Phosphate determination by enzymatic colorimetric assay

A direct colorimetric assay for inorganic phosphate in serum is described. The system is based on utilization of the enzymes, purine-nucleoside phosphorylase and xanthine oxidase, to generate superoxide ions. The superoxide is measured in the presence of an electron mediator compound with 3-(4',5'-dimethyl-2-thiazolyl)-2,4-diphenyl-2H-tetrazolium bromide as the chromogen. The high absorbance of this chromogen between 550 and 660 nm affords useful results with a sample/reagent volume ratio as low as 1:100. A single working reagent is used, and the reaction is complete in 15 min at room temperature. The standard curve is linear for inorganic phosphate concentrations as high as 4.9 mmol/liter. Analytical recovery of phosphate in human sera averages 100%. Within-run precision study gives CV less than or equal to 1.0%. The results of this method compare closely (r greater than 0.99) with those obtained by the semidine method (recommended standard). The method lends itself to automation.     

Preparation of human cord sera for enzymatic triglyceride assays: removal of free glycerol by ultrafiltration

Enzymatic triglyceride assays that generate glycerol from triglycerides as a part of the enzymatic process in quantitating serum triglyceride levels give elevated values when external free glycerol is present. Our objective was to develop an ultrafiltration technique that would remove exogenous and/or endogenous free glycerol from small aliquots of human cord sera so that accurate serum triglyceride values could be obtained with the commercially available triglyceride assay kits. Exogenous glycerol was completely removed from cord sera when the samples were washed twice with saline in Amicon Centricon-30 microconcentrators. This ultrafiltration technique lowered cord serum triglyceride levels significantly (P less than 0.001), but had no effect on cord total serum cholesterol levels. A comparison of washed and unwashed cord sera by either polyacrylamide or agarose gel electrophoresis indicated that the serum protein and lipoprotein profiles were not altered by the ultrafiltration process.     

Analysis of enzymatic activities of subcellular and chromatographic fractions by an automated colorimetric microassay system

A simple, automated colorimetric microassay system has been designed to quantitate enzyme activities commonly used as markers for subcellular compartments. This system relies on the spectrophotometric reading of microtiter wells containing the chromophore products. The microassay allows rapid, economical, and quantitative analysis of enzyme activities associated with sucrose or Percoll gradient fractions used for subcellular fractionation studies as well as the screening of a large number of fractions derived from HPLC and other separation columns used for enzyme purification. We describe its use for the quantitation of activities associated with acid and alkaline phosphatases, alkaline phosphodiesterase, beta-glucuronidase, alpha-N-acetylglucosaminidase, alpha-mannosidase, alpha-L-fucosidase, glycosidases, serine esterases, and succinate dehydrogenase, and give the range of their sensitivities. This microassay system has been applied to the isolation of granules of cytolytic lymphocytes and to the identification and purification of a serine esterase from the isolated granules of these cells.     

Modified enzymatic procedure for the routine determination of glycerol and triglycerides in plasma

A modification of Wieland's enzymatic procedure for glycerol analysis is presented. It is simple and precise, and readily applicable to the routine analysis of plasma and tissue glycerol and triglycerides. Optimal precision is obtained in samples containing 0.003-0.400 micromole of glycerol per ml, which in this method is equivalent to plasma levels of 0.011 to 1.40 micromoles/ml.     

Enhanced enzymatic hydrolysis of wheat straw by aqueous glycerol pretreatment

Considering the practical technology-economy of glycerol processing from oleochemicals industry, the ensuing work was proposed to further explore the atmospheric aqueous glycerol autocatalytic organosolv pretreatment (AAGAOP) to improve the enzymatic hydrolysis of lignocellulosic biomass. With the liquid-solid ratio of 20 g g(-1) at 220 degrees C for 3h, the AAGAOP enabled wheat straw to remove approximately 70% hemicelluloses and approximately 65% lignin, with approximately 98% cellulose retention. The pretreated fiber was achieved with approximately 90% of the enzymatic hydrolysis yield after 48 h. At oven-drying, dehydration was likely to cause the hornification of fiber, which was responsible for the low enzymatic hydrolysis of dried fiber. With SEM observations, the AAGAOP disrupted wheat straw into thin and fine fibrils, with a small average size and more surface area. The AAGAOP technique, as a novel strategy, enhanced the enzymatic hydrolysis of lignocellulosic biomass by removing the chemically compositional barrier and altering the physically structural impediment.     

An enzymatic, spectrophotometric glycerol assay with increased basic sensitivity

Glycerol can be assayed with good precision and accuracy in biological samples free of dihydroxyacetone using a series of four enzymatic reactions. Two moles of nicotinamide-adenine dinucleotide are reduced per mole of glycerol, thus doubling the basic sensitivity of existing enzymatic, spectrophotometric methods. The method was developed primarily for use in studies of adipose tissue. No deproteinization was required in samples obtained from incubation of isolated fat cells or pieces of adipose tissue.