2 2 2 Cholesterol O Cholest-4-ene-3-one H O + ⎯ ⎯ ⎯ → H O ADHP (non-fluorescent) resorufin (fluorescent) A simple and sensitive enzymatic method for cholesterol quantifi cation in macrophages and foam cells (original) (raw)
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Determination of cholesterol in sera
Clinica Chimica Acta, 1983
Two methods are described for the determination of cholesterol in sera. The principle of these methods is that hydrogen peroxide, liberated by action of cholesterol esterase and cholesterol oxidase on cholesterol esters (or free cholesterol), is coupled under the catalytic effect of peroxidase with 4-hydroxyphenylacetic acid to form a fluorescent product, and with 3-methylbenzothiazoline-Zone hydrazone and 3-dimethylaminobenzoic acid to obtain a coloured compound. The main advantages of these procedures over comparable methods are their short reaction time, high sensitivity and low cost of the tests.
Studies of cell cholesterol efflux to human serum: relationship to serum components
Atherosclerosis, 1994
A cell culture system was employed to test a large number of samples of human serum for the ability to stimulate the efflux of cell cholesterol. The extent of efflux obtained with each specimen was correlated with the serum concentrations of cholesterol, triglycerides, apoprotein (apo) B, apo A-I, apo A-II, and lipoprotein subfractions (ie, high-density lipoprotein 2 [HDL 2 ], HDL 3 , lipoprotein [Lp] A-I, and LpA-I:A-II). In addition, the subsequent esterification of the released cholesterol and the distribution of the synthesized exogenous cholesteryl esters between HDL and low-density lipoprotein/ very-low-density lipoprotein provided estimates of the lecithin:cholesterol acyltransferase (LCAT) and cholesteryl ester transfer protein (CETP) activities of each serum. The values for these activities were analyzed for correlations with cell efflux and the various serum parameters. Cell cholesterol efflux best correlated with serum total HDL cholesterol values. HDL 2 and HDL 3 correlated about equally well with efflux, whereas LpA-I demonstrated a much greater association with
Journal of Lipid Research, 1996
Oxysterols arising from the diet or through lipid peroxidation may be important in the modulation of cellular cholesterol metabolism. In this study, the metabolism of one of the oxysterols, 25-hydroxycholestero1(250HC), was examined in 5774 and mouse peritoneal macrophages. Uptake of 250HC from serum was rapid and substantial. Esterification of the cellular 250HC was also rapid as was hydrolysis of pre-formed esters. Like cholesterol, 250HC was removed from cells by an extracellular acceptor such as high density lipoprotein. Unlike cholesterol, 250HC was also rapidly and extensively removed from cells by serum albumin, but not by ovalbumin. The differential removal of oxysterols and cholesterol from cells by albumin allows separation of cellular effects due to oxysterols and cholesterol. In order to understand more about this differential efflux of sterols, a computer model for sterol mass transport in cells was used to compare intracellular trafficking of cholesterol and 250HC. The rate constants determined by this model for movement of sterols between cytoplasm and plasma membrane were similar for both cholesterol and 250HC, whereas those for esterification and ester hydrolysis as well as those for bidirectional movement between plasma membrane and extracellular medium were greater for 250HC than for cholesterol. I For both sterols, the rate-limiting step for removal of cellular esters appeared to be the rate of cytoplasmic ester hydrolysis. As 250HC and cholesterol differ significantly in aqueous solubility, the similarity in their rate constants for movement between cytoplasm and plasma membrane is consistent with facilitation of transport between these two loci.
