气相色谱法测定发酵液中胆固醇的含量

气相色谱法测定了发酵液中胆固醇的含量。采用Rtx-1（100%二甲基聚硅氧烷）毛细管色谱柱、FID检测器进行分析测定。当胆固醇浓度在0～200μg/mL范围内与色谱峰面积呈良好线性关系（r=0.9981）。方法的平均回收率为99.38%,相对标准偏差为0.75%（n=8）,最低检出限可达到0.816μg/mL。气相色谱法作为发酵液中胆固醇含量的快速测定方法,具有试剂用量少、操作简单、结果准确等特点。     

A fluorometric assay for cholesterol reductase activity.

A fluorometric method for the assay of cholesterol reductase activity from pea leaves (Pisum sativum) is presented. This method is based on the decrease in relative fluorescence occurring as a result of the oxidation of NADH when cholesterol is reduced catalytically to coprostanol by cholesterol reductase. The reaction mixture consisted of micellar cholesterol, NADH, and cytosol of pea leaves in a phosphate buffer. After incubation for 1 h, the reaction mixture were diluted with 2-(N-cyclohexylamino)ethanesulfonic acid buffer (50 mM, pH 10.0) to an appropriate concentration for NADH quantification. The relative fluorescence was measured at an excitation wavelength of 360 nm and at an emission wavelength of 460 nm. This fluorometric method is relatively rapid, simple, and inexpensive. The results obtained show close correlation (R = 0.997) with those obtained by the more time-consuming and expensive radiometric method for assay of cholesterol reductase activity. Results suggest that the fluorometric method is useful for the accurate determination of cholesterol reductase activity in biological specimens.     

Production of calcium-mobilizing metabolites by a novel member of the ADP-ribosyl cyclase family expressed in Schistosoma mansoni

ADP-ribosyl cyclases are structurally conserved enzymes that are best known for catalyzing the production of the calcium-mobilizing metabolite, cyclic adenosine diphosphate ribose (cADPR), from nicotinamide adenine dinucleotide (NAD(+)). However, these enzymes also produce adenosine diphosphate ribose (ADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP(+)), both of which have been shown to modulate calcium mobilization in vitro. We have now characterized a new member of the cyclase family from Schistosoma mansoni, a member of the Platyhelminthes phylum. We show that the novel NAD(P)(+) catabolizing enzyme (NACE) expressed by schistosomes is structurally most closely related to the cyclases cloned from Aplysia but also shows significant homology with the mammalian cyclases, CD38 and CD157. NACE expression is developmentally regulated in schistosomes, and the GPI-anchored protein is localized to the outer tegument of the adult schistosome. Importantly, NACE, like all members of the cyclase family, is a multifunctional enzyme and catalyzes NAD(+) glycohydrolase and base-exchange reactions to produce ADPR and NAADP(+). However, despite being competent to generate a cyclic product from NGD(+), a nonphysiologic surrogate substrate, NACE is so far the only enzyme in the cyclase family that is unable to produce significant amounts of cADPR (<0.02% of reaction products) using NAD(+) as the substrate. This suggests that the other calcium-mobilizing metabolites produced by NACE may be more important for calcium signaling in schistosomes. Alternatively, the function of NACE may be to catabolize extracellular NAD(+) to prevent its use by host enzymes that utilize this source of NAD(+) to facilitate immune responses.     

人血清中胆固醇近红外光谱快速检测初步研究

以全自动生化分析仪测定结果为参考值,采用傅利叶变换近红外透射光谱技术,结合偏最小二乘法,建立人血清中胆固醇和甘油三酯的定标模型。利用内部交叉验证和自动优化功能对预测模型进行了优化,确定了最优建模参数。模型对人血清中胆固醇和甘油三酯定标样品集的预测值与参考值的相关系数r分别为0．9011、0．9593,预测校正标准误RMSECV分别为15．0mg／dL,21．6mg／dL。表明利用近红外光谱分析技术实现血清中胆固醇和甘油三酯快速检测是可行的。     

