| For medical practitioners and the general public - Cholesterol Journal Article Catalog. |
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| Determination of cholesterol in atherosclerotic plaques using near infrared diffuse reflection spectroscopy Jaross, W., V. Neumeister, et al. (1999), Atherosclerosis 147(2): 327-37. Abstract: The aim of this investigation was to examine whether near infrared diffuse reflection spectroscopy is an acceptable tool for the determination of cholesterol content in atherosclerotic plaques. Using an FT-spectrophotometer (lambda=1000-2500 nm) and fiberoptic systems (d=4 mm), the cholesterol content could be determined in mixtures of the primary compounds of the aortic wall with acceptable precision. Considering the inhomogeneous distribution of cholesterol and cholesterol esters in atherosclerotic plaques the determination of total cholesterol using this method is of acceptable efficacy, even though the calibration procedure did not reflect the composition correctly. Using an energy dose of less than 100 mW/cm(2) to avoid damage to endothelial cells, arterial tissue of about 170-200 microm thickness attenuates the reflected NIRS signal by up to 50%. Cholesterol levels could be determined accurately in atherosclerotic lesions in human aortic specimens obtained by autopsy. The correlation coefficient between the NIRS results and those of HPLC analysis calculated in the investigation of 82 different areas of 18 human aortic specimens was 0.926 (y=0.869x+0. 771, external validation). Acceptable results were also achieved by means of a coronary-catheterlike fiberoptic strand (d=l mm), despite the worsened signal/noise ratio. The results show that the development of a coronary catheter using NIRS appears to be possible in principle. |
| Determination of cholesterol in lipoprotein fractions by agarose gel electrophoresis Yan, S. K., F. Q. Ren, et al. (2001), Zhongguo Yi Xue Ke Xue Yuan Xue Bao 23(1): 93-6. Abstract: OBJECTIVE: To evaluate a single step electrophoresis for quantitative determination of cholesterol of high-, low-, very-low-density lipoprotein(HDL, LDL, VLDL) and fast pre-beta lipoprotein lipoprotein (a), Lp(a). METHODS: Quantification of lipoprotein cholesterol was performed by enzymatic staining of cholesterol in a new agarose gel electrophoresis method that allows the separation of LDL, VLDL, HDL, and Lp(a) by Helena REP system. The results of electrophoresis method were compared with those by traditional method like PTA-Mg2+ precipitation method for HDL-C, PVS precipitation method for LDL-C, and Immunoturbidimetric assay(ITA) method for Lp(a). RESULTS: Within-runs CV were 5.16%-7.46%, 1.26%-3.28% and 3.78%-5.86% for VLDL-C, LDL-C and HDL-C, respectively. Between-runs CV were 8.35%-11.25%, 2.78%-4.08% and 4.23%-6.36%, respectively. The linearity of this method was up to 10.35 mmol/L total cholesterol. The recoveries were 90.3%, 94.3% and 89.6%, respectively. No interference were observed when bilirubin(< 342 mumol/L), hemoglobin(< 20 g/L) or triglyceride(< 11.0 mmol/L) were added to pooled serum, respectively. There was good agreement between methods, with r = 0.9557 for HDL-C(electrophoresis method vs PTA-Mg2+ precipitation method), r = 0.9609 for LDL-C(electrophoresis method vs PVS precipitation method) and r = 0.9235 for Lp(a)-C (electrophoresis method) vs Lp(a) (ITA method). CONCLUSIONS: The electrophoresis method offers a simple and inexpensive means of simultaneously measuring HDL-C, VLDL-C, Lp(a)-C and LDL-C. |
| Determination of cholesterol in natural bezoar by gas chromatography Zhang, Q., K. Yan, et al. (1991), Zhongguo Zhong Yao Za Zhi 16(7): 426-8, 448. Abstract: A gas chromatographic method for the determination of free and total cholesterol in natural bezoar has been established in this report. The method is simple, specific and accurate. The free and total cholesterol contents in three kinds of bezoar are between 0.072% to 0.214% and 0.546% to 0.608% respectively. |
