Derivatization-independent cholesterol analysis in crude lipid extracts by liquid chromatography/mass spectrometry: applications to a rabbit model for atherosclerosis

Direct measurement of various sterols in crude lipid extracts in a single experiment from limited biological samples is challenging. Current mass spectrometry (MS) based approaches usually require chemical derivatization before subjecting to MS analysis. Here, we present a derivatization-independent method for analyzing various sterols, including cholesterol and its congeners, using liquid chromatography and atmospheric pressure chemical ionization mass spectrometry. Based on the specific tandem mass spectrometry pattern of cholesterol, multiple reaction monitoring (MRM) transitions were used to quantify free cholesterol and its fatty acyl esters. Several cholesterol oxidation products could also be measured using the upfront liquid chromatography separation and specific MRM transitions. The method was validated alongside established enzymatic assays in measuring total cholesterol. As a proof of concept, we analyzed plasma sterols in rabbits administrated with a high cholesterol diet (HCD) which is a classical atherosclerotic model. Free cholesterol, cholesterol esters, 7-hydroxycholesterol, and 7-ketocholesterol were elevated in plasma of rabbits on HCD. This method could also serve as an excellent tool for quantitative analysis of other sterols such as ergosterol and sitosterol in other organisms beside mammalian. In Saccharomyces cerevisiae, our results indicated dramatic increases of the ratio of ergosterol esters to free ergosterol in both yeh2Δ and tgl1Δ cells, which are consistent with the function of the respective enzymes.     

Usefulness of gas chromatographic profiles of plasma total lipids in diagnosis of phytosterolemia

A Cambodian male (aged 5 years and 9 months) presented with subcutaneous and tendon xanthomas in association with hypercholesterolemia. He was erroneously diagnosed as having familial hypercholesterolemia and treated with a low cholesterol diet (+/- cholestyramine) to which he did not respond. A determination of plasma total lipid profile by high-temperature gas chromatography revealed elevated plasma levels of free and esterified plant sterols along with the hypercholesterolemia. Introduction and maintenance of a diet low in cholesterol and plant sterols resulted in significant reduction in the blood concentration of these sterols, which returned to pretreatment level upon discontinuation of the low sterol regimen. The rapid identification and quantitation of the plant sterols by high-temperature gas chromatography provides a sensitive means of distinguishing phytosterolemia, which might be more common than previously suspected, from other forms of dyslipidemia, and for following the course of any treatment.     

Differential effects of cholesterol, ergosterol and lanosterol on a dipalmitoyl phosphatidylcholine membrane: a molecular dynamics simulation study

Lipid raft/domain formation may arise as a result of the effects of specific sterols on the physical properties of membranes. Here, using molecular dynamics simulation, we examine the effects of three closely-related sterols, ergosterol, cholesterol, and lanosterol, at a biologically relevant concentration (40 mol %) on the structural properties of a model dipalmitoyl phosphatidylcholine (DPPC) membrane at 309 and 323 K. All three sterols are found to order the DPPC acyl tails and condense the membrane relative to the DPPC liquid-phase membrane, but each one does this to a significantly different degree. The smooth alpha-face of ergosterol, together with the presence of tail unsaturation in this sterol, leads to closer interaction of ergosterol with the lipids and closer packing of the lipids with each other, so ergosterol has a higher condensing effect on the membrane, as reflected by the area per lipid. Moreover, ergosterol induces a higher proportion of trans lipid conformers, a thicker membrane, and higher lipid order parameters and is aligned more closely with the membrane normal. Ergosterol also positions itself closer to the bilayer/water interface. In contrast, the rough alpha-face of lanosterol leads to a less close interaction of the steroid ring system with the phospholipid acyl chains, and so lanosterol orders, straightens, and packs the lipid acyl chains less well and is less closely aligned with the membrane normal. Furthermore, lanosterol lies closer to the relatively disordered membrane center than do the other sterols. The behavior of cholesterol in all the above respects is intermediate between that of lanosterol and ergosterol. The findings here may explain why ergosterol is the most efficient of the three sterols at promoting the liquid-ordered phase and lipid domain formation and may also furnish part of the explanation as to why cholesterol is evolutionarily preferred over lanosterol in higher-vertebrate plasma membranes.     

