| For medical practitioners and the general public - Cholesterol Journal Article Catalog. |
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| Detergent insolubility of alkaline phosphatase during biosynthetic transport and endocytosis. Role of cholesterol Cerneus, D. P., E. Ueffing, et al. (1993), J Biol Chem 268(5): 3150-5. Abstract: Alkaline phosphatase is anchored to the outer leaflet of the plasma membrane by a covalently attached glycosyl-phosphatidylinositol anchor. We have studied the biosynthetic transport and endocytosis of alkaline phosphatase in the choriocarcinoma cell line BeWo, which endogenously expresses this protein. It was demonstrated that the protein was synthesized as a Triton X-100-soluble precursor. During transport to the cell surface the enzyme was converted in a mature form, which was insoluble in Triton X-100 at 0 degrees C. Once at the cell surface 85% of alkaline phosphatase remained in the detergent-insoluble form. Under steady state conditions 15% of alkaline phosphatase was endocytosed. Most interestingly, this fraction of internalized alkaline phosphatase was completely soluble in Triton X-100 at 0 degrees C. After depletion of membrane cholesterol by saponin, alkaline phosphatase became completely soluble in Triton X-100 at 0 degrees C, suggesting that cholesterol plays a critical role in the formation and maintenance of Triton X-100-resistant membrane domains. |
| Detergent resistant domains in erythrocyte membranes survive after cell cholesterol depletion: an EPR spin label study Rivas, M. G. and A. M. Gennaro (2003), Chem Phys Lipids 122(1-2): 165-9. Abstract: We use electron paramagnetic resonance (EPR) with liposoluble spin labels in order to study the lipid structures obtained after Triton X-100 extraction of erythrocyte membranes. The apparent order profile in these detergent resistant membranes (DRM) is very similar to that of the parent membrane, although with higher absolute values, consistent with a liquid-ordered state. DRM could also be obtained from erythrocytes previously depleted in a 40% of their membrane cholesterol, in apparent opposition to the phenomenon of raft disruption reported by other authors. However, the protein profile of these samples showed important differences with that of DRM from untreated cells. The analysis of our results suggests that the effect of Triton X-100 on cholesterol depleted erythrocytes is limited to the solubilization of raft proteins, without disrupting the lipid matrix of DRM. |
| Detergent-insoluble glycosphingolipid/cholesterol microdomains of the myelin membrane Taylor, C. M., T. Coetzee, et al. (2002), J Neurochem 81(5): 993-1004. Abstract: Glycosphingolipids and cholesterol form lateral assemblies, or lipid 'rafts', within biological membranes. Lipid rafts are routinely studied biochemically as low-density, detergent-insoluble complexes (in non-ionic detergents at 4 degrees C; DIGs, detergent-insoluble glycosphingolipid/cholesterol microdomains). Recent discrepancies recommended a re-evaluation of the conditions used for the biochemical analysis of lipid rafts. We have investigated the detergent insolubility of several known proteins present in the glycosphingolipid/cholesterol-rich myelin membrane, using four detergents representing different chemical classes (TX-100, CHAPS, Brij 96 and TX-102), under four conditions: detergent extraction of myelin either at (i) 4 degrees C or (ii) 37 degrees C, or at 4 degrees C after pre-extraction with (iii) saponin or (iv) methyl-beta-cyclodextrin (MbetaCD). Each detergent was different in its ability to solubilize myelin proteins and in the density of the DIGs produced. Brij 96 DIGs floated to a lower density than other detergents tested, possibly representing a subpopulation of DIGs in myelin. DIGs pre-extracted with saponin were denser than DIGs pre-extracted with MbetaCD. Furthermore, pre-extraction with MbetaCD solubilized proteolipid protein (known to associate with cholesterol), whereas pre-extraction with saponin did not, suggesting that saponin is less effective as a cholesterol-perturbing agent than is MbetaCD. These results demonstrate that DIGs isolated by different detergents are not necessarily comparable, and that these detergent-specific DIGs may represent distinct biochemical, and possibly physiological, entities based on the solubilities of specific lipids/proteins in each type of detergent. |