| Determination of cholesterol in sub-nanomolar quantities in biological fluids by high-performance liquid chromatography Murata, M. and T. Ide (1992), J Chromatogr 579(2): 329-33. Abstract: A highly sensitive method for the determination of cholesterol in biological fluids is described. Unsaponifiable lipids from rat serum and thoracic duct lymph chylomicron samples were treated with cholesterol oxidase. The product of the enzymatic reaction, delta 4-cholestenone, was analysed by normal-phase high-performance liquid chromatography (HPLC) using hexane-isopropanol (95:5, v/v) as a mobile phase and detected with a UV spectrophotometer at 240 nm. When the standard samples containing varying amounts of cholesterol (0.15-3 nmol) were treated with cholesterol oxidase and analysed by HPLC (injected amounts 0.09-1.8 nmol of cholesterol), the peak areas increased proportionally with the amounts of authentic cholesterol with a correlation coefficient of 0.996. The values in these biological fluids determined by the HPLC method were identical to those obtained by enzymatic-colorimetric or gas chromatographic methods. Moreover, the detection limit (0.09 nmol) of the present method (0.15 nmol are required for the sample preparation) is lower than those of conventional methods (approximately 30 nmol). Because of the excellent sensitivity and reproducibility, this method is well suited for the determination of cholesterol in biological fluids where cholesterol concentration is low. |
| Determination of cholesterol levels Azeveda Ade, C. (1990), Arq Bras Cardiol 54(4): 245-6. |
| Determination of cholesterol oxidation products in human plasma by isotope dilution-mass spectrometry Dzeletovic, S., O. Breuer, et al. (1995), Anal Biochem 225(1): 73-80. Abstract: A method based on isotope dilution-mass spectrometry was developed for the determination of nine cholesterol oxidation products in human plasma. The cholesterol oxidation products determined were cholest-5-ene-3 beta,7 alpha-diol, cholest-5-ene-3 beta,7 beta-diol (7 alpha- and 7 beta-hydroxycholesterol, respectively), 3 beta-hydroxycholest-5-en-7-one(7-oxocholesterol),5,6 alpha-epoxy-5 alpha- cholestan-3 beta-ol (cholesterol-5 alpha,6 alpha-epoxide),5,6 beta-epoxy-5 beta-cholestan-3 beta-ol (cholesterol-5 beta,6 beta-epoxide), (cholesterol-5 beta,6 beta-epoxide), cholestane-3 beta,5 alpha,6 beta-triol, cholest-5-ene-3 beta,24-diol (24-hydroxycholesterol), cholest-5-ene-3 beta,25-diol (25-hydroxycholesterol), and cholest-5-ene-3 beta,27-diol (27-hydroxycholesterol). A corresponding deuterium-labeled internal standard, containing 3 to 6 deuterium atoms, was synthesized for each cholesterol oxidation product except 5 beta,6 beta-epoxycholesterol which was determined using the internal standard for 5 alpha,6 alpha-epoxycholesterol. Plasma from 31 healthy volunteers was analyzed by the new method and 27-, 24-, and 7 alpha-hydroxycholesterol were the most abundant cholesterol oxidation products (mean values 154, 64, and 43 ng/ml, respectively). The other oxysterols determined were present in concentrations lower than 30 ng/ml. Males had higher 27-hydroxycholesterol concentrations in plasma than females. The 5,6-oxygenated products were present mainly unesterified while the other oxidation products were mostly in esterified form. |
| Determination of cholesterol oxidation products in milk powder and infant formulas by gas chromatography and mass spectrometry Przygonski, K., H. Jelen, et al. (2000), Nahrung 44(2): 122-5. Abstract: In this paper a method for the cholesterol oxidation products (oxysterols) determination in milk powder and infant formulas has been presented. In the sample preparation step lipids transesterification has been performed. The recoveries of oxysterols have been determined and ranged from 94.2% to 99.9% for all but 20a-hydroxy cholesterol (74.2%). Detection limits were 0.018-0.034 ppm and the relative standard deviations (RSD) values were 4.6%-18.3%. The method has been utilized for the determination of oxysterols in milk-based infant formulas and milk powder available on the market. The concentration of oxysterols was between 0.04 and 4.20 ppm of a lipid extract fraction. |
