Rapid quantification of free and esterified phytosterols in human serum using APPI-LC-MS/MS

A novel analytical platform based on liquid chromatography and tandem mass spectrometry using atmospheric pressure photoionization was applied for the simultaneous quantification of free and esterified beta-sitosterol, campesterol, brassicasterol, and stigmasterol. The total time for sample pretreatment and analysis could be reduced from approximately 3 h [gas chromatography-mass spectrometry (GC-MS)] to 15 min. The detection limits of the different phytosterols ranged between 0.25 and 0.68 microg/l. Linear ranges were between 1 and 1,000 microg/l. The within-run and between-run variabilities ranged between 1.4% and 9.9%. The analytical sensitivity was at least 150-fold higher compared with GC-MS. Our new method allows a rapid and simultaneous determination of free and esterified phytosterols in serum.     

Inhibitors of sterol synthesis. Chemical syntheses and activities of new derivatives of 15-oxygenated sterols

The chemical syntheses of 5α-cholestane-3β,14α,15β-triol, 5α-cholestane-14α-ol-3,15-dione, 5α-cholestane-3β,14α-diol-15-one, 14α,15α-epoxy-5α-cholestan-3β-ol, and 5α-cholest-8(14)-en-3β-ol-15-one oxime are described. All of these compounds were found to be potent inhibitors of sterol synthesis in cultured mouse L cells. However, the former three compounds had little or no effect on the levels of 3-hydroxy-3-methylgutaryl (HMG)-CoA reductase in the same cells. In contrast, in the case of the latter two compounds, the concentrations required to cause a 50% inhibition of the synthesis of digitonin-precipitable sterols were comparable to those required to cause a 50% reduction in the levels of HMG-CoA reductase in the same cells. 5α-Cholest-8(14)-en-3β-ol-15-one oxime had no effect on serum cholesterol levels when administered to male rats at a level of 0.15% in a cholesterol-free diet.     

The N-terminal His-tag affects the triglyceride lipase activity of hormone-sensitive lipase in testis

The sterility of hormone-sensitive lipase (HSL) knockout mice clearly shows the link between lipid metabolism and spermatogenesis. However, which substrate or product of this multifunctional lipase affects spermatogenesis is unclear. We found that an HSL protein with a His-tag at the N-terminus preserved the normal hydrolase activity of cholesteryl ester (CE) but the triglyceride lipase (TG) activity significantly decreased in vitro. Therefore, mice with this functionally incomplete HSL (His-HSL) were produced on a background of HSL deficiency (HSL^−/−h). As a result, HSL^−/−h testis has an 8.65-fold higher CE activity than wild-type testis but a twofold higher TG activity than wild-type testis. To compare His-HSL and wild-type HSL in vitro and in vivo, we confirmed that the His-tag significantly suppressed HSL TG activity. From our results, we believe that TG activity was affected by the His-tag insertion, but CE activity was not influenced. Furthermore, the His-tag protected HSL from binding to the inhibitor BAY. From our study, TG activity and BAY binding sites were affected by N-terminal His-tag insertion.     

Synthesis and antifungal activity of oxygenated cholesterol derivatives

A series of oxygenated cholesterol derivatives were prepared from new synthetic methods and evaluated for their in vitro antimicrobial properties against human pathogens. The activity was highly dependent on the structure of the different compounds involved. The best results were obtained with hydroxy ketones 2, 4 and 5 and diketone 7 exhibiting activities against S. cerevisiae (ATCC 28383) and Candida albicans (CIP 1663-86). For example, compound 2 exhibited high activities against C. albicans (CIP 1663-86) and Amphotericine B and miconazole resistant strain C. albicans (CIP 1180-79) at a concentration of 1.5 microg/mL.     

The 220 MHz NMR spectra of phytosterols

The 220MHz NMR spectra of forty two steroids are reported. Eight pairs of C-24 epimers (24α- and 24β) and two pairs of double bond isomers (cis and trans) can be distinguished by this technique. The influence of substituents, solvents and stereochemistry on methyl group chemical shifts is discussed.     

Altered renal lipid metabolism and renal lipid accumulation in human diabetic nephropathy

Animal models link ectopic lipid accumulation to renal dysfunction, but whether this process occurs in the human kidney is uncertain. To this end, we investigated whether altered renal TG and cholesterol metabolism results in lipid accumulation in human diabetic nephropathy (DN). Lipid staining and the expression of lipid metabolism genes were studied in kidney biopsies of patients with diagnosed DN (n = 34), and compared with normal kidneys (n = 12). We observed heavy lipid deposition and increased intracellular lipid droplets. Lipid deposition was associated with dysregulation of lipid metabolism genes. Fatty acid β-oxidation pathways including PPAR-α, carnitine palmitoyltransferase 1, acyl-CoA oxidase, and L-FABP were downregulated. Downregulation of renal lipoprotein lipase, which hydrolyzes circulating TGs, was associated with increased expression of angiopoietin-like protein 4. Cholesterol uptake receptor expression, including LDL receptors, oxidized LDL receptors, and acetylated LDL receptors, was significantly increased, while there was downregulation of genes effecting cholesterol efflux, including ABCA1, ABCG1, and apoE. There was a highly significant correlation between glomerular filtration rate, inflammation, and lipid metabolism genes, supporting a possible role of abnormal lipid metabolism in the pathogenesis of DN. These data suggest that renal lipid metabolism may serve as a target for specific therapies aimed at slowing the progression of glomerulosclerosis.     

