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.

Methodological Aspects of Phytosterol Measurements in Biological Samples.

Phytosterol measurement has gained a lot of interest during the last two decades after foods and supplements with added 4-desmethyl phytosterols were recognized and used as effective and safe non-pharmacologic hypocholesterolemic agents, and also after the mechanisms of intestinal absorption and hepatic excretion of sterols were unraveled. In addition, the wide use of serum phytosterols as biomarkers of cholesterol absorption has increased the interest in their measurement. In this review, the basic methods are discussed without going into details of the practical operations. The analysis includes first lipid extraction and saponification from various biologic matrices such as serum/plasma, feces, or tissues, after which the individual sterols are separated by adsorption chromatography (gas-liquid or liquid or high performance liquid chromatography) based on the polarity of the various sterols. We also deal with some specific aspects of phytosterol measurements in biological samples such as the need of harmonization of their analysis in biological samples, the discrepancies in the results of sitosterol and campesterol concentrations between different studies, and what is known about their biological day-to-day fluctuation. Phytosterols have a remarkable role in human health, so that their complicated and time consuming measurements call attention to routine ways of standardization between the sterol research laboratories.

Phytosterol-mediated inhibition of intestinal cholesterol absorption in mice is independent of liver X receptor.

SCOPE: Previous studies have proposed that phytosterols activate liver X receptors (LXR) in the intestine, thereby reducing intestinal cholesterol absorption and promoting fecal cholesterol excretion. METHODS AND RESULTS: In the present study, we examined the effects of dietary phytosterol supplementation on intestinal cholesterol absorption and fecal neutral sterol excretion in LXRαß-deficient mice, and wild-type mice treated with synthetic high-affinity LXRαß agonists. LXRαß deficiency led to an induction of intestinal cholesterol absorption and liver cholesterol accumulation. Phytosterol feeding resulted in an approximately 40% reduction of intestinal cholesterol absorption both in wild-type and LXRαß-deficient mice, reduced dietary cholesterol accumulation in liver and promoted the excretion of fecal cholesterol-derived compounds. Furthermore, phytosterols produced additive inhibitory effects on cholesterol absorption in mice treated with LXRαß agonists. CONCLUSIONS: Our data confirm the effect of LXR in regulating intestinal cholesterol absorption and demonstrate that the cholesterol-lowering effects of phytosterols occur in an LXR-independent manner.

Chronic intermittent psychological stress promotes macrophage reverse cholesterol transport by impairing bile acid absorption in mice.

Psychological stress is a risk factor for atherosclerosis, yet the pathophysiological mechanisms involved remain elusive. The transfer of cholesterol from macrophage foam cells to liver and feces (the macrophage-specific reverse cholesterol transport, m-RCT) is an important antiatherogenic pathway. Because exposure of mice to physical restraint, a model of psychological stress, increases serum levels of corticosterone, and as bile acid homeostasis is disrupted in glucocorticoid-treated animals, we investigated if chronic intermittent restraint stress would modify m-RCT by altering the enterohepatic circulation of bile acids. C57Bl/6J mice exposed to intermittent stress for 5 days exhibited increased transit through the large intestine and enhanced fecal bile acid excretion. Of the transcription factors and transporters that regulate bile acid homeostasis, the mRNA expression levels of the hepatic farnesoid X receptor (FXR), the bile salt export pump (BSEP), and the intestinal fibroblast growth factor 15 (FGF15) were reduced, whereas those of the ileal apical sodium-dependent bile acid transporter (ASBT), responsible for active bile acid absorption, remained unchanged. Neither did the hepatic expression of cholesterol 7α-hydroxylase (CYP7A1), the key enzyme regulating bile acid synthesis, change in the stressed mice. Evaluation of the functionality of the m-RCT pathway revealed increased fecal excretion of bile acids that had been synthesized from macrophage-derived cholesterol. Overall, our study reveals that chronic intermittent stress in mice accelerates m-RCT specifically by increasing fecal excretion of bile acids. This novel mechanism of m-RCT induction could have antiatherogenic potential under conditions of chronic stress.

Osbpl8 deficiency in mouse causes an elevation of high-density lipoproteins and gender-specific alterations of lipid metabolism.

OSBP-related protein 8 (ORP8) encoded by Osbpl8 is an endoplasmic reticulum sterol sensor implicated in cellular lipid metabolism. We generated an Osbpl8(-/-) (KO) C57Bl/6 mouse strain. Wild-type and Osbpl8KO animals at the age of 13-weeks were fed for 5 weeks either chow or high-fat diet, and their plasma lipids/lipoproteins and hepatic lipids were analyzed. The chow-fed Osbpl8KO male mice showed a marked elevation of high-density lipoprotein (HDL) cholesterol (+79%) and phospholipids (+35%), while only minor increase of apolipoprotein A-I (apoA-I) was detected. In chow-fed female KO mice a less prominent increase of HDL cholesterol (+27%) was observed, while on western diet the HDL increment was prominent in both genders. The HDL increase was accompanied by an elevated level of HDL-associated apolipoprotein E in male, but not female KO animals. No differences between genotypes were observed in lecithin:cholesterol acyltransferase (LCAT) or hepatic lipase (HL) activity, or in the fractional catabolic rate of fluorescently labeled mouse HDL injected in chow-diet fed animals. The Osbpl8KO mice of both genders displayed reduced phospholipid transfer protein (PLTP) activity, but only on chow diet. These findings are consistent with a model in which Osbpl8 deficiency results in altered biosynthesis of HDL. Consistent with this hypothesis, ORP8 depleted mouse hepatocytes secreted an increased amount of nascent HDL into the culture medium. In addition to the HDL phenotype, distinct gender-specific alterations in lipid metabolism were detected: Female KO animals on chow diet showed reduced lipoprotein lipase (LPL) activity and increased plasma triglycerides, while the male KO mice displayed elevated plasma cholesterol biosynthetic markers cholestenol, desmosterol, and lathosterol. Moreover, modest gender-specific alterations in the hepatic expression of lipid homeostatic genes were observed. In conclusion, we report the first viable OsbplKO mouse model, demonstrating a HDL elevating effect of Osbpl8 knock-out and additional gender- and/or diet-dependent impacts on lipid metabolism.