| Determination of cholesterol oxides in processed food using high-performance liquid chromatography-mass spectrometry with atmospheric pressure chemical ionisation Razzazi-Fazeli, E., S. Kleineisen, et al. (2000), J Chromatogr A 896(1-2): 321-34. Abstract: The present work describes the development and application of an on-line atmospheric pressure ionisation (APCI) LC-MS interface for the simultaneous determination of seven toxicologically relevant cholesterol oxides (7alpha-hydroxycholesterol, 7beta-hydroxycholesterol, 25-hydroxycholesterol, 7-ketocholesterol, 5,6alpha-, 5,6beta-epoxycholesterol and cholestan-3beta,5alpha,6beta-triol). The HPLC method has been optimised to reach better separation of all tested compounds. The influences of APCI parameters (nebulising temperature, cone voltage, source temperature) on signal intensity and fragmentation pattern were investigated for all tested cholesterol oxides compounds. This is the first report on optimisation and determination of two compounds 7alpha-hydroxycholesterol and 5,6beta-epoxycholesterol in processed food using LC-MS. After extraction with hexane, clean-up was carried out using solid-phase extraction on a silica column. For the chromatographic separation of cholesterol oxides an Aquasil C18 column was used with acetonitrile-methanol (60:40) as mobile phase. For the first time we report the use of such a C18 column with a relatively hydrophilic nature for the separation of cholesterol oxides. APCI-MS detection was then applied in selected ion monitoring and positive ion modes by using the molecular ions and the main fragments. The developed method shows good linearity, high repeatability and good recovery for all tested cholesterol oxides. The method was applied for determination of seven selected cholesterol oxidation products in different foodstuffs such as butter, butteroil, lard and egg powder. |
| Determination of cholesterol-lowering potential of minor dietary components by measuring apolipoprotein B responses in HepG2 cells Kurowska, E. M. (2001), Methods Enzymol 335: 398-404. |
| Determination of hepatic acyl-coenzyme A-cholesterol acyltransferase activity in LPN hamsters: a model for cholesterol gallstone formation Smith, J. L. and C. Lutton (1997), J Gastroenterol Hepatol 12(12): 877-86. Abstract: Acyl-coenzyme A-cholesterol acyltransferase (ACAT) catalyses the esterification of cholesterol with long-chain fatty acyl-coenzyme A derivatives and has been implicated in the development of cholesterol gallstones. In this study we have examined several key components of the hepatic ACAT assay in order to develop a reliable and sensitive ACAT assay for LPN hamsters, a breed of golden Syrian hamster which has been characterized recently by this laboratory as a particularly good model for studying the pathogenesis of cholesterol gallstones. The newly developed ACAT assays were subsequently used to examine whether hepatic ACAT activity is altered in this animal model. Important new methodological findings were: (i) ACAT activity displayed two pH optima, one at 7.0 when assayed using endogenous cholesterol as substrate, and the other at about pH 8.5-9.0 when assayed in the presence of exogenous cholesterol; (ii) ACAT activity increased markedly when exogenous cholesterol was delivered to ACAT in Tween 80 (125-fold) or hydroxypropyl-beta-cyclodextrin (200-fold) in contrast to the use of cholesterol/phosphatidylcholine liposomes (9-fold); (iii) the addition of dithiothreitol, but not reduced glutathione, to the assay mixture resulted in a marked decrease in ACAT activity. Using the optimal assay conditions (exogenous cholesterol added), hepatic ACAT activity was shown to be significantly reduced in hamsters fed a high sucrose lithogenic diet compared with controls (587+/-42 vs 737+/-44 pmol/min per mg; P=0.025). In contrast, ACAT activity measured using endogenous cholesterol as a substrate was greater in sucrose-fed hamsters compared with controls (22.3+/-2.5 vs 13.2+/-2.9 pmol/min per mg; P= 0.030). These results highlight the importance of using an ACAT activity assay which has been well characterized and supports the hypothesis that the pathogenesis of cholesterol gallstones in LPN hamsters is related to an altered hepatic cholesterol metabolism. |
| Determination of membrane cholesterol in normal and pathological red blood cells Macchia, T., R. Mancinelli, et al. (1991), Clin Chim Acta 199(1): 59-67. |