The Bioavailability and Biological Activities of Phytosterols as Modulators of Cholesterol Metabolism

Phytosterols are natural sterols widely found in plants that have a variety of physiological functions, and their role in reducing cholesterol absorption has garnered much attention. Although the bioavailability of phytosterols is only 0.5–2%, they can still promote cholesterol balance in the body. A mechanism of phytosterols for lowering cholesterol has now been proposed. They not only reduce the uptake of cholesterol in the intestinal lumen and affect its transport, but also regulate the metabolism of cholesterol in the liver. In addition, phytosterols can significantly reduce the plasma concentration of total cholesterol, triglycerides, and low-density lipoprotein cholesterol (LDL-C), with a dose-response relationship. Ingestion of 3 g of phytosterols per day can reach the platform period, and this dose can reduce LDL-C by about 10.7%. On the other hand, phytosterols can also activate the liver X receptor α-CPY7A1 mediated bile acids excretion pathway and accelerate the transformation and metabolism of cholesterol. This article reviews the research progress of phytosterols as a molecular regulator of cholesterol and the mechanism of action for this pharmacological effect.     

Alteration of Lipid Membrane Rigidity by Cholesterol and Its Metabolic Precursors

Caused by biosynthesis defects, cholesterol deficiency can lead to developmental disorders and malformations, with possible implication of lipid membrane properties. We show that modification of sterol chemical structure alters membrane physical properties significantly. By X-ray diffraction and osmotic stress, we measure changes in the bending rigidity of bilayers containing either cholesterol or one of its metabolic precursors. Membrane elasticity differs dramatically between slightly different sterols and varies in the sequence lanosterol < 7-dehydrocholesterol < lathosterol < cholesterol. We interpret the results in terms of sterol location within lipid structures and modification of lateral stress, a structural feature relevant to interactions within biological membranes. We find that cholesterol is most efficient in enhancing membrane rigidity, a possible clue to why depletion or replacement with other sterols can affect cellular structures.     

膳食纤维降血脂作用及其机制的研究进展

综述了近年膳食纤维降脂作用及其机制的研究进展.膳食纤维能使血清中胆固醇、甘油三酯、低密度脂蛋白水平降低,高密度脂蛋白水平升高.其主要机制为通过减少膳食中胆固醇的吸收、影响机体中胆固醇的代谢、促进胆固醇的排泄等降低血浆中胆固醇水平;通过增加食物在肠道内的过渡时间、延缓胃排空、减缓或降低脂肪的吸收等机制降低血浆中甘油三酯水...     

The use of stable and radioactive sterol tracers as a tool to investigate cholesterol degradation to bile acids in humans in vivo

Alterations of cholesterol homeostasis represent important risk factors for atherosclerosis and cardiovascular disease. Different clinical-experimental approaches have been devised to study the metabolism of cholesterol and particularly the synthesis of bile acids, its main catabolic products. Most evidence in humans has derived from studies utilizing the administration of labeled sterols; these have several advantages over in vitro assay of enzyme activity and expression, requiring an invasive procedure such as a liver biopsy, or the determination of fecal sterols, which is cumbersome and not commonly available. Pioneering evidence with administration of radioactive sterol derivatives has allowed to characterize the alterations of cholesterol metabolism and degradation in different situations, including spontaneous disease conditions, aging, and drug treatment. Along with the classical isotope dilution methodology, other approaches were proposed, among which isotope release following radioactive substrate administration. More recently, stable isotope studies have allowed to overcome radioactivity exposure. Isotope enrichment studies during tracer infusion has allowed to characterize changes in the degradation of cholesterol via the "classical" and the "alternative" pathways of bile acid synthesis. Evidence brought by tracer studies in vivo, summarized here, provides an exceptional tool for the investigation of sterol metabolism, and integrate the studies in vitro on human tissue.     

