = 92) controls. RESULTS: Individuals with T2DM showed a 51% increase in lathosterol and a 65% increase in lanosterol compared to lean controls. Similarly, obese individuals showed a 31% increase in lathosterol compared to lean controls. Lathosterol and lanosterol were positively correlated with body mass index, fasting insulin and glucose, serum triglycerides, and C-reactive protein, and negatively correlated with HDL-cholesterol. In contrast, campesterol and ß-sitosterol were not altered in individuals with T2DM. Moreover, campesterol and ß-sitosterol were negatively correlated with body mass index, fasting insulin, and C-reactive protein and were positively correlated with HDL-cholesterol. CONCLUSIONS: Adolescents and young adults with T2DM show evidence of increased cholesterol synthesis compared to non-diabetic lean controls. These findings suggest that T2DM may promote cardiovascular disease by increasing cholesterol synthesis, and provide additional rationale for the use of cholesterol synthesis inhibitors in this group.

A case of ezetimibe-effective hypercholesterolemia with a novel heterozygous variant in ABCG5.

Sitosterolemia is caused by homozygous or compound heterozygous gene mutations in either ATP-binding cassette subfamily G member 5 (ABCG5) or 8 (ABCG8). Since ABCG5 and ABCG8 play pivotal roles in the excretion of neutral sterols into feces and bile, patients with sitosterolemia present elevated levels of serum plant sterols and in some cases also hypercholesterolemia. A 48-year-old woman was referred to our hospital for hypercholesterolemia. She had been misdiagnosed with familial hypercholesterolemia at the age of 20 and her serum low-density lipoprotein cholesterol (LDL-C) levels had remained about 200-300 mg/dL at the former clinic. Although the treatment of hydroxymethylglutaryl-CoA (HMG-CoA) reductase inhibitors was ineffective, her serum LDL-C levels were normalized by ezetimibe, a cholesterol transporter inhibitor. We noticed that her serum sitosterol and campesterol levels were relatively high. Targeted analysis sequencing identified a novel heterozygous ABCG5 variant (c.203A>T; p.Ile68Asn) in the patient, whereas no mutations were found in low-density lipoprotein receptor (LDLR), proprotein convertase subtilisin/kexin type 9 (PCSK9), or Niemann-Pick C1-like intracellular cholesterol transporter 1 (NPC1L1). While sitosterolemia is a rare disease, a recent study has reported that the incidence of loss-of-function mutation in the ABCG5 or ABCG8 gene is higher than we thought at 1 in 220 individuals. The present case suggests that serum plant sterol levels should be examined and ezetimibe treatment should be considered in patients with hypercholesterolemia who are resistant to HMG-CoA reductase inhibitors.

The coronary artery calcium score is linked to plasma cholesterol synthesis and absorption markers: Brazilian Longitudinal Study of Adult Health.

It is controversial whether atherosclerosis is linked to increased intestinal cholesterol absorption or synthesis in humans. The aim of the present study was to relate atherosclerosis to the measurements of plasma markers of cholesterol synthesis (desmosterol, lathosterol) and absorption (campesterol, sitosterol). In healthy male (n=344), non-obese, non-diabetics, belonging to the city of São Paulo branch of the Brazilian Longitudinal Study of Adult Health (ELSA-Brasil), we measured in plasma these non-cholesterol sterol markers, together with their anthropometric, dietary parameters, traditional atherosclerotic risk factors, and blood chemistry, coronary arterial calcium score (CAC), and ultrasonographically measured common carotid artery intima-media thickness (CCA-IMT). Cases with CAC>zero had the following parameters higher than cases with CAC = zero: age, waist circumference (WC), plasma total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-C), and non-high density lipoprotein-cholesterol (non HDL-C). Plasma desmosterol and campesterol, duly corrected for TC, age, body mass index (BMI), waist circumference (WC), hypertension, smoking, and the homeostasis model assessment-insulin resistance (HOMA-IR) correlated with CAC, but not with CCA-IMT. The latter related to increased age, BMI, waist circumference (WC), and systolic blood pressure (SBP). Plasma HDL-C concentrations did not define CAC or CCA-IMT degrees, although in relation to the lower tertile of HDL-C in plasma the higher tertile of HDL-C had lower HOMA-IR and concentration of a cholesterol synthesis marker (desmosterol). Present work indicated that increased cholesterol synthesis and absorption represent primary causes of CAD, but not of the common carotid artery atherosclerosis.

