Determination of Cholesterol

Determination of CholesteroL For meat extraction, the procedures for determining the cholesterol of extracted lipid samples were described Chao et al. (2i). For edible beef tallow extraction, the preparation of samples for cholesterol content was based on the AOAC (22) method Section 28.110. The prepared sample was then injected into a Supelco SPB-1 fused silica capillary column of 30 meters x 032 mm i.d. in a Varian Model 3700 gas chromatograph equipped with dual flame ionization detectors. The initial holdup time was 4 min at 270°C and then programmed to a temperature of 300°C at a ramp rate of 10°C/min. Helium flow rate and split ratio were 13 ml and 50 1, respectively, while the injector/detector temperature was 310°C. 

Welch, M J, Parris, R M, Sniegoski, L T, and May, W E (2001), CCQM-K6 Key Comparison on the determination of cholesterol in serum (Sevres, France International Bureau of Weights and Measures). 

Assays for the determination of cholesterol in routine clinical laboratories include cholesterol esterase and thus quantify total cholesterol (i.e., unesterified and es-terified cholesterol). However, specific assays are also available that lack cholesterol esterase and hence allow the determination of unesterified or free cholesterol. The difference between total and unesterified cholesterol gives the concentration of cholesterol esters. 

Gentner, P. R. and Haasemen, A. 1979. Method for the determination of cholesterol in milk samples by application of a commercially available enzymatic test kit. Milch-wisseasch 34, 344-346. 

The measurement of serum cholesterol is one of the most common tests performed in the clinical laboratory. Hypercholesterolemia (high blood cholesterol levels) can be the result of a variety of medical conditions. Among the conditions implicated are diabetes mellitus, atherosclerosis, and diseases of the endocrine system, liver, or kidney. High blood cholesterol levels do not point to a specific disease determination of cholesterol is used in conjunction with other clinical measurements mainly for confirmation of a particular diseased condition, rather than for diagnosis of a specific ailment. 

In the Basic Protocol, gas-liquid chromatography (GLC) using an open tubular wall, coated, fused silica column with nonpolar liquid phase is described. In Alternate Protocol 1, reversed-phase HPLC (RP-HPLC) is applied for separation and quantification of cholesterol. In Alternate Protocol 2, enzymatic measurement is applied for determination of cholesterol. 

ALTERNATE PROTOCOL 2 ENZYMATIC MEASUREMENT OF CHOLESTEROL Test combination kits for enzymatic determination of cholesterol in food are now commercially available. For the determination of total cholesterol, esterified cholesterol is hydrolyzed to free cholesterol and fatty acid under mild alkaline conditions. Cholesterol oxidase oxidizes free cholesterol to cholest-4-en-3-one to generate hydrogen peroxide, which further oxidizes methanol to formaldehyde. Formaldehyde then reacts with acetyl acetone in the presence of NH4+ ions to form yellow lutidine dye, which is subsequently determined spectrophotometric al 1 y. 

Several methodologies are known as effective tools for the determination of cholesterol in food and foodstuffs, e.g., colorimetry using the Libermann-Burchard reaction (Naito and David, 1984), Iatroscan thin-layer chromato-graphy/flame ionization detection (Indrasena et al., 1991), enzymatic determination (Shen et al., 1982), GC determination (Karkalas et al.,... 

Methanol used for the HPLC measurement should be of HPLC grade with low absorbance at UV wavelengths below 230 nm. Dried-proc-essed foods usually include a relatively large amountof oxidized cholesterol analogs (Smith, 1996). The benzoate derivatives of these oxides do not separate from cholesterol benzoate by HPLC. Therefore, the GC measurement is more suitable for the determination of cholesterol in dried products with a high level of the oxidized cholesterol. 

Naito, H.K. and David, J.A. 1984. Laboratory considerations Determination of cholesterol, triglyceride, phospholipids, and other lipids in blood and tissues. Lab. Res. Methods Biol. Med. 10 1-76. 

Newkirk, D.R. and Sheppard, AJ. 1981. High pressure liquid chromatographic determination of cholesterol in foods. J. Assoc. Off. Anal. Chem. 64 54-57. 

An evaporative light scattering detector was coupled with a UV spectrophotometer, and was applied to HPLC for the quantitative determination of cholesterol oxides in edible oils and fats. 

Fetouris, D.J., Botsoglou, N.A., Psomas, I.E., and Mantis, A.I. 1998. Rapid determination of cholesterol in milk and milk products by direct saponification and capillary gas chromatography.. /. Dairy Sci. 81 2833-2840. 

For determination of cholesterol in milk and milk products, samples are saponified without lipid extraction in capped tubes with 0.5 M methanolic potassium hydroxide solution by heating at 80°C. 

