| For medical practitioners and the general public - Cholesterol Journal Article Catalog. |
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| Quantitation of cholesterol crystallization from supersaturated model bile Portincasa, P., N. G. Venneman, et al. (2002), J Lipid Res 43(4): 604-10. Abstract: Cholesterol crystallization is an essential step in gallstone formation. Although spectrophotometry and nephelometry have been used for quantitation of crystallization, potential effects of crystal size and shape have not been evaluated. We determined crystallization in model biles total lipid concentration 7.3 g/dl, egg yolk Phosphatidylcholine (EYPC)/(EYPC+taurocholate) molar ratio = 0.05, 0.15, or 0.30; cholesterol saturation index (CSI) = 1.2, 1.7, or 2.0; 37 degrees C plotting in the central three-phase (micelles, vesicles, and crystals containing) zone or in the left two-phase (micelles and crystals containing) zone of the equilibrium ternary phase diagram. Extent of crystallization estimated by spectrophotometry and nephelometry was related to chemical determination of crystal mass and to crystal size or shape (by microscopy). With all methods, crystallization was less extensive when vesicles were present (central three-phase zone) and at lower CSIs. In the left two-phase zone, particularly at EYPC/(EYPC+taurocholate), ratio of 0.15, there were strong increases in spectrophotometric and nephelometric readings during the first days of incubation, but decreases at later stages, despite progressive increases in crystal mass by chemical measurement. Initially, there were large numbers of very small crystals (<10 microm) in these biles, which were subsequently replaced by large cholesterol monohydrate crystals. Decreasing sizes of harvested cholesterol monohydrate crystals by sonication increased spectrophotometric and nephelometric values despite identical crystal mass.When cholesterol crystal mass is assayed by indirect methods such as spectrophotometry or nephelometry, results are strongly influenced by crystal size. |
| Quantitation of cholesterol-carrying particles in human gallbladder bile Eckhardt, E. R., B. J. van de Heijning, et al. (1998), J Lipid Res 39(3): 594-603. Abstract: The inter-mixed micellar/vesicular (non-phospholipid-associated) bile salt concentration (IMC) can be rapidly measured in model biles by centrifugal ultrafiltration, thus allowing reliable separation of vesicular and micellar cholesterol carriers by gel filtration with an elution buffer containing bile salts at the correct IMC (Donovan, J. M., and A. A. Jackson. 1993. J. Lipid Res. 34: 1121-1129). We adapted this method to the more complex human gallbladder bile and examined the relationship between cholesterol solubilization and crystallization in gallbladder biles from 10 cholesterol gallstone patients. The IMC (mean +/- SEM) was 9.67 +/- 1.97 (range 3.56-35.02) mM with significant enrichment with hydrophilic bile salt species. Upon gel filtration of these biles with an eluant buffer containing 10 major bile salts at concentrations according to their IMC, cholesterol was found to be solubilized mainly in mixed micelles. Vesicles were detected in all 10 biles after separation by KBr density gradient ultracentrifugation but in only 5 of these biles with the IMC method. Biles without vesicles had a lower CSI (1.15 +/- 0.12 vs. 1.90 +/- 0.28, P < 0.05), a higher total lipid concentration (11.9 +/- 2.3 vs. 5.9 +/- 1.1, P < 0.05), and a higher bile salt/ (bile salt + phospholipid) ratio (0.83 +/- 0.01 vs. 0.74 +/- 0.04, P = 0.07). For both IMC and ultracentrifugation methods, vesicular cholesterol concentration showed a negative correlation with crystal observation time and a positive correlation with cumulative crystal score during 21 days. Our data indicate that methods such as density gradient ultracentrifugation overestimate vesicular cholesterol solubilization in human biles. |
