Fluorometric determination of phosphatidylcholine as a measure of phospholipid methylation.

The successive methylation of phosphatidylethanolamine to phosphatidylcholine (phospholipid methylation) has been measured by the incorporation of S-[methyl-3H]adenosylmethionine or colorimetric assay of phosphatidylcholine extracted from adipocyte plasma membranes. A fluorometric assay for phosphatidylcholine was developed to measure phospholipid methylation. This assay is 10 times more sensitive than the colorimetric assay and demonstrates no significant interference with other methylated phospholipids. The fluorometric assay was used to determine a biphasic insulin dose response in adipocyte plasma membranes. This fluorometric assay for phosphatidylcholine represents an alternative method for monitoring phospholipid methylation, especially when increased sensitivity is required.

Enzymic clearing of lipaemic serum following total parenteral nutrition: determination of neonatal bilirubin as a model.

Bilirubin determinations in the newborn infant are one of the many analytical tests that can yield misleading results when the specimen is either iatrogenically or naturally lipaemic. Incorporation of a recently reported enzymic clarification system into a commercially available test kit enables one to conveniently and accurately quantify both total and direct bilirubin within the present procedural characteristics of the Bilirubin Stat Analyser Photometer. This instrument measures the former directly with bichromatic spectrophotometry and the latter with a conventional diazo-type reaction. The proposed modification of existing reagents allows one to apply the assay to samples with as much as, and possibly more than 16 g/l of triglycerides which has been introduced into the vascular circulation as intravenous total parenteral nutrition.

Stimulation of phosphatidylcholine synthesis by insulin and ATP in isolated rat adipocyte plasma membranes.

Added individually or together, insulin and/or ATP significantly and rapidly increased the concentration of phosphatidylcholine in an enriched plasma membrane preparation from rat adipocytes. The increase in phosphatidylcholine synthesis mediated by insulin or ATP was suppressed by the phospholipid methyltransferase inhibitor, S-adenosylhomocysteine. These results suggest that the activity of phospholipid methyltransferase from adipocyte plasma membranes may be increased by phosphorylation and that insulin may further increase the activity of the phosphorylated phospholipid methyltransferase by an alternative pathway.

On-line clarification for the measurement of serum glucose in hyperlipidemic specimens.

The optical aberrations due to the presence of the turbidity caused by hyperlipidemia has been eliminated in two serum glucose procedures. This has been accomplished by incorporating lipase and alpha-cyclodextrin into the two glucose reagents. The hydrolytic action of the lipase generates water soluble glycerol and insoluble fatty acids. By including the chemical scavenger alpha-cyclodextrin into the reagent the fatty acids are solubilized and thus the production of a second turbidity is avoided. Because the clearing reagents are incorporated into the glucose reagents, this is an on-line process and no additional labor is required to clear the sample-reagent mixture. Furthermore, no additional time is required as the clearing occurs in the same period of time that it takes for the indicator reactions to reach equilibrium.

The turbid specimen as an analytical medium: hemoglobin determination as a model.

The quantitation of chemical constituents in lipemic samples is a major problem confronting the clinical laboratory. Currently, a number of cumbersome and time-consuming methods are used to clarify samples before analysis. However, the use of enzymic hydrolysis of triglycerides along with efficient chemical removal of the formed non-esterified fatty acids is exemplified here as an excellent alternative to the current methods of clarification such as ultracentrifugation, extraction or chemical precipitation of low density and very low density lipoproteins. This method of clarifying milky serum has been used by us to assay hemoglobin in severely lipemic blood samples as an analytical model.

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.

On the use of a sensitive indicator reaction for the automated glucose oxidase-peroxidase coupled reaction.

A study into the substitution of sodium 2-hydroxy-3,5-dichlorobenzenesulfonate for phenol in the indicator reaction for an automated glucose procedure is presented. Apart from having physical properties that are more conducive to easy handling than phenol, the former material affords considerably more sensitivity than does the latter. Results obtained with either of these glucose oxidase-coupled systems demonstrate good correlation not only with each other but also with the glucose oxidase procedure of the Beckman Astra 8.

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.

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".