A rapid prognostic electrophoretic assay for serum lipid conjugates in carcinoma uterine cervix and breast

The levels of cholesterol and lipid associated sialic acid in sera of patients with carcinoma uterine cervix and breast cancer were determined using agarose electrophoresis and precipitation method. Elevated level of lipid associated sialic acid was found, that was specifically reflected only in LDL and VLDL fractions. In carcinoma uterine cervix patients, cholesterol in low density lipoprotein increased with corresponding decrease in high density lipoprotein and there was no change in total as well as VLDL cholesterol content. But breast cancer patients were found to have higher concentrations of both total and high density lipoprotein cholesterol. A good positive correlation was found between electrophoresis and precipitation methods for the lipoprotein analysis. We recommend electrophoretic method for rapid routine analysis.     

Rapid isolation of VLDL subfractions: assessment of composition and susceptibility to copper-mediated oxidation

VLDLs, synthesized and released by the liver, are a heterogeneous group of particles of varying composition and metabolic fates. A method is described for the rapid isolation of VLDL into four subfractions (A-D) and assessment of their susceptibility to oxidation. The total isolation procedure required less than 3.5 h, and was achieved by gradient ultracentrifugation. Each subfraction was assessed for triglyceride, cholesterol, and apolipoprotein B (apoB) composition and for the presence of contaminants such as albumin and urate. The oxidation potential, in the presence of copper ions, of each subfraction was also assessed. This rapid procedure produced VLDL fractions analogous to those produced by a previously reported but more prolonged isolation method. Comparison of the two procedures demonstrated that lipid and apoB were similar, while the rapid procedure produced subfractions void of albumin and urate contamination and lower in preformed hydroperoxides. Compositional changes were found between the subfractions: as the subfractions became smaller and more dense (A-->D), there was a decrease in the ratio of triglyceride to apoB and an increase in the ratio of cholesterol to apoB, also arachidonic acid was increased in subfraction D compared with subfractions A, B, and C. The smaller subfractions were more susceptible to oxidation, a trend similar to that reported previously for the oxidation of LDL subfractions.     

Measurement of oxysterols and alpha-tocopherol in plasma and tissue samples as indices of oxidant stress status

Oxidant stress seems to play a role in several setting of human pathology, such as atherosclerosis, cancer, and aging. The study of oxidant stress in human disease should be based on the evaluation of either sensitive and specific markers of enhanced oxidant stress, such as oxysterols, or antioxidant defense, by measuring alpha-tocopherol. We have developed a rapid method to measure the oxysterols 7beta-hydroxycholesterol and 7-ketocholesterol in plasma (50 healthy subjects) and tissue as an index of oxidant stress in vivo, and from the same sample alpha-tocopherol content. The mean plasma concentration of 7beta-hydroxycholesterol and 7-ketocholesterol was 4.6+/-1.1 and 13.4+/-7.6 ng/mL, respectively. Plasma alpha-tocopherol concentration was 5.8+/-1.0 micromol/mol cholesterol. Samples from atherosclerotic plaques contained 20 times more cholesterol, about 45 times higher oxysterols levels, and 600 times more alpha-tocopherol compared to normal arteries. No significant difference in cholesterol and oxysterol content was observed between cirrhotic and normal liver. However, cirrhotic liver contained significantly smaller concentration of alpha-tocopherol compared to normal liver. In conclusion, we have developed a rapid and reliable method for the assay of cholesterol oxidation products and alpha-tocopherol in plasma and tissue useful for estimation of oxidant stress/antioxidant balance.     

Enzymatic assay of cholesterol by reaction rate measurements

A dynamic method for free and total cholesterol assay based on the oxidation of cholesterol by cholesterol oxidase, and conversion of cholesteryl oleate to cholesterol by cholesterol esterase is discussed in this article. The reaction conditions for total cholesterol assay were a temperature of 310 K and pH of 7.4. For conversion of cholesteryl oleate to cholesterol, the samples were incubated with 0.6 unit/mL of cholesterol esterase and 0.02 g/mL of taurocholate. The determination of initial reaction rates in the oxidation of free cholesterol, which is directly related to the cholesterol concentration, was found to be rapid, reliable, and inexpensive. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 391-396, 1997.     