| Detergent-insoluble glycosphingolipid/cholesterol-rich membrane domains, lipid rafts and caveolae (review) Hooper, N. M. (1999), Mol Membr Biol 16(2): 145-56. Abstract: Within the cell membrane glycosphingolipids and cholesterol cluster together in distinct domains or lipid rafts, along with glycosyl-phosphatidylinositol (GPI)-anchored proteins in the outer leaflet and acylated proteins in the inner leaflet of the bilayer. These lipid rafts are characterized by insolubility in detergents such as Triton X-100 at 4 degrees C. Studies on model membrane systems have shown that the clustering of glycosphingolipids and GPI-anchored proteins in lipid rafts is an intrinsic property of the acyl chains of these membrane components, and that detergent extraction does not artefactually induce clustering. Cholesterol is not required for clustering in model membranes but does enhance this process. Single particle tracking, chemical cross-linking, fluorescence resonance energy transfer and immunofluorescence microscopy have been used to directly visualize lipid rafts in membranes. The sizes of the rafts observed in these studies range from 70-370 nm, and depletion of cellular cholesterol levels disrupts the rafts. Caveolae, flask-shaped invaginations of the plasma membrane, that contain the coat protein caveolin, are also enriched in cholesterol and glycosphingolipids. Although caveolae are also insoluble in Triton X-100, more selective isolation procedures indicate that caveolae do not equate with detergent-insoluble lipid rafts. Numerous proteins involved in cell signalling have been identified in caveolae, suggesting that these structures may function as signal transduction centres. Depletion of membrane cholesterol with cholesterol binding drugs or by blocking cellular cholesterol biosynthesis disrupts the formation and function of both lipid rafts and caveolae, indicating that these membrane domains are involved in a range of biological processes. |
| Detergent-insoluble GPI-anchored proteins are apically sorted in fischer rat thyroid cells, but interference with cholesterol or sphingolipids differentially affects detergent insolubility and apical sorting Lipardi, C., L. Nitsch, et al. (2000), Mol Biol Cell 11(2): 531-42. Abstract: In contrast to Madin-Darby canine kidney cells, Fischer rat thyroid cells deliver the majority of endogenous glycosylphosphatidyl inositol (GPI)-anchored proteins to the basolateral surface. However, we report here that the GPI proteins Placental Alkaline Phosphatase (PLAP) and Neurotrophin Receptor-Placental Alkaline Phosphatase (NTR-PLAP) are apically localized in transfected Fischer rat thyroid cells. In agreement with the "raft hypothesis," which postulates the incorporation of GPI proteins into glycosphingolipids and cholesterol-enriched rafts, we found that both of these proteins were insoluble in Triton X-100 and floated into the lighter fractions of sucrose density gradients. However, disruption of lipid rafts by removal of cholesterol did not cause surface missorting of PLAP and NTR-PLAP, and the altered surface sorting of these proteins after Fumonisin B1 treatment did not correlate with reduced levels in Triton X-100 -insoluble fractions. Furthermore, in contrast to the GPI-anchored forms of both of these proteins, the secretory and transmembrane forms (in the absence of a basolateral cytoplasmic signal) were sorted to the apical surface without association with lipid microdomains. Together, these data demonstrate that the GPI anchor is required to mediate raft association but is not sufficient to determine apical sorting. They also suggest that signals present in the ectodomain of the proteins play a major role and that lipid rafts may facilitate the recognition of these signals in the trans-Golgi network, even though they are not required for apical sorting. |