Association of Dietary Phytosterols with Cardiovascular Disease Biomarkers in Humans.

Cardiovascular disease (CVD) is a leading cause of death worldwide. Elevated concentrations of serum total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) are major lipid biomarkers that contribute to the risk of CVD. Phytosterols well known for their cholesterol-lowering ability, are non-nutritive compounds that are naturally found in plant-based foods and can be classified into plant sterols and plant stanols. Numerous clinical trials demonstrated that 2 g phytosterols per day have LDL-C lowering efficacy ranges of 8-10%. Some observational studies also showed an inverse association between phytosterols and LDL-C reduction. Beyond the cholesterol-lowering beneficial effects of phytosterols, the association of phytosterols with CVD risk events such as coronary artery disease and premature atherosclerosis in sitosterolemia patients have also been reported. Furthermore, there is an increasing demand to determine the association of circulating phytosterols with vascular health biomarkers such as arterial stiffness biomarkers. Therefore, this review aims to examine the ability of phytosterols for CVD risk prevention by reviewing the current data that looks at the association between dietary phytosterols intake and serum lipid biomarkers, and the impact of circulating phytosterols level on vascular health biomarkers. The clinical studies in which the impact of phytosterols on vascular function is investigated show minor but beneficial phytosterols effects over vascular health. The aforementioned vascular health biomarkers are pulse wave velocity, augmentation index, and arterial blood pressure. The current review will serve to begin to address the research gap that exists between the association of dietary phytosterols with CVD risk biomarkers.

Phytosterol, Lipid and Phenolic Composition, and Biological Activities of Guava Seed Oil.

Plant seeds have been found to contain bioactive compounds that have potential nutraceutical benefits. Guava seeds (Psidium guajava) are by-products in the beverage and juice industry; however, they can be utilized for a variety of commercial purposes. This study was designed to analyze the phytochemicals of the n-hexane extract of guava seed oil (GSO), to study its free-radical scavenging activity, and to monitor the changes in serum lipids and fatty acid profiles in rats that were fed GSO. The GSO was analyzed for phytochemicals using chromatographic methods. It was also tested for free-radical scavenging activity in hepatoma and neuroblastoma cells, and analyzed in terms of serum lipids and fatty acids. GSO was found to contain phenolic compounds (e.g., chlorogenic acid and its derivatives) and phytosterols (e.g., stimasterol, ß-sitosterol and campesterol), and exerted radical-scavenging activity in cell cultures in a concentration-dependent manner. Long-term consumption of GSO did not increase cholesterol and triglyceride levels in rat serum, but it tended to decrease serum fatty acid levels in a concentration-dependent manner. This is the first study to report on the lipid, phytosterol and phenolic compositions, antioxidant activity, and the hepato- and neuro-protection of hydrogen peroxide-induced oxidative stress levels in the GSO extract.

Features of Sitosterolemia in Children.

Sitosterolemia is a rare lipid metabolism disease with heterogeneous manifestations. Atherosclerosis can occur in children, and therefore, early detection, diagnosis, and treatment of this disease are important. We studied 18 pediatric patients with sitosterolemia who showed a significant increase in plasma lipid levels and analyzed their clinical, biochemical, and genetic characteristics. We recorded the initial serum lipid results and clinical manifestations of the patients. Lipid and plant sterol levels were measured after homozygous or compound heterozygous mutations of ABCG5 or ABCG8 were identified by genetic testing. Plasma plant sterol levels were analyzed by gas chromatography. Fourteen cases of sitosterolemia were examined by ultrasound and echocardiography. The initial total cholesterol and low-density lipoprotein levels of the children were significantly increased, but then markedly decreased after diet control or drug treatment, and even reached normal levels. Carotid atherosclerosis and aortic valve regurgitation were present in three of 14 patients. Serum lipid levels of children with sitosterolemia and xanthomas were notably higher than those without xanthomas. There were no significant differences in clinical manifestations between patients with different genotypes. In conclusion, sitosterolemia should be considered in children with hyperlipidemia who do not present with xanthomas, especially with a significant increase in total cholesterol and low-density lipoprotein levels. There does not appear to be a correlation between clinical phenotype and genotype.