Measurement of cholesterol of major serum lipoprotein classes by anion-exchange HPLC with perchlorate ion-containing eluent

We have developed a high-performance liquid chromatography (HPLC) method for measurement of cholesterol in the major classes of serum lipoproteins, i.e., HDL, LDL, IDL, VLDL, and chylomicrons. Lipoproteins in serum were separated on a column containing diethylaminoethyl-ligand nonporous polymer-based gel by elution with a step gradient of sodium perchlorate concentration, and detected by post-column reaction with a reagent containing cholesterol esterase and cholesterol oxidase. The within-day assay and between-day assay coefficients of variation for cholesterol concentration in lipoproteins were in the ranges of 0.9-6.4% and 1.1-11.9%, respectively. The correlation coefficients between the values of HDL, LDL, IDL, VLDL, and chylomicron cholesterol measured by the HPLC method and those estimated by an ultracentrifugation method were 0.892, 0.921, 0.840, 0.930, and 0.873, respectively. Values of remnant-like particle cholesterol measured by an immunoseparation technique (Japan Immunoresearch Laboratories, Japan) were significantly correlated with VLDL and chylomicron cholesterol values measured by the HPLC method (r = 0.883 and r = 0.729, respectively).This rapid and accurate HPLC method was successfully applied to the analysis of plasma lipoproteins of patients with hyperlipidemia.     

Schistosoma mansoni NAD+ catabolizing enzyme: Identification of key residues in catalysis

Schistosoma mansoni NAD⁺ catabolizing enzyme (SmNACE), a distant homolog of mammalian CD38, shows significant structural and functional analogy to the members of the CD38/ADP-ribosyl cyclase family. The hallmark of SmNACE is the lack of ADP-ribosyl cyclase activity that might be ascribed to subtle changes in its active site. To better characterize the residues of the active site we determined the kinetic parameters of nine mutants encompassing three acidic residues: (i) the putative catalytic residue Glu202 and (ii) two acidic residues within the ‘signature’ region (the conserved Glu124 and the downstream Asp133), (iii) Ser169, a strictly conserved polar residue and (iv) two aromatic residues (His103 and Trp165). We established the very important role of Glu202 and of the hydrophobic domains overwhelmingly in the efficiency of the nicotinamide–ribosyl bond cleavage step. We also demonstrated that in sharp contrast with mammalian CD38, the ‘signature’ Glu124 is as critical as Glu202 for catalysis by the parasite enzyme. The different environments of the two Glu residues in the crystal structure of CD38 and in the homology model of SmNACE could explain such functional discrepancies. Mutagenesis data and 3D structures also indicated the importance of aromatic residues, especially His103, in the stabilization of the reaction intermediate as well as in the selection of its conformation suitable for cyclization to cyclic ADP-ribose. Finally, we showed that inhibition of SmNACE by the natural product cyanidin requires the integrity of Glu202 and Glu124, but not of His103 and Trp165, hence suggesting different recognition modes for substrate and inhibitor.     

Method for quantitating cholesterol in subfractions of serum lipoproteins separated by gradient gel electrophoresis

Extensive heterogeneity in particle size distribution of serum lipoproteins of baboons was resolved by a procedure that combined Sudan black B prestaining, polyacrylamide gradient gel electrophoresis (GGE), and quantitative densitometry. Each densitometric scan represented a continuous distribution of the relative amount of cholesterol in a serum sample, as a function of the lipoprotein particle size. For analytical purposes, each scan was divided into 12 fractions, representing 12 particle size ranges. The relationship between the estimated cholesterol concentrations in the summed GGE/densitometric fractions corresponding to very low-density lipoproteins (VLDL) + low-density lipoproteins (LDL) and those corresponding to high-density lipoproteins (HDL) and concentrations measured by the heparin-Mn2+ precipitation/enzymatic procedure was linear over a broad range. However, a systematic overestimation of HDL cholesterol concentration and an underestimation of VLDL + LDL cholesterol concentration was apparent. Therefore, correction factors were developed for adjusting the estimates of VLDL + LDL and HDL cholesterol concentrations obtained by the GGE/densitometric method. This analytical method is rapid, repeatable, economical, and useful for genetic and dietary research in which cholesterol concentrations in multiple particle size ranges of lipoproteins must be measured in large numbers of samples. It also is adaptable to immunoblotting procedures for detecting the distribution of specific apolipoproteins among the size-resolved lipoproteins.     