Quantitative analysis of desmosterol, cholesterol and cholesterol sulfate in semen by high-performance liquid chromatography.

A simple, rapid and accurate method to separate and quantify cholesterol, desmosterol and cholesterol sulfate in human spermatozoa and seminal plasma (SP) is described. This high-performance liquid chromatographic procedure is based on reversed-phase chromatography on a Inertsil ODS2 5 microm silica column with a binary gradient of mixtures of chloroform-methanol and chloroform-methanol-water as the mobile phase at a flow-rate of 0.25 ml/min. Sterols are separated with good resolution and high reproducibility. The eluted sterols are quantified using a light-scattering (mass) detector. As little as 64, 64 and 68 pmol of cholesterol, desmosterol and cholesterol sulfate, respectively, can be quantified under these conditions. Cholesterol is the predominant sterol both in spermatozoa (107+/-7 nmol/10(8) spermatozoa) and SP (0.83+/-0.10 micromol/ml) whereas the concentrations of desmosterol were 38+/-6 nmol/10(8) in spermatozoa and 0.18+/-0.02 micromol/ml in SP. Cholesterol sulfate represents about 6% of total cholesterol in the spermatozoa and SP. In conclusion, this method offers interesting perspectives for the quantitative analysis of these sterols not only in semen, but also in other biological samples.     

Lipid metabolite profiling identifies desmosterol metabolism as a new antiviral target for hepatitis C virus

Hepatitis C virus (HCV) infection has been clinically associated with serum lipid abnormalities, yet our understanding of the effects of HCV on host lipid metabolism and conversely the function of individual lipids in HCV replication remains incomplete. Using liquid chromatography-mass spectrometry metabolite profiling of the HCV JFH1 cell culture infection model, we identified a significant steady-state accumulation of desmosterol, an immediate precursor to cholesterol. Pharmacological inhibition or RNAi-mediated depletion of DHCR7 significantly reduced steady-state HCV protein expression and viral genomic RNA. Moreover, this effect was reversed when cultures were supplemented with exogenous desmosterol. Together, these observations suggest an intimate connection between HCV replication and desmosterol homeostasis and that the enzymes responsible for synthesis of desmosterol may be novel targets for antiviral design.     

Significance of cholesterol methyl groups

Cholesterol is an indispensable molecule in mammalian cell membranes. To truly understand its role in the functions of membranes, it is essential to unravel cholesterol's structure-function relationship determined by underlying molecular interactions. For this purpose, we elaborate on this issue by considering the previously proposed idea that cholesterol's effects on a number of physical properties of membranes have been optimized during the evolution by removal of its excess methyl groups from the alpha-face of cholesterol, thus "smoothening" the structure. Consequently, the methyl groups still attached to cholesterol are one of the most intriguing structural features of the molecule. An obvious question arises: Why do these methyl groups still exist, and could cholesterol properties be further optimized by their removal? Because of the nature of the biosynthetic pathways of cholesterol, and the evidence of decreased interactions between sterols and lipid acyl chains when methyl groups are present, it seems plausible that removal of the methyl groups might indeed lead to stronger ordering and condensing effects of the cholesterol molecule. Atomic-scale molecular dynamics simulations of numerous modified sterols embedded in saturated lipid bilayers demonstrate, however, that the issue is more subtle. The analysis reveals a complex interplay between the lipid acyl chains and the structural details of cholesterol. Changes in cholesterol structure typically do not improve its performance in terms of promoting membrane order. This view is substantiated by a detailed analysis of the simulation data. In particular, it highlights the importance of the methyl group C18 for cholesterol properties. The C18 group resides between the third and fourth ring of cholesterol on its "rough" beta-side, and the results provide compelling evidence that C18 is crucial for the proper orientation of the sterol. More generally, the data provide insight into the role of the methyl groups of cholesterol.     