| Determinants of atherosclerosis susceptibility in the C3H and C57BL/6 mouse model: evidence for involvement of endothelial cells but not blood cells or cholesterol metabolism Shi, W., N. J. Wang, et al. (2000), Circ Res 86(10): 1078-84. Abstract: Lipids, monocytes, and arterial wall cells are primary components involved in atherogenesis. Using the inbred mouse strains C57BL/6J (B6) and C3H/HeJ (C3H), which have been extensively studied as models of the genetic control of diet-induced atherosclerosis, we examined which of these components determine genetic susceptibility. To test whether dietary responsiveness is involved, a congenic strain of C3H carrying an apoE-null allele (apoE(-/-)) was constructed. Although C3H.apoE(-/-) mice had higher plasma cholesterol levels, they developed much smaller lesions than their B6.apoE(-/-) counterpart on either chow or Western diets. Reciprocal bone marrow transplantation between the strains, with congenics carrying the same H-2 haplotype, was performed to examine the role of monocytes. The atherosclerosis susceptibility was not altered in the recipient mice, indicating that variations in monocyte function were not involved. Endothelial cells isolated from the aorta of B6 mice exhibited a dramatic induction of monocyte chemotactic protein-1, macrophage colony-stimulating factor, vascular cell adhesion molecule-1, and heme oxygenase-1 in response to minimally modified LDL, whereas endothelial cells from C3H mice showed little or no induction. In a set of recombinant inbred strains derived from the B6 and C3H parental strains, endothelial responses to minimally modified LDL cosegregated with aortic lesion size. These data provide strong evidence that endothelial cells, but not monocytes or plasma lipid levels, account for the difference in susceptibility to atherosclerosis between the 2 mouse strains. |
| Determinants of change in total cholesterol and HDL-C with age: the Framingham Study Wilson, P. W., K. M. Anderson, et al. (1994), J Gerontol 49(6): M252-7. Abstract: OBJECTIVE: The purpose of the study was to assess the determinants of change of total cholesterol and high density lipoprotein cholesterol (HDL-C) change in an adult population. METHODS. The prospective cohort was examined at baseline and eight years later. A total of 2,222 men and 2,677 women age 20-79 years at baseline were included. Analyses were performed in 15-year age groups, and persons with cardiovascular disease or cancer during the observation period were excluded. RESULTS. In longitudinal analyses, body mass index (BMI) and plasma total cholesterol levels of each rose in concert among younger age groups, whereas levels declined in older individuals. Mean levels of BMI and total cholesterol peaked at a later age in women than in men. The corresponding changes in HDL-C were negative at all ages, and greater declines were seen in the elderly. A decrease in plasma total cholesterol was highly associated with greater age and a decrease in body mass index over the study interval, whereas the decline in HDL-C was proportional to change in body mass index. These changes remained significant after adjustment for baseline age and change in alcohol intake, cigarette consumption, diuretic use, and oral estrogen use. CONCLUSIONS. The rise in plasma total cholesterol among apparently healthy young men and women and its fall in the elderly are significantly associated with similar trends for obesity. The key determinants of a decline in HDL-C are an increase in obesity and advancing age itself. A decline in total cholesterol and in HDL-C is particularly common among the elderly, and it can be expected to occur without specific dietary or pharmacologic intervention. |
| Determinants of compliance with statin therapy and low-density lipoprotein cholesterol goal attainment in a managed care population Schultz, J. S., J. C. O'Donnell, et al. (2005), Am J Manag Care 11(5): 306-12. Abstract: OBJECTIVE: To identify determinants of medication compliance and low-density lipoprotein cholesterol goal attainment. Study Design: This retrospective analysis used claims data from a large, national, employment-based independent practice association database. Subjects were identified based on the existence of a filled prescription for statin therapy between April 1, 1999, and June 30, 2001. Subjects had to be 18 years or older, continuously enrolled in the health plan for 2 years, and new users of statin therapy. METHODS: Multivariate logistic regression models were used to identify predictors of compliance and goal attainment in high-risk subjects. RESULTS: As the mean copayment for statins increased, there was a decrease in the likelihood of compliance. Of the subjects with laboratory results, 50.7% attained their low-density lipoprotein cholesterol goal level established by National Cholesterol Education Program Adult Treatment Panel III guidelines. Older individuals and men were more likely to reach their low-density lipoprotein cholesterol target goal, as were individuals who were compliant with their statin therapy. CONCLUSIONS: Compliance with statin therapy in the managed care setting remains poor. Of particular concern is the lower level of compliance among women and younger high-risk patients, along with patients who have fewer outpatient visits associated with hyperlipidemia and lower incidences of cholesterol testing. |