Assay of cholesterol 7 alpha-hydroxylase activity in rat hepatocytes in primary monolayer culture

A sensitive and precise method is described to assay cholesterol 7 alpha-hydroxylase activity in homogenates of rat hepatocytes cultured in monolayers for up to 76 h. The assay is based on measurement of the amount of radioactive cholesterol converted into 7 alpha-[14C]-hydroxycholesterol. Since no subcellular fractionation was applied to measure enzyme activity, this method is rapid and can be performed with cell protein, corresponding to as little as 1 to 2 million hepatocytes. Optimal assay conditions were determined and the reproducibility of this cholesterol 7 alpha-hydroxylase determination was established. Exogenous cholesterol (105 microM), solubilized in Tween 80, was added to saturate the enzyme, giving an apparent Km of 56 microM. Under these conditions, 70% of the cholesterol present in the homogenates is directly accessible to the cholesterol 7 alpha-hydroxylase. The detection limit of the assay was found to be about 10 pmol per incubation. A time course of the cholesterol 7 alpha-hydroxylase activity in cultured hepatocytes revealed that after an initial loss of approximately 60% of the activity as compared with 287 pmol/h/mg for freshly isolated cells, the enzyme activity was increased to the initial level in hepatocytes cultured for 52 h. This result and the finding that the cholesterol 7 alpha-hydroxylase activity was diminished by 94% after a 24-h incubation with 5 microM cycloheximide suggest that the enzyme activity is associated with de novo protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microquantification of cholesterol and cholesteryl esters in rat peritoneal macrophages by reverse-phase high-performance liquid chromatography

A simple and rapid method for the microquantification of cholesterol and cholesteryl esters by reverse-phase high performance liquid chromatography has been established. Comparison of elution patterns of authentic cholesterol and cholesteryl esters revealed that a mu Bondasphere reverse-phase C8 (300-A) column was more suitable than a corresponding reverse-phase C4 or C18 column in terms of rapidity and sensitivity. Recovery of cholesterol and cholesteryl esters from a C8 column was greater than 98% when determined either by radioactive cholesterol and cholesteryl oleate or by cholesteryl heptadecanoate. The sensitivity of the quantification ranged from 5 ng to 50 micrograms for both cholesterol and cholesteryl esters. This method was applied to determination of cellular cholesterol and cholesteryl esters of rat peritoneal macrophages. Lipid extracts of these cells were found to contain 38.01 +/- 2.60 micrograms of cholesterol and 3.18 +/- 0.36 micrograms of cholesteryl esters per milligram of cell protein. When the cells were loaded with cholesteryl esters by incubation for 24 h with various concentrations of acetylated low-density lipoprotein, a cellular level of cholesteryl esters showed a dose-dependent increase and reached a maximal level of 106.60 +/- 3.05 micrograms/mg cell protein. Thus, the present method is useful for the microquantification of cholesterol and cholesteryl esters from lipid extracts of biological samples.     