Determination of some hydroxycholesterols in human serum samples

The simultaneous determination of some hydroxycholesterols in human serum samples is described. The procedure is based on hydrolysis and extraction of these compounds in serum samples, followed by removal of especially cholesterol (making use of reversed-phase high-performance liquid chromatography) and derivatization of the purified compounds to their trimethylsilyl ethers and subsequent gas chromatography using flame ionization detection. Serum levels of 7 alpha-hydroxycholesterol, 7 beta-hydroxycholesterol and 26-hydroxycholesterol were determined in several groups of patients: normals, untreated patients suffering from cerebrotendinous xanthomatosis, patients suffering from cerebrotendinous xanthomatosis and treated with either chenodeoxycholic acid or cholic acid in an effective dose, patients suffering from cerebro-hepato-renal syndrome, patients suffering from hypercholesterolemia and treated with cholestyramine for prolonged periods and one patient presumed to be suffering from an inborn error of metabolism in bile acid synthesis. It can be concluded that the 7 alpha-hydroxycholesterol concentration in serum is a good parameter for establishing disorders involving the metabolic conversion of 7 alpha-hydroxycholesterol towards bile acids. In addition, 26-hydroxycholesterol levels in patients suffering from cerebrotendinous xanthomatosis are beyond detectable limits, even during treatment with bile acids in an effective dose, whereas in all other conditions this compound is substantially present.     

Metabolomics analysis of the therapeutic mechanism of Semen Descurainiae Oil on hyperlipidemia rats using 1H‐NMR and LC–MS

Semen descurainiae oil (SDO) is an important traditional Chinese medicine that was recently discovered to have the function of reducing blood lipids. Metabolomics analyses of plasma, liver and kidney in rats were performed using ¹H‐NMR and LC–MS to illuminate the lower blood lipid concentration effect of SDO, and niacin was considered as the active control. The measure of total cholesterol (TC) and low‐density‐lipoprotein cholesterol (LDL‐C) in plasma showed that SDO treatment decreased significantly the content of TC and LDL‐C. An orthogonal partial least squares–discriminant analysis approach was applied to identify the different metabolic profiles of plasma, liver and kidney in rats and to detect related potential biomarkers. The results suggested that the metabolic profiles of the control group and hyperlipidemia group showed significant difference and the SDO and niacin group had effective anti‐hyperlipidemia function. The biomarkers primarily concern lipid metabolism, amino acid metabolism and glycometabolism, and the change in biomarkers indicated that hyperlipidemia could cause the unbalance of these metabolic pathways in vivo. SDO reduced blood lipids by repairing amino acid and lipid metabolism.     

Hypolipidemic effects of hydrolyzed xyloglucan

Xyloglucan is found in the primary cell walls of higher plants, where it plays important biological roles. It also exists in certain seeds, for example tamarind, as a reserve polysaccharide. Some xyloglucan oligosaccharides possess plant growth promotion activity. We prepared hydrolyzed xyloglucan from the tamarind seed xyloglucan using endo-(1 → 4)- β -glucanase and examined its effects on lipid metabolism in rats fed a high fat diet. Among the plasma lipids, total lipid, cholesterol, triglyceride and β - lipoprotein were each reduced 14 - 17% by hydrolyzed xyloglucan. Hydrolyzed xyloglucan significantly reduced both adipose tissue weight and plasma GOT. Among the hepatic lipids, total lipid, cholesterol, triglyceride and phospholipid were significantly reduced by hydrolyzed xyloglucan. These hypolipidemic effects may be important for the treatment and prevention of geriatric diseases, including diabetes and cardiac disorders.     