| Determination of mRNA levels of cholesterol biosynthesis enzymes and LDL receptor using ribonuclease protection assay Shimokawa, T., Y. Kawabe, et al. (1995), J Lipid Res 36(9): 1919-24. Abstract: We designed a rapid method for determining mRNA content of cholesterol biosynthesis enzymes and LDL receptor (LDLR) using a ribonuclease protection assay (RPA). 32P-labeled cRNA fragments for genes of human LDLR and the enzymes HMG-CoA synthase (HMGS), HMG-CoA reductase (HMGR), mevalonate kinase (MK), farnesyl pyrophosphate synthase (FPPS), and squalene synthase (SQS) were prepared by in vitro transcription. Total RNA prepared from HepG2 cells was hybridized with the cRNA probe and the hybridized mRNA was determined under protection from RNase digestion. Probe content used in this assay was excess in determining the desired mRNA in total RNA, and surplus probes were completely digested using RNase under standard conditions. When cells were cultured in DMEM supplemented with 10% fetal calf serum (FCS), mRNA levels of FPPS, SQS, and LDLR were about 4- to 7-fold higher than those of HMGS, HMGR, and MK. On incubation with DMEM supplemented with 10% lipoprotein-deficient serum (LPDS) for 8 h, all messenger RNA levels increased 1.5- to 3.5-fold. In addition, when the HMG-CoA reductase inhibitor compactin was added to 10% LPDS-DMEM, these levels increased even further and the change in mRNA level seemed to differ between the enzymes and LDLR. From these results, we conclude that RPA is a useful method for determining the very small amount of mRNA level of cholesterol biosynthesis enzymes and LDLR in the cell. |
| Determination of plasma free fatty acids, free cholesterol, cholesteryl esters, and triacylglycerols directly from total lipid extract by capillary gas chromatography Lohninger, A., P. Preis, et al. (1990), Anal Biochem 186(2): 243-50. Abstract: An accurate capillary gas chromatographic method using different internal standards for determining free fatty acids, cholesterol, cholesteryl esters, and triacylglycerols in plasma and other biological sources is described. It is designed to give information about species composition and, consequently, more detailed information about changes in lipid metabolism of patients suffering from metabolic disorders. After plasma extraction the lipids, except phospholipids, are directly examined without any further derivatization. For free fatty acid determination the programmed temperature vaporizer (PTV) injector was heated from 40 degrees C (sample introduction) to 190 degrees C. In a second gas chromatographic run the PTV-injector system was heated from 60 degrees C (sample introduction) to 400 degrees C, enabling the determination of free cholesterol, cholesteryl esters, and triacylglycerol species, differing in the number of carbon atoms. Evaluation of the values obtained resulted in coefficients of variation (%) of 1.0-2.8, 2.0, 1.29-2.24, and 2.8, for free fatty acid standards, plasma free fatty acids, cholesterol and cholesteryl ester standards, and plasma total cholesterol, respectively. Free fatty acids, cholesterol, and cholesteryl esters were not influenced by storage of plasma at -24 degrees C up to 4 days prior to extraction. The results of the gas chromatographic method and the enzymatic methods correlated well. Determination by gas chromatography yielded higher total cholesterol and lower triacylglycerol values than those values obtained by enzymatic methods. |
| Determination of serum cholesterol by isotope dilution mass spectrometry with a benchtop capillary gas chromatograph/mass spectrometer: comparison with the National Reference System's Definitive and Reference Methods Eckfeldt, J. H., L. A. Lewis, et al. (1991), Clin Chem 37(7): 1161-5. Abstract: We developed an isotope dilution mass spectrometric cholesterol method with 25,26,27-13Ccholesterol as internal standard and a benchtop gas chromatograph/mass spectrometer (GC/MS) that is much easier and less time consuming than previously described Reference and Definitive Methods for cholesterol. The internal standard, cholesterol standards, and unknown specimen are delivered volumetrically with an automated dilutor and the