| Determinants of HDL-cholesterol and the HDL-cholesterol/total cholesterol ratio. Results of the Lubeck Blood Pressure Study Chambless, L., A. Doring, et al. (1990), Int J Epidemiol 19(3): 578-85. Abstract: Results are presented here on the relationship, in a large city in the Federal Republic of Germany, between HDL-cholesterol (HDL-C) or the HDL-cholesterol/total cholesterol ratio (HDL-C/TC) and the variables smoking, alcohol consumption, Body Mass Index (BMI), and age. The strength of each relationship is estimated and compared with the findings of other similar studies. Careful attention is given to the interactive effects of the variables. Evidence is presented to support, in a European setting, previous findings which suggest negative relationships between HDL-C or HDL-C/TC and certain lifestyle factors, namely BMI and cigarette smoking, and a positive relationship with alcohol consumption. In general, no relationship with oral contraceptive use or age is found. Several interactions among the various lifestyle factors and their relationships to HDL-C are seen, including a confirmation of a smoking/alcohol interaction found earlier. |
| Determinants of plasma cholesterol responsiveness to diet Cobb, M. M. and H. Teitlebaum (1994), Br J Nutr 71(2): 271-82. Abstract: Plasma cholesterol change, or 'responsiveness', to dietary saturated fat modification has long been acknowledged. The present study sought to determine the specific, predicted response of each cholesterol subfraction to known dietary manipulations. Two metabolically controlled diets, one with a low polyunsaturated:saturated fat (low P:S) ratio, and one with a high P:S ratio were fed in a crossover design to sixty-seven normolipidaemic subjects pooled from six foregoing metabolic studies. A series of statistical analyses was performed to identify the lipids and subfractions independently affected by the diet crossover. Multivariate analysis of variance revealed that the changes in total cholesterol (delta TC), low-density-lipoprotein-cholesterol (delta LDL-C), and high-density-lipoprotein-cholesterol (delta HDL-C) were the only statistically significant diet-specific 'responsive' lipids. Multiple regression was performed to identify the independent predictors of delta TC, delta LDL-C and delta HDL-C. It was found that age (years), extent of change in dietary saturated fat, and baseline LDL-C (mg/l) levels determine LDL-C change, while extent of change in saturated and polyunsaturated fat, and baseline HDL-C (mg/l) levels can predict HDL-C change. A series of equations to predict lipoprotein responsiveness to diet are derived for potential use in clinical practice. |
| Determinants of plasma HDL-cholesterol in hypertriglyceridemic patients. Role of cholesterol-ester transfer protein and lecithin cholesteryl acyl transferase Tato, F., G. L. Vega, et al. (1997), Arterioscler Thromb Vasc Biol 17(1): 56-63. Abstract: Hypertriglyceridemic patients commonly have low levels of HDL cholesterol. Elevated triglycerides per se may be one cause of low HDL levels, but other factors also may be involved. The current study was designed to define the role of cholesterol-ester transfer protein (CETP) in causation of a low HDL cholesterol in hypertriglyceridemic patients; in addition other factors-lecithin cholesterol acyl transferase (LCAT), hepatic triglyceride lipase (HTGL), and lipoprotein lipase (LPL)-were examined. Plasma activities of CETP and LCAT were measured in 137 male patients with moderate hypertriglyceridemia (plasma triglycerides TGs 200 to 500 mg/dL and LDL cholesterol < 160 mg/dL). Results were compared with those from 50 normolipidemic men of similar age and body habitus. In addition, lipase activities in postheparin