Vesicle aggregation in model systems of supersaturated bile: relation to crystal nucleation and lipid composition of the vesicular phase

The presence of small vesicles composed of phospholipid and cholesterol has recently been demonstrated in super-saturated model and in dilute native human biles by several groups using differing methods. Among compositional factors shown to favor spontaneous vesicle formation and prolong the cholesterol monohydrate nucleation time in model bile systems are dilution, a raised cholesterol saturation index (CSI), and a low bile salt/phospholipid ratio. Time-lapse video-enhanced microscopy of a series of model bile systems representing systematically designed variations in the above factors revealed strong evidence for an essential linkage between antecedent vesicle aggregation and subsequent crystal nucleation. Stability of vesicles was inversely related to their degree of cholesterol saturation, i.e., the greater the degree of vesicular cholesterol saturation, the less their stability (metastability). Instability of vesicles was reflected by their early aggregation followed by rapid cholesterol crystal nucleation. The lowest degree of vesicular cholesterol saturation was found in dilute systems which also exhibited the greatest metastability despite a high degree of cholesterol solubility (raised CSI). Conversely, the more concentrated and least metastable systems exhibited both rapid vesicle aggregation and rapid onset of crystal nucleation. These systems, while influenced by the other compositional factors, were found to have a high degree of vesicular cholesterol saturation, i.e., cholesterol/phospholipid molar ratio = 2.0. An additional finding was the extreme variability in the proportionate distribution of total solution cholesterol distributed to the vesicular phase, i.e., from zero to as high as 37%. Higher solute concentration, raised bile salt/lecithin ratio, and raised CSI were interactive and almost equally capable of increasing the proportionate amount of cholesterol in the vesicular phase. In conclusion, lipid compositional differences in model bile systems drastically affect the cholesterol saturation of spontaneously formed phospholipid-cholesterol vesicles. This effect, in turn, exerts a potent influence upon the metastability of vesicles, subsequently affecting the cholesterol crystal nucleation time.     

Amperometric detection of cholesterol using an indirect electrochemical oxidation method

The indirect electrochemical oxidation method using bromine species in an organic media for measuring cholesterol was developed. The electrochemical behaviors of cholesterol were examined by cyclic voltammetry in a potential range of −0.5 to 2.0 V (vs. Ag/AgCl/saturated KCl). The polarization curve of the steady-state current in the applied potential range of 0-2.0 V is reported. The obtained kinetic parameters for the catalytic oxidation of cholesterol support the assumption that positive bromine species can be generated from bromine by undergoing two consecutive electrochemical oxidation steps. The positive bromine acts both as electron mediators and as electrocatalysts. Amperometric detection with an anodic current was investigated, and the calibration curve exhibited a linear relationship between the steady-state current and the concentration of cholesterol in the range of 30-200 μM, from which the sensitivity was calculated to be about 0.2 μA/cm²/μM. Moreover, the amperometric current followed Michaelis-Menten’s enzymatic model for cholesterol concentrations in the range of 30 μM to 5 mM, which can be applied for cholesterol rapid detection in processed foods.     

Regulation of fibroblast mitochondrial 27-hydroxycholesterol production by active plasma membrane cholesterol

Side chain oxysterols are cholesterol derivatives thought to signal the abundance of cell cholesterol to homeostatic effector proteins. Here, we investigated how plasma membrane (PM) cholesterol might regulate 27-hydroxycholesterol (HC) biosynthesis in cultured fibroblasts. We showed that PM cholesterol was a major substrate for 27-HC production. Biosynthesis commenced within minutes of loading depleted cells with cholesterol, concurrent with the rapid inactivation of hydroxy-3-methylglutaryl CoA reductase (HMGR). 27-HC production rose ∼30-fold in normal and Niemann-Pick C1 fibroblasts when PM cholesterol was increased by ∼60%. 27-HC production was also stimulated by 1-octanol, which displaces PM cholesterol from its phospholipid complexes and thereby increases its activity (escape tendency) and elevates its intracellular abundance. Conversely, lysophosphatidylserine and U18666A inhibited 27-HC biosynthesis and the inactivation of HMGR, presumably by reducing the activity of PM cholesterol and, therefore, its circulation to mitochondria. We conclude that, in this in vitro system, excess (active) PM cholesterol rapidly reaches mitochondria where, as the rate-limiting substrate, it stimulates 27-HC biosynthesis. The oxysterol product then promotes the rapid degradation of HMGR, along with other homeostatic effects. The regulation of 27-HC production by the active excess of PM cholesterol can thus provide a feedback mechanism in the homeostasis of PM cholesterol.     