| Quantitation of pre beta-HDL-dependent and nonspecific components of the total efflux of cellular cholesterol and phospholipid Kawano, M., T. Miida, et al. (1993), Biochemistry 32(19): 5025-8. Abstract: Both receptor-mediated and diffusional processes have been proposed as mechanisms for the efflux of cellular cholesterol to plasma. The depletion of a minor high-density lipoprotein subfraction (pre beta-1-HDL) from plasma by incubation was associated with a proportional reduction in up to 58% of cholesterol and lecithin efflux from cultured fibroblasts. Pre beta-HDL-dependent efflux was blocked by protease pretreatment of the cells, while residual ("nonspecific") efflux was protease-insensitive. The whole of cholesterol efflux from blood erythrocytes was both pre beta-1-HDL-and protease-independent. These data suggest that two distinct pathways contribute to total efflux from fibroblast monolayers; one of these is directly proportional to plasma pre beta-1-HDL concentration and may involve a cell-surface protein. |
| Quantitation of the pool of cholesterol associated with acyl-CoA:cholesterol acyltransferase in human fibroblasts Lange, Y. and T. L. Steck (1997), J Biol Chem 272(20): 13103-8. Abstract: The esterification of cholesterol in homogenates of human fibroblasts was explored as a means of estimating the size of the pool of cholesterol associated with the endoplasmic reticulum (ER) in vivo. The rationale was that the acyl-coenzyme A:cholesterol acyltransferase (ACAT) in homogenates should have access only to cholesterol associated with the (rough) ER membrane fragments in which it resides. Reacting whole homogenates to completion with an excess of 14Coleoyl-CoA converted approximately 0.1-2% of total cell-free cholesterol to 14Ccholesteryl esters. Control studies indicated that membranes not associated with ACAT did not contribute cholesterol to this reaction. The extent of in vitro cholesterol esterification varied with pretreatment of the cells. Exposing intact cells to serum lipoproteins, oxysterols, or sphingomyelinase increased cholesterol esterification in homogenates severalfold; exposing the cells to mevinolin or cholesterol oxidase had the opposite effect. The variation in cholesterol esterification did not correlate with either the total cellular cholesterol or the intrinsic activity of ACAT, neither of which was changed significantly by the pretreatments. Rather, the total amount of cholesterol esterified in homogenates paralleled the rate of cholesterol esterification in the corresponding intact cells. The pool of cholesterol esterified in vitro therefore appears to reflect that associated with the ER in vivo. Since several of the mechanisms keeping cell cholesterol under tight feedback control are themselves located in the ER, this pool might not only be regulated physiologically, but could, in turn, help to regulate homeostatic effector pathways. |
| Quantitation of two pathways for cholesterol excretion from the brain in normal mice and mice with neurodegeneration Xie, C., E. G. Lund, et al. (2003), J Lipid Res 44(9): 1780-9. Abstract: Although the pool of cholesterol in the adult central nervous system (CNS) is large and of constant size, little is known of the process(es) involved in regulation of sterol turnover in this pool. In 7-week-old mice, net excretion of cholesterol from the brain equaled 1.4 mg/day/kg body weight, and from the whole animal was 179 mg/day/kg. Deletion of cholesterol 24-hydroxylase, an enzyme highly expressed in the CNS, did not alter brain growth or myelination, but reduced sterol excretion from the CNS 64% to 0.5 mg/day/kg. In mice with a mutation in the Niemann-Pick C gene that had ongoing neurodegeneration, sterol excretion from the CNS was increased to 2.3 mg/day/kg. Deletion of cholesterol 24-hydroxylase activity in these animals reduced net excretion only 22% to 1.8 mg/day/kg. Thus, at least two different pathways promote net sterol excretion from the CNS. One uses cholesterol 24-hydroxylase and may reflect sterol turnover in large neurons in the brain. The other probably involves the movement of cholesterol or one of its metabolites across the blood-brain barrier and may more closely mirror sterol turnover in pools such as glial cell membranes and myelin. |
