A universal reagent for phospholipid analysis.

To establish optimum conditions for creatine kinase (EC 2.7.3.2) activity measurement with the creatine phosphate in equilibrium creatine reaction, we re-examined all kinetics factors relevant to an optimal and standardized enzyme assay at 30 and 25 degrees C. We determined the pH optimum in vaious buffers, considering the effect of the type and concentration of the buffer, as well as the influence of various buffer anions on the activity. The relation between activity and substrate concentration was shown and the apparent Michaelis constants of creatine kinase for creatine phosphate and ADP were evaluated. We tested the effect on creatine kinase measurement of the concentration of substrates (glucose and NADP+) in the auxillary and indicator reactions, especially the influence of the added auxiliary (hexokinase) and indicator (glucose-6-phosphate dehydrogenase) enzymes on the lag phase, at different temperatures. The NADP+ concentration proved to be the factor limiting the duration of constant reaction rate. We studied the inhibition of creatine kinase and adenylate kinase by AMP and established a convenient AMP concentration. For reactivation of creatine kinase, N-acetyl cysteine as sulfhydryl compound was introduced. Finally, we examined the relationship between activity and temperature.

Creatine kinase in serum: 1. Determination of optimum reaction conditions.

This paper describes a paper-based microfluidic device that measures two enzymatic markers of liver function (alkaline phosphatase, ALP, and aspartate aminotransferase, AST) and total serum protein. A device consists of four components: (i) a top plastic sheet, (ii) a filter membrane, (iii) a patterned paper chip containing the reagents necessary for analysis, and (iv) a bottom plastic sheet. The device performs both the sample preparation (separating blood plasma from erythrocytes) and the assays; it also enables both qualitative and quantitative analysis of data. The data obtained from the paper-microfluidic devices show standard deviations in calibration runs and “spiked” standards that are acceptable for routine clinical use. This device illustrates a type of test useable for a range of assays in resource-poor settings.

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Measuring Markers of Liver Function Using a Micropatterned Paper Device Designed for Blood from a Fingerstick

Previously we have demonstrated that systemic activation of the complement system after intravenous injection of cobra venom factor (CVF) results in acute lung injury as reflected by increases in the vascular permeability of the lung as well as by morphologic evidence of damage to lung vascular endothelial cells. In using the vascular permeability of the lung as the reference, the current studies show a quantitative correlation between lung injury and the appearance in plasma of lipid peroxidation products (conjugated dienes) as well as increased concentrations of lactic dehydrogenase (LDH) and one of its isoenzymes (LDH-4). After injection of CVF, extracts of lungs also showed elevated levels of conjugated dienes, whereas no elevations were found in extracts of liver, kidney, and spleen. There was no evidence in CVF-injected rats of renal or hepatic injury as reflected by the lack of development of proteinuria and the failure to detect increased serum levels of liver-related enzymes. Other peroxidation products identified in plasma of CVF-injected rats involved hydroperoxides and fluorescent compounds with features of Schiff bases. Not surprisingly, malondialdehyde was not found to be a reliable plasma indicator of lipid peroxidation associated with oxygen radical-mediated lung vascular injury. In using a model of oxygen radical-independent lung injury induced by oleic acid, although large amounts of LDH and LDH-4 were found in the plasma, no increases in plasma levels of conjugated dienes were detected. In CVF-injected animals treated with interventions protective against lung injury (neutrophil depletion, catalase, hydroxyl radical scavengers, or iron chelators), there were striking reductions in the plasma levels of conjugated dienes, hydroperoxides, and fluorochromic products. Morphometric analysis of lung sections revealed that the protective interventions did not interfere with the accumulation of neutrophils in lung interstitial capillaries after systemic activation of complement. In vitro studies with phorbol-stimulated neutrophils failed to demonstrate appearance of conjugated dienes, suggesting that the dienes appearing in plasma of CVF-injected animals are not the result of autotoxic changes in neutrophils. The data presented in this paper suggest that acute lung injury mediated by oxygen radicals derived from phagocytic cells can be monitored by the appearance in plasma of products of lipid peroxidation.

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Systemic complement activation, lung injury, and products of lipid peroxidation.

Pancreatic duct obstruction triggers acute necrotizing pancreatitis in the opossum

Data are presented from the study of a new systematized, kinetic method for the determination of alkaline phosphatase* which was run concurrently in three geographically separated laboratories. The data include estimates of within-day and day-to-day precision, and laboratory-to-laboratory reproducibility. Comparisons are made with three other alkaline phosphatase procedures. An estimated range of normal values is given for adult males and females.

Seymour Winsten

Papers

Evaluation of a New System For the Kinetic Measurement of Serum Alkaline Phosphatase

A rapid micro diazo technique for measuring total bilirubin.

Ascorbic Acid in Blood and Urine

Laboratory evaluation of a chromogenic amylase method.

Large-volume continuous-flow electrophoresis of serum proteins with glass microbeads.