A reversed-phase HPLC method was used for determination of cholesterol in dairy products, it evaluated that a more complete evaluation of cholesterol contents in dairy products is necessary when the vegetables and fruits are present. 

Prygonski, K., Jelen, H., and Wasowicz, E. 2000. Determination of cholesterol oxidation products in milk powder and infant formulas by gas chromatography and mass spectrometry. Nahrung 44 122-125. 

PA Marshall, JM Vandepeer, I Pant, VC Trenerry, P Scheelings, DR Buick. The development and evaluation of secondary food reference materials for the determination of cholesterol, fatty acids and selected water-soluble vitamins in foods. Food Chem 58 269-276, 1997. 

N. Pena, G. Ruiz, A.J. Reviejo and J.M. Pingarron, Graphite-teflon composite bienzyme electrodes for the determination of cholesterol in reversed micelles. Application to food samples, Anal. Chem., 73(6) (2001) 1190-1195. 

The determination of cholesterol is important for the diagnosis and prevention of a number of clinical disorders such as hypertension, arteriosclerosis, cerebral thrombosis and coronary heart disease. As the majority of cholesterol in human blood is present in an esterified form, a separate saponification step is required to obtain a total cholesterol analysis early methods for this involved caustic and toxic reagents, long analysis times and a relatively large sample volume. Free cholesterol can be determined chromatographically, although this requires cumbersome and expensive laboratory-based equipment. Modern methods use the enzyme cholesterol esterase to release esterified cholesterol which is then oxidised by a second enzyme, cholesterol oxidase (ChOx, Fig. 23.3) [48]. 

X.S. Miao, C.D. Metcalfe, Determination of cholesterol-lowering statin drugs in aqueous samples using liquid chromatography-electrospray ionization tandem mass spectrometry, J. Chromatogr. A 998 (2003) 133-141. 

Martin, S.P., D.J. Lamb, J.M. Lynch, et al. 2004. Enzyme-based determination of cholesterol using the quartz crystal acoustic wave sensor. Anal. Chim. Acta 487 91-100. 

A method for the determination of cholesterol has been adapted. The color developed in the presence of ferric chloride, sulfuric acid, and glacial acetic acid has been utilized for the analysis. The use of such strong reagents has required employment of tetrafluorethylene tubing. These acid reagents are not pumped directly from reagent bottles but are displaced from reservoirs by fluid pumped into the reservoir behind a flexible diaphragm. 

AOAC (Association of Official Analytical Chemists) (1995b) AOAC Official Method 970.51 Fats (Animal) in Vegetable Fats and Oils (Determination of Cholesterol), in Official Methods of Analysis of the Association of Official Analytical Chemists, 16th edn, Arlington, Virginia, USA. 

Morgan and Deeming, in a benchmark paper which describes the method,19) due in the first instance to Nelder and Mead 20), found that only 26 experiments were required to optimise a complicated spectrophotometric method for the determination of cholesterol in blood serum, for which five variables had to be taken into account. 

Angulo, A.J., Romera, J.M., Ramirez, M., Gil, A. 1997. Determination of cholesterol oxides in dairy products. Effect of storage conditions. 1. Agric. Food Chem. 45, 4318 1323. 

Guardiola, F., Boatella, J., Codony, R. 2002. Determination of cholesterol oxidation products by gas chromatography. In Cholesterol and Phytosterol Oxidation Products Analysis, Occurrence, and Biological Effects (F. Guardiola, P.C. Dutta, R. Codony, G.P. Savage, eds.), pp. 50-65, AOCS Press, Champaign, IL. 

Sandhoff R, Briigger B, Jeckel D, Lehmann WD, Wieland FT. Determination of cholesterol at the low picomole level by nano-electrospray ionization tandem mass spectrometry. J. Lipid Res. 1999 40 126-132. 

Raith K, Brenner C, Farwanah H, Muller G, Eder K, Neubert RH. A new LC/APCl-MS method for the determination of cholesterol oxidation products in food. J. Chromatogr. A 2005 1067 207-211. 

FIbronectIn The values of fibronectin in malignant ascites are significantly higher than in cases of portal ascites. Given a threshold value of 7.5 mg/dl, it is possible to differentiate between the two forms of ascites with a sensitivity of 100% and a specificity of more than 95%. The combined determination of cholesterol and fibronectin allows malignant ascites to be identified reliably. (33, 39)... 

Determination of cholesterol in milk fat by reversed-phase high-performance liquid chromatography and evaporative light-scattering detection G. A. Spanos and S. J. Schwartz, LC-GC 10(10) 774 (19XX)... 

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