plasma were measured in 118 of the subjects with hypertriglyceridemia. The activities of CETP and LCAT were 17% (P <.01) and 7% (P <.05), respectively, higher in the hypertriglyceridemic group than in control subjects. By stepwise regression analysis CETP appeared to contribute 15.2% and LCAT 9.8% to variation in HDL-cholesterol levels. Activities of LPL and HTGL together contributed an additional 14.1% to HDL-cholesterol variation. In contrast, levels of plasma TG accounted for only 5.4% of the variation. There were no differences in relative contributions of these parameters in patients with and those without coronary heart disease. This study indicates that several factors contribute to the variation in HDL-cholesterol levels in hypertriglyceridemic patients, and five factors-CETP, LCAT, HTGL, LPL, and triglyceride levels-account for almost half of this variation. |
| Determinants of postprandial lipemia in men with coronary artery disease and low levels of HDL cholesterol Syvanne, M., P. J. Talmud, et al. (1997), J Lipid Res 38(7): 1463-72. Abstract: We studied the determinants of postprandial lipemia in 49 post-coronary-bypass men with low HDL cholesterol (< or = 1.1 mmol/l at screening). The subjects were given a mixed meal containing 63 g fat and 150,000 IU vitamin A. Serum was obtained before and 3, 4, 5, 6, and 8 h after the meal. S(f) > 400 and S(f) 12-400 lipoproteins, LDL, and HDL were separated by ultracentrifugation; and triglyceride (TG), retinyl ester (RE), and apolipoprotein (apo)E concentrations were measured. The associations of 15 potential predictor variables with measures of postprandial lipemia were evaluated in univariate and multivariate models. Fasting TG concentration was the most important determinant of postprandial lipid and apoE concentrations. In univariate analyses, neither apoE phenotype nor common genetic polymorphisms in the apoB gene (XbaI and apoB signal peptide length polymorphisms), lipoprotein lipase gene (Hind III polymorphism), or apoC-III gene (C1100 to T sequence change) significantly predicted the magnitude of postprandial lipemia. In multivariate linear regression analyses, fasting TG concentration (P< 0.001) and postheparin plasma hepatic lipase activity (P = 0.023) were directly, and body mass index (P = 0.007) and the presence of apoE2 (P = 0.029) allele inversely related to the TG increment in S(f) >400 lipoproteins. Fasting TG was associated with a high (P < 0.001) and presence of the SP24 allele of the apoB signal peptide gene with a low (P = 0.014) S(f) 12-400 TG response. Fasting TG concentrations alone predicted 35%, 10%, and 34% of the variability in postprandial S(f) >400 responses of TG, RE, and apoE; multivariate models improved this predictive power to 40-50%. Even multivariate models were poor predictors of postprandial responses in S(f) 12-400 lipoproteins (0-26%). Much of the interindividual variation in the magnitude of postprandial lipemia remained unexplained in the present study. |
| Determinants of total high density lipoprotein cholesterol and high density lipoprotein subfraction levels among Hispanic and non-Hispanic white persons with normal glucose tolerance: the San Luis Valley Diabetes Study Fulton-Kehoe, D. L., R. H. Eckel, et al. (1992), J Clin Epidemiol 45(11): 1191-200. Abstract: Determinants of total high-density lipoprotein cholesterol (HDL-C) and HDL subfractions were assessed in Hispanic and non-Hispanic white persons (n = 932), aged 20-74 years, in the San Luis Valley, Colorado. Using multiple regression, BMI was negatively associated with HDL-C, HDL2-C, and HDL3-C in men and HDL-C and HDL3-C in women. Among females, current smokers had lower HDL-C and subfractions. Women on beta-blockers had lower HDL3-C levels. For both sexes, a positive association was observed between age and HDL-C and subfractions and physical activity with HDL-C and HDL3-C. Drinking alcohol (> or = 50 g/week) was associated with higher HDL-C and HDL3-C in both sexes and HDL2-C in women. The positive association of age and negative associations of the subscapular/triceps ratio and fasting insulin had consistent relationships with HDL-C, HDL2-C, and HDL3-C in men and women. Ethnicity was not significantly associated with HDL-C or subfractions after controlling for body fat distribution or fasting insulin. |