saponifying reagent. After saponification, extraction, and derivatization, specimens are injected into a benchtop quadrupole MS with an autosampler. Unknown cholesterol concentrations are calculated automatically by comparing the peak area ratio of the m/z = 368, 371 ion pair with the ratios for the cholesterol standards (0 to 12.93 mmol/L). We found within-run and day-to-day (overall) imprecision of 0.44% and 0.95%, respectively, when specimens were assayed singly. In several lyophilized and frozen Standard Reference Material (SRM) pools, cholesterol results with our GC/MS method averaged 0.4% less than the National Institute for Standards and Technology definitive GC/MS result performed about three years earlier. Our GC/MS results averaged 1.3% and 2.0% less than results by the National Reference System (NRS) Abell-Levy-Brodie-Kendall (ALBK) results from clinical specimens and the SRM pools, respectively. These results are consistent with the previously reported bias between the NRS Reference and Definitive Methods and the 0.1% per year decrease in cholesterol concentrations in SRM pools as determined by GC/MS analysis. These results further emphasize the small but consistent bias between cholesterol results by isotope dilution mass spectrometry and the ALBK Reference Method, the latter being the basis for the National Cholesterol Education Program guidelines and population reference values. |
| Determination of serum cholesterol by stable isotope dilution method using discharge-assisted thermospray liquid chromatography/mass spectrometry Takatsu, A. and S. Nishi (1993), Biol Mass Spectrom 22(4): 247-50. Abstract: A discharge-assisted thermospray (plasmaspray) liquid chromatography/mass spectrometric method for the determination of total serum cholesterol is described. The method incorporates stable isotope dilution using (3,4-13C)cholesterol as an internal standard. Liquid chromatographic separation is performed using methanol as a flow solvent and effluents are directly introduced to the mass spectrometer. MH-H2O+ ions are monitored during liquid chromatography/mass spectrometry using the selected ion monitoring method. Satisfactory agreement between the analytical result and the certified value of the National Institute of Standards and Technology (formerly National Bureau of Standards) standard reference material serum is obtained with a relative standard deviation of 0.6%. |
| Determination of serum cholesterol concentration in the presence of ascorbate Lumb, P. J. and B. M. Slavin (1993), J Clin Pathol 46(3): 283-4. Abstract: Pretreatment of a serum or plasma sample with ascorbate oxidase removed interfering ascorbate and allowed the determination of cholesterol to be carried out by a current enzymatic cholesterol method available in kit form. The Cobas-Fara was programmed to carry out pretreatment of the sample with ascorbate oxidase before addition of the cholesterol colour reagent. |
| Determination of the cholesterol-collagen ratio of arterial atherosclerotic plaques using near infrared spectroscopy as a possible measure of plaque stability Neumeister, V., M. Scheibe, et al. (2002), Atherosclerosis 165(2): 251-7. Abstract: Particular danger associated with an arteriosclerotic plaque consists in the possible rupture of its cap, dependent on the thickness of the cap covering the lipid core, its composition and different inflammatory changes. The purpose of this study was to compare the total cholesterol and collagen contents of arterial walls, both measured by near infrared spectroscopy (NIRS), and to test whether the ratios of cholesterol to collagen correlate with histochemical parameters possibly being indicators for plaque stability. NIR spectra of 118 sections from 36 human aortas were measured at 1000-2500 nm. Evaluation was performed by the partial least squares method (PLS), the chemical reference analysis by HPLC. Acceptable results were achieved for calibrations. With these calibrations 38 further aortic sections taken at autopsy were NIR-spectroscopically analysed and ordered in relation to histological findings of fatty deposits, cap thickness over the lipid core, and the ratio of fatty deposits to cap thickness. Correlations were found to exist between the spectroscopically determined total cholesterol concentrations and the histologically estimated fatty deposits (r=0.887), between the spectroscopically determined collagen concentrations and the cap thickness over the lipid core (r=0.441), and between the ratios total cholesterol to collagen and the ratios fatty deposits to cap thickness (r=0.575). |
