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M W McGowan

Publications and source records attributed to M W McGowan.

7 recordsLinked to original sources

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.

Blood Chemical Analysis↗

Enzymic colorimetric determination of phosphatidylglycerol in amniotic fluid.

We describe a procedure for the enzymic, colorimetric determination of phosphatidylglycerol in amniotic fluid. After extraction into chloroform:methanol (2:1 by vol) and evaporation, the phospholipid-containing residue is redissolved in a non-ionic detergent, which thus provides an aqueous sample. The subsequent enzymic reaction sequence involves phospholipase-catalyzed hydrolysis of glycerol from its phospholipid. Subsequent enzyme-catalyzed reactions phosphorylate this glycerol and oxidize the resulting glycerol phosphate to produce hydrogen peroxide, which is reacted to produce an intense red chromogen in the peroxidase-catalyzed coupling of 4-aminoantipyrine and 2-hydroxy-3,5-dichlorobenzenesulfonate. When used in conjunction with previously reported enzymic techniques for determination of lecithin and sphingomyelin, this procedure may provide an accurate and precise "lung profile" for assessment of fetal lung maturity.

Amniotic Fluid↗

A peroxidase-coupled method for the colorimetric determination of serum triglycerides.

We describe an enzymatic method for rapid, precise measurement of serum triglycerides with use of sample:reagent ratios as large as 1:200. Hydrolysis of triglycerides is catalyzed by lipase to produce glycerol and free fatty acids. The glycerol generated is then phosphorylated by adenosine 5'-triphosphate in the presence of glycerol kinase. Oxidation of the resulting glycerol 3-phosphate to produce hydrogen peroxide is catalyzed by L-alpha-glycerophosphate oxidase. An intense red chromogen is produced by the peroxidase-catalyzed coupling of 4-aminoantipyrene and sodium 2-hydroxy-3,5-dichlorobenzenesulfonate with hydrogen peroxide. This sensitive chromogen system not only permits use of unusually small sample volumes, it also facilitates a linear response to serum triglyceride concentrations up to at least 10 g/L while displaying good Ringbom (measure of accuracy) characteristics.

Adenosine Triphosphate↗

Enzymatic colorimetry of lecithin and sphingomyelin in aqueous solution.

A procedure for the enzymatic determination of lecithin and sphingomyelin in aqueous solution is described. The phospholipids are first dissolved in chloroform:methanol (2:1 by vol), the solvent is evaporated, and the residue is redissolved in an aqueous zwitterionic detergent solution. The enzymatic reaction sequences of both assays involve hydrolysis of the phospholipids to produce choline, which is then oxidized to betaine, thus generating hydrogen peroxide. The hydrogen peroxide is subsequently utilized in the enzymatic coupling of 4-aminoantipyrine and sodium 2-hydroxy-3,5-dichlorobenzenesulfonate, an intensely red color being formed. The presence of a non-reacting phospholipid enhances the hydrolysis of the reacting phospholipid. Thus we added lecithin to the sphingomyelin standards and sphingomyelin to the lecithin standards. This precise procedure may be applicable to determination of lecithin and sphingomyelin in amniotic fluid.

Alkaline Phosphatase↗

A procedure for the kinetic colorimetric determination of serum cholinesterase activity.

A choline oxidase-peroxidase coupled enzyme procedures is proposed for the determination of cholinesterase activity in human serum. This system is not only kinetic and colorimetric but is also relatively quick and simple to perform. The initial comparisons suggest that this method correlates well with a commonly used propionylthiocholine-dinitrobis-(nitro-benzoic acid) technique. Large amounts of bilirubin in the sample appear to have only minor deleterious effects on the assay. Since there are only two reagents that may be premixed, the procedure appears to be amenable to automation. The use of a mixture of sodium 2-hydroxy-3,5-dichlorobenzenesulfonate and 4-aminoantipyrene in the peroxidase catalyzed indicator reaction provides for a marked increase in sensitivity over previously reported 4-aminoantipyrene-phenol systems. This augmented sensitivity provides for a relatively large reagent to sample ratio. In addition, the reagents lend themselves toward lyophilization or "dry-fill".

Cholinesterases↗

Proteolytic analysis of the topological arrangement of red cell phosphoproteins.

The topology of human erythrocyte membrane phosphoproteins was determined by using protease digestion and selective solubilization. The distribution of 32P among the membrane polypeptides in intact cells differs from the pattern found in isolated ghosts. The following membrane skeleton polypeptides, the nomenclature of Steck [Steck, T.L. (1972a) J. Mol. Biol. 66, 295-305] being used, were phosphorylated: 2, 2.1, 4.1, 4.5b, and 4.9, together with two polypeptides at 105000 and 110000 daltons that are heavily phosphorylated. Among integral proteins, band 3 and glycophorins A and B are phosphorylated. Glycophorin C appears to have little turnover of phosphate. Autoradiograms of trypsin-treated membranes permitted identification of the transmembrane proteolytic fragment of glycophorin A as a dimer of 38000 daltons. The band 5 region contained two phosphoproteins, the peripheral protein 4.9 (48 kdaltons) and PAS-2. Similarly, band 7 resolved into an integral phosphoprotein and a nonphosphorylated protein associated with the membrane skeleton.

Electrophoresis, Polyacrylamide Gel↗

A procedure for the determination of high-density lipoprotein choline-containing phospholipids.

A procedure for the determination of serum high-density lipoprotein choline-containing phospholipids is described. The choline-containing phospholipids represent 91-97% of the total serum and high-density lipoprotein phospholipids, which have previously been determined in relation to liver disease, neoplastic disorders, diabetes mellitus and atherosclerosis. The enzymatic assay is quick, simple and precise. No interference was found from the precipitating agents used to isolate the high-density lipoprotein fraction. Several serum samples were assayed for high-density lipoprotein cholesterol and choline-containing phospholipids. From this preliminary data there would appear to be a correlation between these two lipid classes.

Cholesterol↗