Accumulation of cholesterol precursors and plant sterols in human stenotic aortic valves

The pathogenesis of aortic valve stenosis (AS) is characterized by the accumulation of LDL-derived cholesterol in the diseased valves. Since LDL particles also contain plant sterols, we investigated whether plant sterols accumulate in aortic valve lesions. Serum samples were collected from 82 patients with severe AS and from 12 control subjects. Aortic valves were obtained from a subpopulation of 21 AS patients undergoing valve surgery and from 10 controls. Serum and valvular total cholesterol and noncholesterol sterols were measured by gas-liquid chromatography. Noncholesterol sterols, including both cholesterol precursors and sterols reflecting cholesterol absorption, were detected in serum samples and aortic valves. The higher the ratios to cholesterol of the cholesterol precursors and absorption markers in serum, the higher their ratios in the stenotic aortic valves (r=0.74, P<0.001 for lathosterol and r=0.88, P<0.001 for campesterol). The valvular ratio to cholesterol of lathosterol correlated negatively with the aortic valve area (r= -0.47, P=0.045), suggesting attenuation of cholesterol synthesis with increasing severity of AS. The higher the absorption of cholesterol, the higher the plant sterol contents in stenotic aortic valves. These findings suggest that local accumulation of plant sterols and cholesterol precursors may participate in the pathobiology of aortic valve disease.     

The fate of radiolabelled C28 and C29 phytosterols in the honey bee

The fate of radiolabelled campesterol, sitosterol and 24-methylenecholesterol fed in chemically-defined diets to honey bee (Apis mellifera L.) workers was determined. At various intervals, sterols of prepupae, newly emerged adults and queens were analyzed qualitatively, quantitatively and radiochemically and it was determined that there was not sufficient radioactivity associated with cholesterol and/or desmosterol in any of the samples to verify that any of the three C₂₈ and C₂₉ sterols was dealkylated and converted to cholesterol. Similarly, there was no evidence for the conversion of campesterol or sitosterol to 24-methylenecholesterol. It was concluded that the major portion of the sterols incorporated into the tissues of the brood larvae originated from the worker bees used to establish the colony. There is good evidence supporting the premise that the workers can make available sterols from their endogenous pools to the nutrient in the hive and that they can replenish these sterols with those from the artificial diet. The queen is also able to replenish sterols utilized in egg production from those obtained by the workers from the artificial diet, and at the end of nine weeks queens contained more than four times as much sterol, on a ‘μg sterol per g fresh weight’ basis, than was found in fertile queens at the beginning of the test period.     

Mixtures of lecithin with polymerizable derivatives of cholesterol

One of the best investigated binary lipid mixtures is the lecithin-cholesterol system. We show here that it is possible to modify the cholesterol in such a way that it can be polymerized without changing its behaviour in mixtures with lecithin. The polymerizable derivatives exhibit a very similar phase diagram in the mixture with dipalmitoyl-phosphatidylcholine as the cholesterol itself. This is demonstrated by filmbalance measurements.     

Trace formation of progesterone from 22-dehydro phytosterols during storage

Progesterone has been isolated from different samples of 22-dehydro phytosterots of previously known composition, kept in a laboratory collection for periods from 10 to 18 years. It is not present in detectable amount in cholesterol, fucosterol or desmosterol, or in freshly prepared samples of stigmasterol. The possibility of progesterone being an autoxidation artifact is thus emphasized, in particular in the environment, and hence, in concentrating organisms.     

In vivo conversion of [4-14C]sitosterol and [22,23-3H]sitosterol to stigmasterol in barley seedlings

Six-day-old barley seedlings were allowed to take up [4-¹⁴C]sitosterol and [22, 23-³H]sitosterol for 2.5 hr and the incorporation into the sterol fractions was determined after 0, 6, 12 and 24 hr. Sitosterol was readily incorporated into every sterol class. The ³H/¹⁴C ratio in the free forms dropped when compared with the ³H/¹⁴C ratio of the administered sitosterol. In the free sterol, radioactive stigmasterol, showing a ³H/¹⁴C ratio half that of the sitosterol ³H/¹⁴C ratio, was isolated and its radiochemical purity established by dilution with carrier material and crystallization to constant specific activity.     

