M.W. McGowan

Sensitive high-density lipoprotein cholesterol assay

Abstract A procedure has been described in which a currently acceptable simple procedure for the precipitation of low-density and very-low-density lipoproteins is paired with a modified sensitized enzyme reagent system containing sodium 2-hydroxy-3,5-dichlorobenzenesulfonate for the determination of cholesterol by equilibrium reaction with the high density lipoprotein fraction of serum. The resulting increase in absorbances by using the auxochromic derivative of phenol is some four to five times that of the phenol system and therefore makes it possible to discriminate with more assurance within the critical ±50 mg/liter concentration difference. Factors such as comparative calibration slopes for the phenol reaction and its auxochrome substitute, the water displacement error, the presence of bilirubin, and the contamination by LDL-VLDL precipitating reagents are considered and discussed.

Description of analytical problems arising from elevated serum solids.

There has always been a problem with the collection of data and interpretation of the results obtained from any biological fluid in which the solids content was increased to a great extent. Of these solids, the triglycerides of the lipids may cause a plasma (serum) to vary in appearance from opalescent to milky. This condition of the specimen and the concomitant turbidity upon its addition to reagents creates the well-documented optical aberrations of spectrophotometric measurements. In addition, the lipids, in conjunction with the proteins, can act as diluents when they are elevated, thereby decreasing what might be termed the residual true plasma volume. Thus the water content of an aliquot sampled for a particular analytical procedure is diminished, and by that means a situation is created in which a short sample is drawn. This dilution effect by the solids results in a lowering of the assay values obtained for the measured constituents of such a serum sample. An associated phenomenon of high concentrations of solids, especially proteins, is the increase in viscosity of a specimen, a condition that also causes an error of short sampling when certain peristaltic pumping devices are used. This review considers several aspects of problems encountered when dealing with a number of circumstances that are critical to the measurement of analytes in severely hyperlipemic and/or hyperproteinemic specimens. These include the problems of short sampling; the potential amelioration of the problem by corrective mathematics, extraction of the lipids, or ultracentrifugation of the true plasma from the lipids; the important need to include most analytes into our considerations; the difference in reference base values for the calculation of concentrations of lipids of serum versus other analytes; the concept of the use of ratios when the reference base values differ, numerator analyte from denominator analyte; and the problems of using serum blanks when necessary corrective action for the solids volume is neglected. Thus, in the final analysis, problems with underestimated volumes of samples used for many spectrophotometric determinations are considered here along with the other difficulties encountered when the need to measure analytes in serums with extremely high solids content presents.

Determination of free, esterified, and total serum cholesterol using a sensitive color reaction

Abstract A method is described for the enzymatic determination of free and total cholesterol sequentially in either whole serum or the HDL fraction. Measurement of low levels of free cholesterol is facilitated by the substitution of sodium 2-hydroxy-3,5-dichlorobenzene-sulfonate for phenol in the peroxidase-catalyzed indicator reaction. Both free and total cholesterol may be measured in the same tube or separately. These alternatives may appeal to investigators who for various reasons may prefer to perform these determinations either sequentially or separately. The sequential approach is a means of conserving the relatively expensive enzymatic reagents.

Spectrophotometric study on minimizing bilirubin interference in an enzyme reagent mediated cholesterol reaction

Abstract A spectrophotometric study was carried out which describes the effect of the inclusion of potassium ferrocyanide into an enzyme reagent system as a protective device against the competitive interaction of bilirubin in the indicator reaction. A previously described system for depicting how bilirubin interacts competitively in a modified 4-aminoantipyrene-phenolic peroxidase-peroxide coupled reaction was used as a template to judge the effectiveness of the protective action. The results obtained indicate that the protection is not total, but that the close approximation may make this a worthwhile modification in procedure to consider for the determination of total, free, and/or high density lipoprotein cholesterol. At the same time it might be inferred that the interference of bilirubin can be diminished chemically for other oxidase-peroxidase coupled systems. A search for even better solutions to this interference problem may be possible. For us, this lead has pointed the way in other similar directions. In addition, this study reports on a preliminary investigation as to the site of coupling of phenolic compounds with 4-aminoantipyrene.

Interference study on precipitating reagents used in a sensitive color reaction for high-density lipoprotein cholesterol determination☆

Abstract The use of a combination of divalent cations and polyanions to isolate the HDL fraction of serum by precipitation of the other lipoprotein classes is quite popular. However, some of the combinations used have been reported to falsely elevate the apparent HDL-C value, perhaps due to interference from the precipitating reagents or imperfect lipoprotein precipitation ( 10, 12 ). In this article, we have isolated an interfering effect of precipitating reagents on the equilibrium reaction used for the determination of cholesterol by the use of pure aqueous standards. Of the most common precipitating reagents used, it was found that Mg/dextran and Mg/phosphotungstate caused little or no chemical interference in the color reaction itself, while polyethylene glycol elevated cholesterol values slightly and Mn/heparin caused a measureable increase in the cholesterol value observed.

Enzymatic determination of the major serum lipids with a common indicator reaction

Abstract Enzymatic, colorimetric assays for serum cholesterol, choline-containing phospholipids, and triglycerides were developed with the intent of being able to specifically determine these individual lipids after isolation of serum lipoproteins by a technology such as ultracentrifugation that is already developed, electrophoresis that is already available for cholesterol and awaits the other lipid stains, or chemical fractionation. Once the relative amounts of the lipids in each lipoprotein fraction are known, the absolute amounts in each separated fraction can then be calculated using the total serum concentrations. As a secondary purpose, the measurement and arithmetic summation of serum cholesterol, choline-containing phospholipids, and triglycerides concentrations appears to provide a close estimate of the major serum total lipids. This summation technique results in apparent lower serum total lipids values than are obtained by a conventional turbidimetric method. This bias appears to be due to a combination of overestimation by the turbidimetric method and underestimation by the summation technique. However, this summation technique appears to be able to provide more information for specific lipid measurements on chemical, ultracentrifugal, or electrophoretic separation of lipoproteins by revealing which lipid class(es) is/are present in an abnormal concentration. It could also prove useful in providing major total lipid information for nonspecific staining of electrophoretic zones where fat-soluble stains such as Sudan black B are presently used. In the latter case, cholesterol and triglycerides are already determined and only the choline-containing phospholipid assay is additional.