Oligosaccharides might contribute to the antidiabetic effect of honey: a review of the literature

Evidence shows that honey improves glycemic control in diabetes mellitus. Besides its hypoglycemic effect, studies indicate that honey ameliorates lipid abnormalities in rats and humans with diabetes. The majority of these studies do not examine the mechanisms by which honey ameliorates glycemic and/or lipid derangements. The gut microbiota is now recognized for its ability to increase energy harvest from the diet and alter lipid metabolism of the host. Recently available data implicate a causal role of these gut microbes in the pathophysiology of obesity, insulin resistance, and diabetes mellitus. In this review, we present some of the latest findings linking gut microbiota to pathogenesis of obesity, insulin resistance, and diabetes mellitus. The review also underlines data that demonstrate the beneficial effects of oligosaccharides on various abnormalities commonly associated with these disorders. Based on the similarities of some of these findings with those of honey, together with the evidence that honey contains oligosaccharides, we hypothesize that oligosaccharides present in honey might contribute to the antidiabetic and other health-related beneficial effects of honey. We anticipate that the possibility of oligosaccharides in honey contributing to the antidiabetic and other health-related effects of honey will stimulate a renewed research interest in this field.     

Triple-Knockout,Synuclein-Free Mice Display Compromised Lipid Pattern

Recent studies have implicated synucleins in several reactions during the biosynthesis of lipids and fatty acids in addition to their recognised role in membrane lipid binding and synaptic functions. These are among aspects of decreased synuclein functions that are still poorly acknowledged especially in regard to pathogenesis in Parkinson’s disease. Here, we aimed to add to existing knowledge of synuclein deficiency (i.e., the lack of all three family members), with respect to changes in fatty acids and lipids in plasma, liver, and two brain regions in triple synuclein-knockout (TKO) mice. We describe changes of long-chain polyunsaturated fatty acids (LCPUFA) and palmitic acid in liver and plasma, reduced triacylglycerol (TAG) accumulation in liver and non-esterified fatty acids in plasma of synuclein free mice. In midbrain, we observed counterbalanced changes in the relative concentrations of phosphatidylcholine (PC) and cerebrosides (CER). We also recorded a notable reduction in ethanolamine plasmalogens in the midbrain of synuclein free mice, which is an important finding since the abnormal ether lipid metabolism usually associated with neurological disorders. In summary, our data demonstrates that synuclein deficiency results in alterations of the PUFA synthesis, storage lipid accumulation in the liver, and the reduction of plasmalogens and CER, those polar lipids which are principal compounds of lipid rafts in many tissues. An ablation of all three synuclein family members causes more profound changes in lipid metabolism than changes previously shown to be associated with γ-synuclein deficiency alone. Possible mechanisms by which synuclein deficiency may govern the reported modifications of lipid metabolism in TKO mice are proposed and discussed.     

Determination of serum cholestanol by semi‐micro high‐performance liquid chromatography with electrochemical detection

A simple and sensitive method that dose not require derivatization for determining cholestanol has been developed using HPLC with electrochemical detection (HPLC‐ECD). The current peak height was linearly related to the amount of cholestanol injected, ranging from 1 to 200 μM (r = 0.999). The detection limit (S/N = 3) of cholestanol was 0.23 μM (1.2 pmol). Total cholestanol in control human and mouse serum was determined by the present method with a recovery rate of more than 90% and an RSD (n = 5) of less than 7.3%. Further, this method was successfully applied to monitor experimental hypercholestanolemia in mice fed a high‐cholestanol diet, an animal model of cerebrotendinous xanthomatosis (CTX). In conclusion, we found this method to be both simple and useful for the determination of cholestanol in serum, helping in the diagnosis of CTX. Copyright © 2009 John Wiley & Sons, Ltd.     

The role of microscopy in understanding atherosclerotic lysosomal lipid metabolism.