Marked differences in cholesterol synthesis between neurons and glial cells from postnatal rats

Neurons have a high demand for cholesterol to develop and maintain membrane-rich structures like axons, dendrites and synapses, but it remains unclear, whether they can satisfy their need by costly de novo synthesis. To address this, we compared cholesterol synthesis in serum-free cultures of highly purified CNS neurons and glial cells from postnatal rats. We observed marked cell-specific differences: Compared with glial cells, neurons showed different profiles of biosynthetic enzymes, post-squalene precursors and cholesterol metabolites, and they produced cholesterol less efficiently, possibly because of very low levels of lanosterol-converting enzymes. Astrocytes responded to inhibition of cholesterol synthesis with a much stronger up-regulation of biosynthetic enzymes than neurons. Our results support the idea that neurons cannot produce cholesterol efficiently and that they depend on an external source of this lipid.     

脂质代谢在乳腺癌发病中的作用

乳腺癌已经成为全球第一大癌症,其发病机制及治疗方法的探索越来越受到人们重视。脂质代谢异常是癌细胞中最突出的代谢改变之一,探索乳腺癌细胞中脂质代谢的改变,以寻找新的诊断指标和治疗靶点是至关重要的。本文从脂肪酸代谢、甘油三酯代谢、胆固醇代谢和脂质代谢信号通路4个方面介绍脂质代谢异常在乳腺癌中的研究进展,为靶向脂质代谢治疗乳腺癌提供新思路和新方法。     

26-Hydroxycholesterol and cholest-4-en-3-one,the first metabolites of cholesterol in potato plants

Etiolated potato sprouts convert administered cholesterol-4-¹⁴C to radioactive 26-hydroxycholesterol and cholest-4-en-3-one. These two steroids must be the first products of cholesterol metabolism in potato plants.     

前蛋白转化酶枯草溶菌素9的生物学功能

前蛋白转化酶枯草溶菌素9(proprotein convertase subtilisin/kexin type 9,PCSK9)基因属于前蛋白转化酶(PC)家族,是一个新发现不久的与胆固醇代谢相关基因.近年来,PCSK9在其生物学效应及疾病中的作用越来越受到重视.大量的研究表明,除通过调节低密度脂蛋白受体(LDLR)影响胆固醇代谢外,PCSK9还参与细胞凋亡,促进肝发育、再生,促进神经系统发育,影响神经系统分化并且与炎症过程以及糖尿病相关.本文对PCSK9功能方面最新研究进展进行了综述。     

Suppression of 5-lipoxygenase activity by anionic cholesterol derivatives

5-Lipoxygenase (5-LO) is a key enzyme involved into biosynthesis of leukotrienes (LTs), mediating the host defense system, and acting simultaneously as inflammatory agents. In this work the effect of anionic cholesterol derivatives on 5-LO activity has been investigated. Cholesterol sulfate activates human polymorphonuclear leukocytes (PMNL) and stimulates their adhesion to endothelium and collagen. Cholesterol sulfate and cholesterol phosphate suppressed leukotriene production in PMNL and in rat basophil leukemia (RBL-1) cell line as well as in homogenates of these cells. Kinetic characteristics of the effect of anionic cholesterol derivatives on leukotriene synthesis have been obtained. In all experiments cholesterol phosphate (charge-2) was shown to be more potent inhibitor than cholesterol sulfate (charge-1). We believe that this fact highlights the importance of negatively charged ester groups for suppression of 5-LO activity.     

长链非编码RNA在脂质代谢中的作用

脂质代谢是人体三大代谢之一，在激素等信号分子的调控下，脂质代谢处于稳定平衡的状态。当稳态被打破，血液中甘油三酯（triglyceride，TG）、胆固醇等水平发生变化，最终引起动脉粥样硬化（atherosclerosis，AS）、肥胖等脂质代谢疾病。长链非编码RNA（long noncoding RNA，lncRNA）是一组不具备蛋白质编码能力，长度大于200个核苷酸的RNA，近来研究发现，lncRNA与机体代谢、炎症和免疫系统以及血管功能的调控密切相关。大量文献表明，lncRNA参与脂质代谢调控，因而有望成为一些脂质代谢疾病的潜在治疗靶点。     

锌α2糖蛋白生物学功能

锌α2糖蛋白(zinc alpha-2 glycoprotein,ZAG)是一种42 kD可溶性蛋白,广泛存在于人体液中.自从ZAG被发现以来,有许多关于其结构及功能的报道.最初ZAG被认为与生殖及脂代谢有关,后又发现ZAG具有载体蛋白、核糖核酸酶的活性等功能.ZAG也参与免疫控制、细胞黏着、调节黑色素生成等.近年来,多项研究表明,ZAG与促进骨骼肌合成、抑制肿瘤细胞增殖、诊断早期癌症、抗糖尿病有关.本文从ZAG分子结构出发,对ZAG生物学功能的研究进展进行综述.