| Determination of alpha-tocopherol, free cholesterol, esterified cholesterols and triacylglycerols in human lipoproteins by high-performance liquid chromatography Seta, K., H. Nakamura, et al. (1990), J Chromatogr 515: 585-95. Abstract: The determination of alpha-tocopherol, free cholesterol, esterified cholesterols and triacylglycerols in human plasma and in fractions containing individual lipoproteins was achieved by reversed-phase high-performance liquid chromatography (HPLC). The lipoprotein fractions, such as chylomicron, VLDL, LDL, HDL2 and HDL3, were collected by ultracentrifugation of human plasma. The chromatographic separation was accomplished with a column packed with Hitachi Gel 3057, which is a spherical octadecylsilica of particle size 3 microns. The mobile phase was acetonitrile-2-propanol (75:25, v/v), and the eluate was monitored with ultraviolet absorption and fluorescence detectors connected in series. Qualitative analysis of the main chromatographic peaks collected during the HPLC of a plasma sample was done with the use of field-desorption mass spectrometry. The determination analysis of alpha-tocopherol, free cholesterol and esterified cholesterols was effected with a single chromatographic run with n-hexane extracts of plasma or lipoprotein fraction. The separation and determination of these fat-soluble components required as little as 5 microliters of plasma or lipoprotein fraction. |
| Determination of changes in serum lathosterol during treatment with simvastatin to evaluate the role of lathosterol as a parameter for whole body cholesterol synthesis De Cuyper, I., B. G. Wolthers, et al. (1993), Clin Chim Acta 219(1-2): 123-30. Abstract: Serum levels of cholesterol and the cholesterol precursor lathosterol were determined in five healthy volunteers who took 20 mg simvastatin daily during 1 week. During this period and for the following 5 days blood samples were collected. Five days after ingestion of simvastatin, serum lathosterol had already reached a steady-state level and its concentration decreased by 55-73%. In contrast, the cholesterol concentration decreased only by 17-29% and did not reach a steady-state level even after 7 days of treatment. After withdrawal of simvastatin, serum lathosterol quickly rose to pretreatment values. From the data a mean half-life of lathosterol could be calculated of 23.5 +/- 6.6 h during treatment with simvastatin and of 28.7 +/- 15.1 h after its withdrawal, taking, respectively, the decrease and increase of serum lathosterol into account. From these data it can be concluded that serum lathosterol is also a good parameter for determining whole body cholesterol synthesis during non-steady-state conditions, although plasma mevalonic acid, another (early) cholesterol precursor, is preferred owning to its much shorter reported half-life. |
| Determination of cholesterol absorption in humans: from radiolabel to stable isotope studies Pouteau, E., C. Piguet-Welsch, et al. (2003), Isotopes Environ Health Stud 39(4): 247-57. Abstract: Hypercholesterolemia is a major health risk. Dietary cholesterol absorption is one important factor affecting levels of plasma and tissue cholesterol. Considerable effort has thus been devoted to develop reliable in vivo clinical methodologies to determine dietary cholesterol absorption in humans. The present paper summarises radiolabelled experiments and major advances in stable isotope technologies to determine cholesterol absorption. Initially, direct methods employing gastro-intestinal intubation were developed. Later, indirect methods using oral-faecal cholesterol balance permitted calculation of cholesterol mass absorption. Once the use of radiolabelled 3H, 14Ccholesterol balance was developed in healthy humans, it was finally possible to distinguish exogenous and endogenous cholesterol. Non-invasive and safer stable isotope (2H, 13C, 18O) labelled cholesterol tracers then replaced radioisotopes for use in infants and adults. Stable isotopes and radioisotopes showed identical cholesterol kinetics. The most promising contemporary stable isotope assessment of cholesterol absorption is a dual stable isotope dual tracer approach based on simultaneous administration of oral and intravenous differentially labelled cholesterol tracers, followed by plasma sampling for 3-4 d. Online GC/Combustion/IRMS and GC/Pyrolysis/IRMS allow minimal amounts of dual stable isotope cholesterol tracers to be detected. Using the dual stable isotope dual tracer approach, the percent cholesterol absorption in adult volunteers has been determined to be 50-70%. |