Reference Intervals of Serum Non-Cholesterol Sterols by Gender in Healthy Japanese Individuals.

AIMS: The present study was conducted to establish a practical method for measuring non-cholesterol sterols and reference intervals of serum levels. METHODS: Healthy subjects (109 men and 151 women), four patients with sitosterolemia, and 10 heterozygous mutation carriers of ABCG5/ABCG8 genes were investigated. Then, three non-cholesterol sterols (sitosterol, campesterol, and lathosterol) of fasting serum samples were measured via a practical and highly sensitive gas chromatography (GC) method with 0.2 µg/mL as the lower limit of quantification. The coefficient of variation (CV) values for within-run reproducibility were 3.06%, 1.89%, and 1.77% for lathosterol, campesterol, and sitosterol, respectively. The CV values for between-run reproducibility were 2.81%, 2.06%, and 2.10% for lathosterol, campesterol, and sitosterol, respectively. RESULTS: The serum levels of sitosterol and campesterol were significantly higher in women than in men, whereas the serum levels of lathosterol were significantly higher in men than in women. Because of these gender difference, the determination of reference intervals of the three sterol values was performed by considering gender. The reference intervals of sitosterol, campesterol, and lathosterol were 0.99-3.88, 2.14-7.43, and 0.77-3.60 µg/mL in men and 1.03-4.45, 2.19-8.34, and 0.64-2.78 µg/mL in women, respectively. The serum levels of sitosterol and campesterol were higher in patients with sitosterolemia (94.3±47.3 and 66.3±36.6 µg/mL, respectively) than in healthy subjects. CONCLUSION: These results demonstrate a practical and highly sensitive GC method to measure non-cholesterol sterol levels and gender-segregated reference intervals of sitosterol, campesterol, and lathosterol in Japanese healthy subjects.

Phytosterolemia and γ-glutamyl transferase in adults with parenteral nutrition: Fish versus vegetal lipids: A randomized clinical trial.

OBJECTIVES: Fish oil (FO)-based lipid emulsions (LEs) have been reported to prevent hepatic dysfunction in patients treated with parenteral nutrition (PN). We studied patients with alterations of γ-glutamyl transferase (GGT) associated with the administration of PN containing olive/soybean (O/S)-based LE. The aim of this study was to determine whether the strategy of reducing the lipid dose by 50%, by changing to an FO-based LE, reduced plasma levels of phytosterols (PS) and GGT more effectively and safely, than the strategy of reducing lipid contribution by 50% while maintaining the same LE composition. METHODS: A randomized double-blind clinical trial was carried out in patients with normal initial GGT, who after a minimum of 1 wk of daily PN (0.8 g/kg of O/S-based LE) presented with GGT values twice the upper normal value. At the time of randomization 1:1, lipids were reduced to 0.4 g/kg daily. Group A maintained O/S LE and group B changed to FO LE. The primary endpoints were reduction of plasmatic PS and GGT on day 7 after randomization, performed in the study population per protocol by Student's t test and simple linear regression. Secondary outcomes included alkaline phosphatase (AP), alanine transaminase (ALT), and total bilirubin (BIL), and safety variables. RESULTS: Nineteen patients were included. On day 7 after randomization, GGT and AP values were higher in the O/S group (n = 10; GGT: median [Med], 4.99; interquartile range [IQR], 4.09; AP: Med, 2.59 µkat/L; IQR 1.74) than in the FO group (n = 9; GGT: Med, 2.26 µkat/L; IQR, 1.07; AP: Med, 1.2 µkat/L; IQR 1.44). Although there were no differences in ALT and BIL values, the ALT decrease was larger and more statistically significant in the FO group than in the O/S group (P = 0.009). Total PS (Med, 21.10 µg/mL; IQR, 5.50) in the O/S group was higher than in the FO group (Med, 13.4 µg/mL; IQR, 10.65; P = 0.002). Significant decreases in PS and their fractions were observed, with the exception of campesterol and stigmasterol. CONCLUSION: Plasma accumulation of PS and high values of GGT, AP, and ALT can be prevented with the exclusive administration of FO-based LE.

