Cholesterol determination

Hydrogen peroxide produced as a result of reactions of oxidase enzymes with analyte substrates can be sensitively determined, both directly by luminol ECL and indirectly by Ru(bpy)32+ ECL. For the latter, hydrogen peroxide is detected on the basis of its ability to diminish the ECL reaction between Ru(bpy)32+ and added oxalate, by reacting with, and depleting the concentration of, oxalate. Thus ECL intensity is inversely proportional to the concentration of analyte. This principle has been used, for example, to determine cholesterol [70],... 

Bioanalyzer can be considered another version of commercial biosensors for off-line analysis. It was developed to have capabilities of complete analysis, short response time, specificity, and sensitivity that allows a quick clinical test. Abbott Vision, Boehringer-Mannheim Reflectron, and Kodak Ektachem DT60 (IBI Biolyzer is the new name) are used for cholesterol measurement in doctors offices. Bioanalyzer consists of biological and transducing component that are not physically connected. The uniqueness of this separation provides the versatility of analysis, i.e., use of disposable and different biological component for multi-components measurements. In authors laboratory, Kodak Ektachem DT60 was used successfully to determine cholesterol in some food matrices as well as in off-line process control. The analysis time was only 10 minutes compared to 1-2 days for the GC and HPLC methods. Complicated... 

Allen MP, DeLizza A, Ramel U, Jeong H, Singh P. A non-instrumental quantitative test system and its application for determining cholesterol concentration in whole blood. Chn Chem 1990 36 1591-7. 

Preparation of Extracts. Leffler (L2) and others have previously used isopropanol as a lipid solvent for determining cholesterol. Connerty et al. (C6) and Lofland (L4) also showed that triglycerides and phospholipids are extracted quantitatively. Under our extraction conditions, complete recovery of these lipid fractions is obtained at a serum-to-solvent ratio of 1 10 through 1 25. We routinely use 1 20 dilution (0.5 ml of sera+ 9.5 ml of isopropanol). We find that the addition of Lloyd s reagent removes bilirubin and other chromogenic material present in serum, as well as approximately 80% of the phospholipids present. 

As the electrode moves closer to the cell surface, the amperometric current rises (Figure 17.1.3C). When the electrode is placed in contact with the cell surface, cholesterol is extracted from the membrane and moves across the thin membrane layer and partitions into the electrode-supported enzyme-modified lipid bilayer. Subsequent enzyme catalyzed oxidation of cholesterol by molecular oxygen produces hydrogen peroxide which is oxidized at the platinum electrode and detected. This is an efficient and simple method to determine cholesterol levels in the plasma membrane of cells which should find applicability in determining the role cholesterol plays in the organization of cellular membranes (51). 

Stable isotope methodology to determine cholesterol absorption has been described by Bosner et al. [93,94] applying administration of cholesterol orally and Cs-cholesterol intravenously and measurement of the isotope... 

The first application of the Gaussian distribution is in medical decision making or diagnosis. We wish to determine whether a patient is at risk because of the high cholesterol content of his blood. We need several pieces of input information an expected or normal blood cholesterol, the standard deviation associated with the normal blood cholesterol count, and the blood cholesterol count of the patient. When we apply our analysis, we shall anive at a diagnosis, either yes or no, the patient is at risk or is not at risk. 

Cholesterol was isolated m the eighteenth century but its structure is so complex that Its correct constitution was not determined until 1932 and its stereochemistry not verified until 1955 Steroids are characterized by the tetracyclic ring system shown m Figure 26 9a As shown m Figure 26 9b cholesterol contains this tetracyclic skeleton modified to include an alcohol function at C 3 a double bond at C 5 methyl groups at C 10 and C 13 and a C Hn side chain at C 17 Isoprene units may be discerned m var lous portions of the cholesterol molecule but the overall correspondence with the iso prene rule is far from perfect Indeed cholesterol has only 27 carbon atoms three too few for It to be classed as a tnterpene... 

As part of a collaborative study of a new method for determining the amount of total cholesterol in blood, two samples were sent to ten analysts with instructions to analyze each sample one time. The following results, in milligrams of total cholesterol per 100 mb of serum, were obtained... 

Unfortunately, excess consumption of fatty foods has been correlated with serious human disease conditions. Effects on cardiovascular disease (95), cancer (96), and function of the immune system (97) have been shown. Numerous studies have been conducted to determine the effects of saturated, monounsaturated, and polyunsaturated fatty acids on semm cholesterol and more recently high density Hpoprotein (HDL) and low density Hpoprotein... 