Combination of an enzymatic method and HPLC for the quantitation of cholesterol in cultured cells.

The study of the cellular events that lead to the foam cell formation requires the development of fast, accurate, and sensitive methods to quantify cholesterol in cultured cells. Here we describe a procedure that allows the rapid determination of free and total cholesterol in a reduced number of cells, which makes it very suitable for cholesterol determination in cell cultures. The method consists of the enzymatic conversion of cholesterol to cholest-4-ene-3-one by cholesterol oxidase followed by the analysis of the sample by high performance liquid chromatography (HPLC) to detect this oxidized product. Due to the relatively high wavelength at which cholest-4-ene-3-one has its maximum absorption (240 nm), other cellular components do not interfere with the chromatographic procedure and prior lipid extraction is not required. Moreover, the duration of each chromatogram is about 3 min, contributing to the celerity of the method. All the cholesteryl esters used (oleate, palmitate, stearate and linoleate) were quantitatively hydrolyzed by incubation with cholesterol esterase; this was observed to occur with both pure standards and in cell homogenates. Sensitivity is enough to allow the determination of free and total cholesterol in less than 5 x 10(3) cells. We have applied this method to human monocyte-derived macrophages and the values obtained for free and total cholesterol are in close agreement with published data.     

A rapid radioassay procedure for plasma lecithin-cholesterol acyltransferase

A rapid and accurate single step procedure is described for the assay of lecithin-cholesterol acyltransferase activity. After incubation, using radiolabeled cholesterol as the substrate, an ethanolic solution of digitonin is added directly to the incubation mixture to extract the lipids. Excess cholesterol is then added, and the labeled cholesterol-digitonide along with denatured proteins are sedimented by low speed centrifugation, leaving the labeled esterified cholesterol in solution. An aliquot of the supernatant is counted in an aqueous scintillation mixture. The method correlates well with the established thin-layer chromatographic procedure using either lecithin-cholesterol vesicles or heat-inactivated plasma as the substrate for lecithin-cholesterol acyltransferase.     

Method for rapid separation of liposome-associated doxorubicin from free doxorubicin in plasma

To understand and predict the efficacy and/or toxicity of liposomal drugs in vivo, it is essential to have rapid, reliable methods of separating and quantitating both the free and the liposomal forms of the drug. A method using solid-phase extraction chromatography columns was developed to separate and quantitate unencapsulated doxorubicin and liposome-associated doxorubicin in plasma following the intravenous injection of liposomal doxorubicin. The method facilitated the recovery and quantitation of free and liposomal drug. The separation and recovery of doxorubicin were linear across the entire range of possible mixtures (0 to 100%) of the two forms of the drug in plasma. Free drug and liposomal drug were readily separated for liposomal doxorubicin systems varying in size (0.1-1.0 microns) and lipid composition (egg yolk phosphatidylcholine/cholesterol and distearylphosphatidylcholine/cholesterol). The method is rapid and allows for multiple samples to be processed simultaneously.     

SELECTIVE LABELLING OF TWO PHASES OF AXONAL TRANSPORT OF CHOLESTEROL IN THE CHICK OPTIC SYSTEM

Abstract— In the chick optic system cholesterol is axonally transported in two phases which appear to take their cholesterol from different cellular pools. The intraocular injection of radioactire cholesterol results in the specific labelling of the slow phase which carries cholesterol in the unesterificd form and appears to move at the same rate as the slow phase of protein transport (R ostas et al. , 1975). The intraocular injection of radioactive mevalonic acid, a metabolic precursor of cholesterol, results in the preferential labelling of a more rapid phase of axonal transport which also carries cholesterol in the unesterified form and is first detected at the optic tectum 10 h after the injection. It is likely that this rapid phase travels at the same rate as the rapid phase of protein transport and that the delayed arrival at the tectum is due to a lag time in the retina caused by the synthesis of cholesterol and its packaging for transport. Because the individual pools for the two transport phases can be selectively labelled, the retina and optic nerve provide a unique model system in which the metabolic turnover, intracellular compartmentalization and intracellular transport of cholesterol can be studied.