| Quantitative analyses of lesion areas of coronary atherosclerosis in cholesterol-fed rabbits Kitajima, S., S. Sakuma, et al. (1996), J Vet Med Sci 58(9): 855-60. Abstract: A simple method to quantitatively evaluate atherosclerosis in the rabbit coronary arteries by measuring macroscopic lesion areas (%) was attempted in the present study. Sixteen rabbits were fed a 0.5% cholesterol diet for 15 weeks and then 9 rabbits were sacrificed whereas the remaining 7 rabbits were maintained for further 9 weeks on a normal chow (at week 24). The left circumflex coronary arteries (LCX) were excised from the rabbit hearts under stereoscopic observation. The prepared arterial strips of LCX were 38.7 +/- 7.1 mm long and all of them reached the cardiac apex from the orifice. At week 15, the lesion area in LCX was negligible (3.2 +/- 0.4%) whereas the aortic lesions significantly developed (50.0 +/- 7.6%). At week 24, atherosclerotic lesions in both LCX and aortas increased to 32.8 +/- 9.2% and 85.9 +/- 5.6%, respectively. This is the first report that determined the luminal surface areas of atherosclerotic lesions in rabbit coronary arteries. This method may be more practical and useful for quantitative evaluation of coronary atherosclerosis in a large number of rabbits than histological observations of serial sections of rabbit hearts. |
| Quantitative analysis of antiatherosclerotic effect of nifedipine in cholesterol-fed rabbits Ohta, Y., N. Higuchi, et al. (1990), Cardiovasc Drugs Ther 4 Suppl 5: 1021-6. Abstract: Reports concerning the effect of slow calcium-channel blockers on experimental atherosclerosis are controversial. We examined the antiatherosclerotic effect of nifedipine (40 mg/day for 16 weeks) on aorta of rabbits on diets containing 0.3%, 0.5%, and 1.0% cholesterol. There were no significant differences in levels of serum lipids with or without nifedipine in the same cholesterol-fed rabbits. The results obtained show that nifedipine suppressed the extent of lipid deposition and surface involvement (S.I.) in aorta in 0.3% cholesterol-fed rabbits, whereas nifedipine only tended to suppress S.I. in 0.5% cholesterol-fed rabbits and had no effect in 1.0% cholesterol-fed rabbits. The log dose-response relationship of S.I. was obtained by plotting the concentration of cholesterol in the feed or the "integrated value" of the total serum cholesterol (TC), i.e., the cumulative sum of the serum TC values obtained at each week. The log dose-response curve was shifted in parallel with the right in nifedipine groups. The Lineweaver-Burk plot constructed from the dose-response curve had the same points crossing the ordinate with or without nifedipine. These results suggested that nifedipine suppressed S.I. in a competitive manner with cholesterol on the specific binding site of lipid deposition. Electron-microscopic findings also demonstrated that fat droplets in smooth muscle cells, extracellular matrix containing collagen, and elastic fibers decreased in nifedipine-treated rabbits. |
| Quantitative analysis of cholesterol and cholesteryl esters in human atherosclerotic plaques using near-infrared Raman spectroscopy Weinmann, P., M. Jouan, et al. (1998), Atherosclerosis 140(1): 81-8. Abstract: Raman spectroscopy is a non-destructive analytical technique and previous results have shown that qualitative analysis of the lipid component of human atheromatous arteries is feasible. In this paper, we describe a quantitative analytical method for cholesterol and cholesteryl esters in human atherosclerotic plaques, combined with Raman spectroscopic results, using partial least-squares (PLS) regression, a statistical multivariate method based on factorial analysis. Twenty-nine human atherosclerotic pooled samples were studied and the results of Raman spectroscopy coupled with the PLS method were compared to biochemical results. The standard error of prediction was 16.1, 13.6, 1.9, 3.3 and 3.4 mg/g for total cholesterol, free cholesterol, palmitate cholesteryl, oleate cholesteryl and linoleate cholesteryl, respectively. The repeatability of Raman spectroscopy was found to be excellent. Our results show that Raman spectroscopy is a promising technique to obtain a consistent and non-destructive quantitative analysis of cholesterol and cholesteryl esters in human atherosclerotic lesions. In situ and in vivo analysis is a possibility in the near future. |
| Quantitative analysis of desmosterol, cholesterol and cholesterol sulfate in semen by high-performance liquid chromatography Sion, B., G. Grizard, et al. (2001), J Chromatogr A 935(1-2): 259-65. Abstract: 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. |