Journal of Pharmaceutical and Biomedical Analysis, 2013
A liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) assay was developed and qualified for analyzing 4-hydroxycholesterol and cholesterol in 5 l of human and mouse plasma. Stable isotope-labeled d7-analogs of both analytes were used as internal standards and 4.2% (w/v) human serum albumin in phosphate-buffered saline was used as the surrogate matrix for preparation of calibration curves and QCs. The assay is capable of quantification of 4-hydroxycholesterol and cholesterol from 5 to 500 ng/ml and 50 to 2000 g/ml, respectively, with acceptable accuracy and precision following evaluation of recovery of analytes, autosampler stability and potential contribution of chemical oxidation to the formation of 4-hydroxycholesterol. The final reconstituted solution was diluted for quantification of cholesterol typically present at 1000 fold higher concentration than 4-hydroxycholesterol in the same samples used for 4-hydroxycholesterol quantification. The successful quantification using a low plasma volume was achieved by quantification of total forms (free and conjugated) of both analytes after alkaline hydrolysis, followed by derivatization to form electrospray ionization-sensitive picolinyl esters, which upon collision-induced dissociation gave high mass precursor-product ion pair for selective detection by multiple reaction monitoring. In addition, chromatographic separation using a 16-min reversed phase gradient elution on a 1.9 m particle size, C18 column, overcame interference from other isobaric plasma oxysterols during detection by multiple-reaction monitoring. This assay was compared to an orthogonal enzymatic assay for cholesterol and all samples, but one, provided values that were within 10% of each other. In addition, this assay passed the incurred sample tests for both analytes in human and mouse plasma samples according to reported acceptance criteria for incurred sample reanalysis. The quantification of both analytes permitted the determination of 4-hydroxycholesterol compared to its ratio to cholesterol as an endogenous biomarker for CYP3A4/5 activity. The LC-ESI-MS/MS assay was also successfully applied to quantification of 4-hydroxycholesterol and cholesterol in plasma samples from untreated human and mice including FRG TM KO C57Bl/6 chimeric mice with humanized livers. The preliminary data indicated that the plasma 4-hydroxycholesterol concentrations or their ratio to cholesterol from mice including chimeric mice were higher than those from human.
Journal of Visualized Experiments, 2012
Cholesterol content of cells must be maintained within the very tight limits, too much or too little cholesterol in a cell results in disruption of cellular membranes, apoptosis and necrosis 1. Cells can source cholesterol from intracellular synthesis and from plasma lipoproteins, both sources are sufficient to fully satisfy cells' requirements for cholesterol. The processes of cholesterol synthesis and uptake are tightly regulated and deficiencies of cholesterol are rare 2. Excessive cholesterol is more common problem 3. With the exception of hepatocytes and to some degree adrenocortical cells, cells are unable to degrade cholesterol. Cells have two options to reduce their cholesterol content: to convert cholesterol into cholesteryl esters, an option with limited capacity as overloading cells with cholesteryl esters is also toxic, and cholesterol efflux, an option with potentially unlimited capacity. Cholesterol efflux is a specific process that is regulated by a number of intracellular transporters, such as ATP binding cassette transporter proteins A1 (ABCA1) and G1 (ABCG1) and scavenger receptor type B1. The natural acceptor of cholesterol in plasma is high density lipoprotein (HDL) and apolipoprotein A-I. The cholesterol efflux assay is designed to quantitate the rate of cholesterol efflux from cultured cells. It measures the capacity of cells to maintain cholesterol efflux and/or the capacity of plasma acceptors to accept cholesterol released from cells. The assay consists of the following steps. Step 1: labelling cellular cholesterol by adding labelled cholesterol to serum-containing medium and incubating with cells for 24-48 h. This step may be combined with loading of cells with cholesterol. Step 2: incubation of cells in serum-free medium to equilibrate labelled cholesterol among all intracellular cholesterol pools. This stage may be combined with activation of cellular cholesterol transporters. Step 3: incubation of cells with extracellular acceptor and quantitation of movement of labelled cholesterol from cells to the acceptor. If cholesterol precursors were used to label newly synthesized cholesterol, a fourth step, purification of cholesterol, may be required. The assay delivers the following information: (i) how a particular treatment (a mutation, a knock-down, an overexpression or a treatment) affects the capacity of cell to efflux cholesterol and (ii) how the capacity of plasma acceptors to accept cholesterol is affected by a disease or a treatment. This method is often used in context of cardiovascular research, metabolic and neurodegenerative disorders, infectious and reproductive diseases.
A simple method for the determination of the cholesterol esterase activity
Acta biochimica Polonica, 2013
The proposed method determines the activity of cholesterol esterase (CEH) and takes advantage of its ability to catalyze the hydrolysis of cholesterol esters naturally present in human serum. The assay is based on Allain's method of spectrophotometric determination of cholesterol by means of cholesterol oxidase, peroxidase, but using 3,5-dichloro-dihydroxybenzenesulfonic acid (DHBS) as phenolic chromogen and human serum as a source of substrate for the CEH as a novelty. Furthermore, it is characterized by low costs and high precision. It can be employed to control the activity of CE preparations used for the preparation of enzymatic kits for the determination of cholesterol or for screening of potential bacterial enzyme producers.