Microscopy has played a critical role in first identifying and then defining the role of lysosomes in formation of atherosclerotic foam cells. We review the evidence implicating lysosomal lipid accumulation as a factor in the pathogenesis of atherosclerosis with reference to the role of microscopy. In addition, we explore mechanisms by which lysosomal lipid engorgement occurs. Low density lipoproteins which have become modified are the major source of lipid for foam cell formation. These altered lipoproteins are taken into the cell via receptor-mediated endocytosis and delivered to lysosomes. Under normal conditions, lipids from these lipoproteins are metabolized and do not accumulate in lysosomes. In the atherosclerotic foam cell, this normal metabolism is inhibited so that cholesterol and cholesteryl esters accumulate in lysosomes. Studies of cultured cells incubated with modified lipoproteins suggests this abnormal metabolism occurs in two steps. Initially, hydrolysis of lipoprotein cholesteryl esters occurs normally, but the resultant free cholesterol cannot exit the lysosome. Further lysosomal cholesterol accumulation inhibits hydrolysis, producing a mixture of cholesterol and cholesteryl esters within swollen lysosomes. Various lipoprotein modifications can produce this lysosomal engorgement in vitro and it remains to be seen which modifications are most important in vivo.     

How do sterols determine the antifungal activity of amphotericin B? Free energy of binding between the drug and its membrane targets

Amphotericin B (AmB) is a well-known polyene antibiotic used to treat systemic fungal infections. It is commonly accepted that the presence of sterols in the membrane is essential for the AmB biological activity, that is, for the formation of transmembrane ion channels. The selective toxicity of AmB for fungal cells is attributed to the fact that it is more potent against fungal cell membranes containing ergosterol than against the mammalian membranes with cholesterol. According to the "primary complex" hypothesis, AmB associates with sterols in a membrane to form binary complexes, which may subsequently assemble into a barrel-stave channel. To elucidate the molecular nature of the AmB selectivity for ergosterol-containing membranes, in the present work, we used computational methods to study the formation of the putative AmB/sterol complexes in a lipid bilayer. The free energy profiles for the AmB-sterol association in phospholipid bilayers containing 30 mol % of sterols were calculated and thoroughly analyzed. The results obtained confirm the formation of specific AmB/ergosterol complexes and are used to determine the energetic and structural origin of the enhanced affinity of AmB for ergosterol than for cholesterol. The significance of this affinity difference for the mechanism of action of AmB is discussed. The data obtained allowed us also to suggest a possible origin of the increased selectivity of a novel class of less toxic AmB derivatives.     

Sterols of grapefruit,orange, and mandarin pulps

The composition and percentage distribution of the sterols in preparations of the free (I), glycosylated (II), and esterified (III) sterols in the pulp of citrus fruits have been studied. In the sterol preparations, cholesterol, campesterol, stigmasterol, and β-sitosterol have been identified and detected, and three other sterols have been detected but not identified. It has been established that the sequences of the relative amounts of sterols in preparations (I), (II), and (III) of the grapefruit and the orange are similar and differ from that of mandarin pulp.     

Association of lipoprotein lipase (LPL) single nucleotide polymorphisms with type 2 diabetes mellitus

The etiology and pathogenesis of type 2 diabetes mellitus (T2DM) are not completely understood although it is often associated with other conditions such as obesity, hypertension, and dyslipidemia. Lipoprotein lipase (LPL) is a key enzyme in human lipid metabolism that facilitates the removal of triglyceride-rich lipoproteins from the bloodstream. LPL hydrolyzes the core of triglyceride-rich lipoproteins (chylomicrons and very low density lipoprotein) into free fatty acids and monoacylglycerol. To gain insight into the possible role of LPL in T2DM, nine single nucleotide polymorphisms (SNPs) of LPL were analyzed for the association with T2DM using 944 unrelated Koreans, including 474 T2DM subjects and 470 normal healthy controls. Of the nine LPL SNPs we analyzed, a significant association with multiple tests by the false discovery rate (FDR) was observed between T2DM and SNP rs343 (+13836C>A in intron 3). SNP rs343 was also marginally associated with some of T2DM-related phenotypes including total cholesterol, high density lipoprotein cholesterol (HDLc), and log transformed glycosylated hemoglobin in 470 normal controls, although no significant association was detected by multiple tests. In total, our results suggest that the control of lipid level by LPL in the bloodstream might be an important factor in T2DM pathogenesis in the Korean population.