| Quantitative analysis of hydrophobic amine inhibition of intracellular cholesterol transport Underwood, K. W., B. Andemariam, et al. (1996), J Lipid Res 37(7): 1556-68. Abstract: U18666A and imipramine are hydrophobic amines that inhibit intracellular cholesterol transport pathways. In this study, we conducted dose-response curves for each of the cholesterol transport pathways. Our analyses indicate that hydrophobic amine inhibition of LDL-stimulated cholesterol esterification is much more sensitive to inhibition than either the combined bulk movement of cholesterol from lysosomes to the plasma membrane and from the plasma membrane to the endoplasmic reticulum. Hydrophobic amines must inhibit a previously uncharacterized pathway from lysosomes to the endoplasmic reticulum or a signaling event that activates acyl CoA:cholesterol acyltransferase. Possible mechanisms for U18666A action were evaluated. The function of p-glycoprotein, which has been implicated in cholesterol transport, was unaffected by U18666A. We have evidence for a specific membrane U18666A binding site, which we hypothesize is involved in the plasma membrane to endoplasmic reticulum cholesterol transport pathway. Identification of the binding site and mechanism of hydrophobic amine action may provide information essential for understanding intracellular cholesterol transport. |
| Quantitative analysis of vitamin E, cholesterol and phospholipid fatty acids in a single aliquot of human platelets and cultured endothelial cells Leray, C., M. Andriamampandry, et al. (1997), J Chromatogr B Biomed Sci Appl 696(1): 33-42. Abstract: A reliable procedure is described for the joint analysis of vitamin E (tocopherols), cholesterol and phospholipids in the same minute sample of human platelets and on human cultured endothelial cells. The whole procedure is based on the extraction of total lipids, thin-layer chromatography of all compounds of interest and microcolumn purification of tocopherols and cholesterol. The combined use of butyl hydroxytoluene and ascorbic acid in the purification steps allowed a complete recovery of the tocopherols analyzed, as well as of cholesterol by high-performance liquid chromatography. The detection of these lipids was performed with fluorometric, spectrophotometric and evaporative light-scattering detectors whose respective sensitivities were compared. The fatty acid composition of phospholipid classes from the same sample, separated on the same silica gel plate, was determined by gas-liquid chromatography. The whole procedure is rapid since it requires about 4 h to analyse tocopherols and cholesterol and to prepare methylated fatty acids, 28 samples being easily completed within one working day. The evaluation of the whole membrane antioxidant status requires as little as one 25 cm2 confluent culture flask (about 0.75 x 10(6) cells) for endothelial cells or two ml of blood (3 x 10(8) platelets). |
| Quantitative assessment of aortic atherosclerosis in APOE*3 Leiden transgenic mice and its relationship to serum cholesterol exposure Groot, P. H., B. J. van Vlijmen, et al. (1996), Arterioscler Thromb Vasc Biol 16(8): 926-33. Abstract: Transgenic mice overexpressing the human dysfunctional apolipoprotein E variant, APOE*3 Leiden, develop hyperlipidemia and are highly susceptible to diet-induced atherosclerosis. In the present study, we investigated the effects of diet composition and feeding period on serum cholesterol exposure and the amount of atherosclerosis in the aortic sinus in these mice, using quantitative image analysis. On each of the three diets tested--a low-fat diet, a high-saturated-fat/cholesterol diet, and a high saturated-fat/high-cholesterol/0.5%-cholate diet--transgenic animals showed a marked hyperlipidemia compared with nontransgenic littermates. Measurement of the atherosclerotic lesion areas in cross sections of the aortic sinus in animals exposed to these three diets for up to 6 months showed a 5 to 10 times greater lesion area in transgenic mice compared with nontransgenic controls. Highly significant positive correlations were found between the log-transformed data on lesion area and serum cholesterol exposure (r =.82 to.85 for the 1-, 2-, and 3-month treatment groups), indicating that the hyperlipidemia is likely to be a major determinant in lesion formation. On the basis of these findings, we suggest that the APOE*3 Leiden mouse represents a promising model for intervention studies with hypolipidemic and antiatherosclerotic drugs. |