High molecular weight oat ß-glucan enhances lipid-lowering effects of phytosterols. A randomised controlled trial.

BACKGROUND & AIMS: Oat ß-glucan (OBG) and phytosterols (PS) are known to lower blood cholesterol levels via different mechanisms. Combination of high molecular weight (MW) OBG and PS in a single functional food could have complementary and/or synergistic effects for optimising heart health. The aim of this study was to investigate the effects of dietary supplementation with high-MW OBG with or without PS on plasma lipids in hypercholesterolaemic individuals. METHODS: In a double-blinded, placebo-controlled, 2 × 2 factorial trial, participants were randomised to receive biscuits fortified with either no PS or OBG (PL, n = 18) or 2 g PS (PS, n = 18), 3 g OBG (OBG, n = 18), or combination of 2 g PS and 3 g OBG (PS-OBG, n = 18) per day for 6 weeks. Primary outcome was fasting plasma total cholesterol (TC) and secondary outcomes were LDL-cholesterol, LDL-C; HDL-cholesterol, HDL-C; triglycerides, TG and TC to HDL-cholesterol (TC:HDL) ratio. RESULTS: TC and LDL-C were significantly lowered following PS (-4.6% and -7.6% respectively; p < 0.05), OBG (-5.7% and -8.6%; p < 0.01) and PS-OBG (-11.5% and -13.9%; p < 0.0001) administration. The reduction in TC in the PS-OBG group was significantly greater compared to PL (p < 0.001) and PS (p < 0.05). PS-OBG group had a significantly greater reduction in LDL-C compared to PL (p < 0.01) but not in comparison to PS or OBG groups. TC:HDL ratio was significantly reduced following PS-OBG (-8.9%; p < 0.01) only, and there was no significant difference found between groups. Plasma TG reduced by 8.4% following PS-OBG, however, this was statistically non-significant. Plasma HDL-C remained unchanged across all groups. CONCLUSIONS: Dietary supplementation with high-MW OBG and PS in a single functional food enhances their lipid-lowering potential. Blood cholesterol lowering by PS and OBG is additive. Delivery of these two bioactive nutrients in a single food allows optimisation of their lipid-lowering effects and may provide added heart health benefits with enhanced compliance. The trial was registered with the Australian New Zealand Clinical Trials Registry at http://www.anzctr.org.au/(ACTRN12618001455257).

Differences in lipid metabolism between anagliptin and sitagliptin in patients with type 2 diabetes on statin therapy: a secondary analysis of the REASON trial.

BACKGROUND: Anagliptin, a dipeptidyl peptidase-4 inhibitor, is reported to reduce the level of low-density lipoprotein cholesterol (LDL-C). The underlying mechanism of this effect and effect on lipid metabolism however remains uncertain. AIM AND METHODS: We therefore evaluate the effects of anagliptin on lipid metabolism-related markers compared with those of sitagliptin. The study was a secondary analysis using data obtained from the Randomized Evaluation of Anagliptin versus Sitagliptin On low-density lipoproteiN cholesterol in diabetes (REASON) trial. This trial in patients with type 2 diabetes at a high risk of cardiovascular events and on statin therapy showed that anagliptin reduced LDL-C levels to a greater extent than sitagliptin. Cholesterol absorption (campesterol and sitosterol) and synthesis (lathosterol) markers were measured at baseline and 52 weeks in the study cohort (n = 353). RESULTS: There was no significant difference in the changes of campesterol or sitosterol between the two treatment groups (p = 0.85 and 0.55, respectively). Lathosterol concentration was increased significantly at 52 weeks with sitagliptin treatment (baseline, 1.2 ± 0.7 µg/mL vs. 52 weeks, 1.4 ± 1.0 µg/mL, p = 0.02), whereas it did not change in the anagliptin group (baseline, 1.3 ± 0.8 µg/mL vs. 52 weeks, 1.3 ± 0.7 µg/mL, p = 0.99). The difference in absolute change between the two groups showed a borderline significance (p = 0.06). CONCLUSION: These findings suggest that anagliptin reduces LDL-C level by suppressing excess cholesterol synthesis, even in combination with statin therapy. Trial registration ClinicalTrials.gov number NCT02330406. https://clinicaltrials.gov/ct2/show/NCT02330406; registered January 5, 2015.