| Determination of total cholesterol content in food by flow injection analysis with immobilized cholesterol oxidase enzyme reactor Baticz, O. and S. Tomoskozi (2002), Nahrung 46(1): 46-50. Abstract: A new analytical method has been developed--including a new sample preparation procedure--for the automatic determination of total (free and bound) cholesterol in food by flow injection analysis (FIA) with immobilized (cholesterol oxidase) enzyme reactor (IMMER). A suitable sample preparation procedure has been applied to eliminate the problems derived from sensitivity of FIA equipment to common organic solvents used for dissolving of cholesterol: after direct saponification the non-saponificable fraction was dissolved in water phase detergent (sodium cholate) solution. The analytical method is based on the oxidation by cholesterol oxidase followed by the photometric determination of hydrogen peroxide (at 500 nm) using the indicator reaction with peroxidase. The new FIA method was tested for commercial food samples such as whole egg powder and dried pasta. The results were compared with data obtained by GC-determination. It was found that this new procedure is suitable for rapid automated measurement of total cholesterol content in foodstuff and consequently, the FIA technique with immobilized enzyme reactor could be an alternative to the widely used gas-chromatographic (GC) method. |
| Determination of total cholesterol in serum by liquid chromatography-isotope dilution mass spectrometry Kock, R., B. Delvoux, et al. (1997), Clin Chem 43(10): 1896-903. Abstract: We have developed a liquid chromatography-isotope dilution mass spectrometry procedure to quantify total cholesterol in serum. A particle-beam interface was used for coupling the liquid chromatograph and the mass spectrometer. After electron impact ionization the ions m/z = 386 and m/z = 389 were used for selective ion monitoring of cholesterol and the internal standard 25,26,27-(13)Ccholesterol. The sample preparation steps required for serum materials are alkaline hydrolysis and an extraction of the cholesterol into the cyclohexane phase. Imprecision for the determination of cholesterol in control materials is typically <1.0%. The deviation from the certified reference values was <0.75% for all control materials tested. A method comparison of the results obtained by this method with those obtained by gas chromatography-isotope dilution mass spectrometry for n = 28 pooled human sera derived from samples analyzed in our routine laboratory did not show differences >2.5%. |
| Determination of triacylglycerol and cholesterol ester hydroperoxides in human plasma by high-performance liquid chromatography with fluorometric postcolumn detection Akasaka, K., H. Ohrui, et al. (1993), J Chromatogr 617(2): 205-11. Abstract: Cholesterol ester (ChE) and triacylglycerol (TG) hydroperoxides in human plasma were determined by high-performance liquid chromatography with postcolumn detection with diphenyl-1-pyrenylphosphine. Human plasma was extracted once with n-hexane. 2,6-Di-tert.-butyl-4-methylphenol and N-stearylcinnamide were added to human plasma before extraction as an antioxidation agent and an internal standard, respectively. The detection limits of both ChE and TG hydroperoxides were 1 pmol. The sample size was minimized to 250 microliters for each run. The recoveries of ChE and TG hydroperoxides from fresh plasma were ca. 90 and 80%, respectively. The relative standard deviations (n = 8) of their values in frozen human plasma were 5.4% (ChE hydroperoxides, 298 nM) and 5.7% (TG hydroperoxides, 267 nM). No TG hydroperoxides and 24.5 +/- 9.6 nM (n = 15) ChE hydroperoxides were detected in fresh human plasma. The relative standard deviation (n = 8) of ChE hydroperoxides values in fresh plasma was 5.8% (27.1 nM). |