Steroids are synthetic products of cholesterol [57-88-5]. The chemical stmcture of a steroid hormone is determined by sequential enzymatic processing of the cholesterol molecule. Steroid products differ among steroid-secreting glands because of differences in enzyme processing, eg, the production of estrogen by the ovary requires enzymatic steps that do not occur in the adrenal cortex. 

Glucose [50-99-7] urea [57-13-6] (qv), and cholesterol [57-88-5] (see Steroids) are the substrates most frequentiy measured, although there are many more substrates or metaboUtes that are determined in clinical laboratories using enzymes. Co-enzymes such as adenosine triphosphate [56-65-5] (ATP) and nicotinamide adenine dinucleotide [53-84-9] in its oxidized (NAD" ) or reduced (NADH) [58-68-4] form can be considered substrates. Enzymatic analysis is covered in detail elsewhere (9). 

Cholesterol The end point for the cholesterol reaction can be determined by following dye formation. Additionally, the amount of oxygen consumed can be measured amperometricaHy by an oxygen-sensing electrode (see Electro analytical techniques). The H2O2 produced by cholesterol oxidase requires phenol to produce dye. 

Free cholesterol can also be determined, if cholesterol esterase is omitted. 

Defects in the LDL receptor have been particularly well explored as a basis of the disease familial hypercholesterolemia (93,111). A number of defects that collectively impair LDL receptor trafficking, binding, or deUvery underHe this disease where LDL and semm cholesterol rise to levels that mediate early cardiovascular mortaUty. Studies of the population distribution of this defect can determine the source of the original mutation. Thus, in Quebec, about 60% of the individuals suffering from familial hypercholesterolemia have a particular 10-kdobase deletion mutation in the LDL gene (112). This may have arisen from an original founder of the French Canadian settiement in the seventeenth century. 

Trace each of the carbon atoms of mevalonate through the synthesis of cholesterol, and determine the source (i.e., the position in the mevalonate structure) of each carbon in the final structure. 

Multidimensional LC has also been used to determine ursodeoxycholic acid and its conjugates in serum (14). These compounds are used in the treatment of cholesterol gallstones, hepatitis and bilary cirrhosis. These authors employed a traditional (10 X 4 mm) pre-column and a micro-bore (35 X 2 mm) analytical column that were interfaced by using a six-port switching valve. 

The polyene macrolide filipin was isolated in 1955 from the cell culture filtrates of Sterptomyces filipinensis, and was later shown to be a mixture of four components [36]. Although too toxic for therapeutic use, the filipin complex has found widespread use as a histochemical stain for cholesterol and has even been used to quantitate cholesterol in cell membranes [37]. The flat structure of filipin III, the major component of the filipin complex, was assigned from a series of degradation studies [38]. Rychnovsky completed the structure determination by elucidating the relative and absolute stereochemistry [39]. The total synthesis plan for filipin III relied heavily on the cyanohydrin acetonide methodology discussed above. 

FIGURE 1 Effect of (sequential) extrusion of MLV dispersions through polycarbonate membrane filters (Unipore) with pore sizes of 1.0, 0.6, 0.4, 0.2, and 0.1 ym on the mean liposome diameter. DXR-containing MLV (phosphatidylcholine/phosphatidylserine/ cholesterol 10 1 4) mean diameter of nonextruded dispersion about 2 ym pH 4. Mean particle size determined by dynamic Light scattering (Nanosizer, Coulter Electronics). (From Crommelin and Storm, 1987.)... 

Solberg and co-workers have applied discriminate analysis of clinical laboratory tests combined with careful clinical and anatomic diagnoses of liver disease in order to determine which combinations of the many dozen liver diagnostic tests available are the bes t ( ). These authors found that the measurement of GPT, GMT, GOT, ALP and ceruloplasmin were the most useful enzymatic tests, when combined with other non-enzymatic tests such as the measurement of bilirubin, cholesterol, hepatitis-B associated Australian antigen, etc. Another group of highly useful enzymes, not discussed in this review, are those clotting factors and the enzyme cholinesterase which are synthesized by the liver cells. 

Although the primary focus of oxidase based enzyme electrodes has been the determination of glucose, the list of extensions to other analytes is considerable. Systems have been described for cholesterol 123,132-136) galactose ii -i35-i37) dd i38.i39) lactatepyruvatecreatinine serum lipaseethanoland amino acids... 

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