| Quantitative assessment of comparative potencies of cholesterol-crystal-promoting factors: relation to mechanistic characterization Nishioka, T., S. Tazuma, et al. (1998), Biochem J 332 (Pt 2): 343-50. Abstract: The crystallization of cholesterol is affected by various factors in bile. The present study evaluated the relative importance of cholesterol-nucleation-promoting factors and partially characterized the mechanisms of their action. Model biles with an identical relative composition of cholesterol, egg-yolk phosphatidylcholine and taurocholate, except for replacing phosphatidylcholine (5-20%) with dilinoleoyl-phosphatidylcholine or taurocholate (10-30%) with taurodeoxycholate. Cholesterol crystallization was quantitatively assessed spectrophotometrically and morphologically estimated by the laser-scattering diffraction analyser and video-enhanced microscopy in the absence and presence of concanavalin A-binding glycoprotein isolated from human bile. In a series of experiments, lipid distribution among particulate species was determined after isolation by FPLC. In all experiments, cholesterol crystallization was dose-dependently enhanced with a rank order of: concanavalin A-binding glycoprotein > dilinoleoyl - phosphatidyl choline> taurodeoxycholate. No morphological alteration was evident for vesicles and crystals, but the cholesterol/phospholipid ratio in vesicles was increased significantly by replacement with dilinoleoyl-phosphatidylcholine and excess cholesterol. A high proportion of relatively hydrophilic phosphatidylcholine species such as dilinoleoyl-phosphatidylcholine and excess cholesterol in bile cause a redistribution of cholesterol to increase a vesicular cholesterol/phospholipid ratio, eventually promoting cholesterol crystallization, whereas concanavalin A-binding glycoprotein acts via differing mechanisms. |
| Quantitative changes in dietary fat intake and serum cholesterol in women: results from a randomized, controlled trial Boyd, N. F., M. Cousins, et al. (1990), Am J Clin Nutr 52(3): 470-6. Abstract: We compared observed and predicted changes in serum cholesterol in women with mammographic dysplasia who participated for 12 mo in a randomized, controlled trial of a low-fat, high-carbohydrate diet, in which total fat intake was reduced from an average of 37% of calories to 21% and carbohydrate intake increased from 44% to 52% of calories. Changes observed in serum cholesterol were greater than those predicted (by the formulas of Hegsted and Keys) for subjects with initial serum cholesterol values in the upper tertile of the population, were not significantly different from those predicted for subjects with baseline values in the middle tertile, and were significantly less than those predicted for subjects with initial values in the lower tertile. These results show that the usefulness of serum cholesterol as a marker of change in dietary fat intake in women depends on the distribution of serum cholesterol values in the population studied. |
| Quantitative characterization of insulin-glucose response in Watanabe heritable hyperlipidemic and cholesterol-fed rabbits and the effect of cilazapril Zhang, B., K. Saku, et al. (1994), Metabolism 43(3): 360-6. Abstract: A great deal of evidence suggests that insulin resistance, via hyperinsulinemia, contributes to hyperlipoproteinemia and coronary atherosclerosis. When Watanabe heritable hyperlipidemic (WHHL) rabbits, an animal model of familial hypercholesterolemia (FH), are compared with normolipidemic Japanese White (JW) rabbits, an elevated fasting plasma insulin level and a heightened plasma insulin response to an intravenous (i.v.) glucose challenge are found. To elucidate the mechanism behind this phenomenon, a two-compartment model of the glucose/insulin system was fitted to empirical time courses of glucose and insulin concentrations during an i.v. glucose tolerance test (IVGTT) by nonlinear least-square regression, and the model parameters such as the glucose utilization