| Determination of cholesterol and alpha-tocopherol in eggs by capillary gas chromatography Jin, M. C., L. Wang, et al. (2001), Se Pu 19(5): 478-80. Abstract: A simple and rapid method has been developed for simultaneous determination of cholesterol and alpha-tocopherol in eggs. The method involves alcoholic KOH saponification of the samples, extraction with petroleum ether, and determination by wide-bore capillary gas chromatography. The recoveries of cholesterol and alpha-VE in eggs were 91.0%-93.3% and 82.5%-95.0%, with RSDs of 2.7%-3.1% and 4.9%-6.9% respectively. |
| Determination of cholesterol and cholesterol ester with novel enzyme microsensors Motonaka, J. and L. R. Faulkner (1993), Anal Chem 65(22): 3258-61. Abstract: Enzyme microsensors using cholesterol oxidase (EC 1.1.3.6) and cholesterol esterase (EC 3.1.1.13) were developed for measuring cholesterol and cholesterol ester. The platinum microsensors (platinum diameter, 50 microns) were etched in hot aqua regia to create a cavity at their tip. A porous composite material prepared from acetylene black and Teflon emulsion was packed into this cavity and the redox mediator Os(bpy)3(PF6)2 was monitored by cyclic voltammetry in the potential range of 200-900 mV. The microsensors were dipped overnight in buffer solution containing the desired enzyme to immobilize it on the tip by adsorption. Calibration curves for measurements of cholesterol and cholesterol ester, the effects of pH, temperature, and concomitant compounds, the lifetime of the microsensors, and their availability for measuring cholesterol and cholesterol ester in urine were examined. Under optimal conditions, the response of the sensors was linear in concentration ranges of 5 microM-0.47 mM cholesterol and 2 microM-1.00 mM cholesterol ester. |
| Determination of cholesterol and cortisone absorption in polyurethane. I. Methodology using size-exclusion chromatography and dual detection Dillon, J. G. and M. K. Hughes (1991), J Chromatogr 572(1-2): 41-9. Abstract: A size-exclusion chromatographic method is described for measuring the absorption of the steroid-based lipids cholesterol and cortisone into Pellethane 2363, a polyurethane used in biomedical implants. The method uses refractometry and ultraviolet diode-array detection, with tetrahydrofuran as the mobile phase. Using an injection volume of 150 microliters, the lower limit of accurate measurement for cholesterol (refractive index detection) was 6 micrograms/ml with a lower limit of detection, based on a 2:1 signal-to-noise ratio, of 0.15 micrograms (1 microgram/ml). For cortisone (ultraviolet detection), the lower accurate limit was 0.6 micrograms/ml with a lower limit of 0.015 micrograms (0.1 micrograms/ml). The results show that after 44 h, 2037 micrograms/g cholesterol and 3131 micrograms/g cortisone were absorbed by the polyurethane. The method eliminates extensive sample manipulation and is sensitive to low levels of lipid in the presence of a high-molecular-mass synthetic polymer. |
| Determination of cholesterol at the low picomole level by nano-electrospray ionization tandem mass spectrometry Sandhoff, R., B. Brugger, et al. (1999), J Lipid Res 40(1): 126-32. Abstract: A mass spectrometric method for the quantification of free cholesterol in cells and subcellular membranes is presented. The method is based on a simple one-step chemical derivatization of cholesterol to cholesterol-3-sulfate by a sulfur trioxide-pyridine complex. Quantification is performed by nano-electrospray ionization tandem mass spectrometry (nanoESI-MS/MS) using a stable isotope labeled internal standard. The determination of free cholesterol is demonstrated in about 250 cells of a Chinese hamster ovary (CHO) cell line. With this method a molar ratio of free cholesterol to total phospholipids of 0.34 mol/mol in CHO cells was determined. In a subcellular membrane fraction enriched in Golgi membranes, a molar ratio of free cholesterol to total phospholipids of 0.57 mol/mol was determined. The method should be of value for quantification of other sterols as demonstrated for ergosterol and stigmasterol. |