rate constant, insulin degradation rate constant, and pancreas sensitivity were determined. WHHL rabbits showed decreased values of glucose utilization and insulin degradation rate constants and slightly higher values of pancreas sensitivity. This suggests that insulin resistance occurs in extrapancreatic tissues, and that this may be attributable to insulin receptor and/or post-insulin receptor abnormalities. Cholesterol feeding did not significantly change glucose tolerance or insulin action in JW rabbits. The effects of an angiotensin-converting enzyme (ACE) inhibitor, cilazapril, on insulin resistance were also examined in WHHL and JW rabbits. A decreased insulin response to an i.v. glucose challenge and increased glucose utilization and insulin degradation rate constants were observed in WHHL rabbits that had been treated with cilazapril, indicating that cilazapril improved insulin resistance in WHHL rabbits, possibly by increasing the number of insulin receptors. No significant differences were found in glucose tolerance and insulin action in JW rabbits before and after cilazapril administration.(ABSTRACT TRUNCATED AT 250 WORDS) |
| Quantitative determination of cholesterol 5alpha-, 7alpha-, and 7beta-hydroperoxides in rat skin Yamazaki, S., N. Ozawa, et al. (1999), Free Radic Biol Med 27(1-2): 110-8. Abstract: An assay method for determination of cholesterol 5alpha-, 7alpha-, and 7beta-hydroperoxides (ChOOHs) in rat skin using high-performance liquid chromatography (HPLC) with a chemiluminescence detector has been developed. In the assay method, free form and free plus ester forms of ChOOHs could be separately determined by HPLC in combination with the treatment of a tissue extract by cholesterol esterase. Lower limits of quantitation for cholesterol 5alpha-, 7alpha-, and 7beta-hydroperoxides were 0.2, 0.1, and 0.5 nmol/g skin, respectively. This assay method showed that (i) good absolute recoveries of ChOOHs from rat skin (80-90% of radiolabeled ChOOHs added to rat skin); (ii) negligible autoxidation of cholesterol caused by the assay procedure (<9.4x10(-5)% of radiolabeled cholesterol added to rat skin); and (iii) good correlation between ChOOHs added to rat skin and ChOOHs determined, indicating this assay method is applicable to quantify ChOOHs in rat skin. By using this assay method, we observed that (i) cholesterol 5alpha-hydroperoxide was detected in skin of rats pretreated with oral doses of pheophorbide a and subsequent visible irradiation; (ii) concentrations of cholesterol 7-hydroperoxides in skin of rats in an ambient light room were not significantly different from those in a dark room for 12 weeks; and (iii) ultraviolet light B irradiation markedly enhanced the concentrations of cholesterol 7-hydroperoxides in the skin of rats. |
| Quantitative determination of cholesterol in lipoprotein fractions by electrophoresis Contois, J. H., R. G. Gillmor, et al. (1999), Clin Chim Acta 282(1-2): 1-14. Abstract: The Helena REP cholesterol profile system (Helena Laboratories, Beaumont, TX) separates VLDL, LDL, HDL and Lp(a) by agarose gel electrophoresis, and quantitates cholesterol by enzymatic staining and densitometry. We compared results by electrophoresis to combined ultracentrifugation/precipitation (beta-quantification, BQ) for VLDL, LDL, and HDL cholesterol and to immunonephelometry for Lp(a) mass (Behring Diagnostics, Westwood, MA) in serum from 64 patients with a variety of lipid disorders. There was good agreement between methods, with a mean bias of -0.19 (-7.3), 0.09 (3.5), and 0.09 (3.4) mmol/l (mg/dl) for VLDL, HDL, and LDL cholesterol for electrophoresis vs. BQ. These differences were significant for HDL and VLDL cholesterol (P < 0.001), but not for LDL cholesterol measurement (P > 0.05). There was also good correlation between methods with coefficients of 0.83, 0.92, 0.91, and 0.97 for VLDL, HDL, Lp(a), and LDL, respectively. Our data indicate that this method can accurately and precisely measure LDL cholesterol directly in fresh serum from patients with a wide range of triglyceride values. However, HDL cholesterol measurement did not meet NCEP guidelines for precision and accuracy. Also, the poor resolution of VLDL and LDL in some specimens is a concern. |
| Quantitative determination of cholesterol in liposome drug products and raw materials by high-performance liquid chromatography Lang, J. K. (1990), J Chromatogr 507: 157-63. Abstract: Most liposomes used as drug delivery systems contain cholesterol as a major structural component. Cholesterol has profound effects on the chemical, physical and metabolic stability of liposomes and liposome drug products and must be accurately monitored during formulation and processing development, stability testing and manufacturing. Before analyzing their components, the liposomes must be disintegrated and solubilized by dilution with methanol or 2-propanol. This high-performance liquid chromatographic assay is applicable to the resulting lipid-rich matrices and allows a direct quantitative analysis of cholesterol. Cholesterol separates well from common ingredients of liposome-based drug products and cholesterol oxidation products. Calibration curves are linear over two orders of magnitude and the cholesterol detection limit is 1.5 microliter/ml. Method precision for an anticancer liposome drug formulation was 0.9% relative standard deviation. The assay is also useful for measuring cholesterol in phospholipid and cholesterol raw materials. |
| Quantitative determination of cholesterol sulphate in plasma by stable isotope dilution fast atom bombardment mass spectrometry Veares, M. P., R. P. Evershed, et al. (1990), Biomed Environ Mass Spectrom 19(10): 583-8. Abstract: A stable isotope dilution assay has been developed for the quantitative determination of cholesterol sulphate in plasma using negative ion fast atom bombardment (FAB) mass spectrometry. The assay is highly selective and avoids problems of contamination from free cholesterol and other conjugates of cholesterol present in plasma. (6,7,7-2H3)Cholesterol sulphate is used as the internal standard and solvent extraction and silica Sep-Paks are employed to isolate plasma cholesterol sulphate. Limited-range acceleration voltage scanning in FAB mass spectrometric analyses leads to sub-microgram detection limits. Comparison of results obtained by FAB mass spectrometry of the intact cholesterol sulphate, and by gas chromatography/mass spectrometry selected ion monitoring of the free cholesterol, released by solvolysis of the cholesterol sulphate, showed that the latter approach probably overestimates plasma levels of cholesterol sulphate. |
| Quantitative determination of cholesterol, sitosterol, and sitostanol in cultured Caco-2 cells by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry Palmgren, J. J., A. Toyras, et al. (2005), J Chromatogr B Analyt Technol Biomed Life Sci 821(2): 144-52. Abstract: In this study, we describe a simple liquid extraction (methanol/choloroform, 1:1, v/v) method for endogenous free cholesterol and administered sterols extracted from cultured Caco-2 cells. To quantify sterol contents in Caco-2 cells, a new HPLC-APCI-MS method was developed. All the sterols were baseline separated using reversed-phase column (C8, 2.1 mm x 150 mm, 3.5 microm) and isocratic conditions (90%, v/v, methanol-water mixture containing 0.2 mM ammonium acetate). The full scan mass spectra of sterols were measured by an ion trap mass spectrometer equipped with an APCI ion source. The intense fragment ions resulting from the loss of water M+H-H2O+ (m/z 369, 395, 397 and 399 for cholesterol, stigmasterol, sitosterol, and sitostanol, respectively) were used for determinations. The absolute extraction recovery of sterols from the spiked cell samples were 109.7+/-26.2, 105.7+/-5.1, 109.8+/-5.0 and 99.0+/-7.0% for cholesterol, stigmasterol, sitosterol, and sitostanol, respectively. Furthermore, no significant matrix effect was observed for the sterols in the cell samples. The sample assay was based on the internal standard method using stigmasterol as an internal standard. The method was linear over the concentration ranges of 0.45-9.0 microM (cholesterol) and 0.225-7.2 microM (sitosterol and sitostanol). The within- and between-day precision was less than 7% and accuracy ranged from 93.51 to 101.77%. The lowest limit of quantitation (LLOQ) was 0.225 microM for sitosterol and sitostanol, and 0.45 microM for cholesterol. The accuracy range was 95-106% and precision was lower than 9% for all LLOQ values. |