Monitoring of unfractionated heparin with the activated partial thromboplastin time: determination of therapeutic ranges.

The purpose of the present study was to determine therapeutic ranges for unfractionated heparin therapy using the activated partial thromboplastin time (APTT) by calibration against anti-Xa concentration. APTT assays were performed locally, i.e. at the institution of blood collection, on fresh plasma samples from patients treated with intravenous unfractionated heparin. The measurements were performed by 25 Dutch clinical laboratories using 11 different APTT reagents and 10 different types of coagulometers. After the local APTT measurement, the samples were frozen and transported to a central laboratory for measurement of anti-Xa activity. The number of samples from the participating laboratories ranged from 10 to 48. Local APTT results were correlated with the central anti-Xa measurements. Orthogonal regression analysis of log-transformed values was used to calculate APTT therapeutic ranges corresponding to anti-Xa concentrations of 0.29-0.47 IU/ml. The calculated APTT ranges were different between laboratories, even when the same reagent was used. In many laboratories, the therapeutic APTT range in use was much wider than the calculated range. Imprecision of the calculated APTT range was influenced by the wide scatter of the measurement points and by the selection of samples for the orthogonal regression equation. The present results show that, if anti-Xa concentrations of 0.29-0.47 IU/ml reflect the true therapeutic range, many laboratories do not use the proper therapeutic APTT range.

Subcellular fractionation of rat cerebellum: separation of synaptosomal populations and heterogeneity of mitochondria.

A crude mitochondrial fraction (M) derived from manually disrupted cerebellar tissue and enriched in choline acetyltransferase (ChAT) activity was fractionated by centrifugation in discontinuous and continuous sucrose gradients. Further purification of 'cholinergic' synaptosomes was achieved (relative specific activity (RSA) of ChAT greater than 3), but the overlap with other synaptosomal populations was still considerable. Hand-homogenized cerebella processed through the full fractionation procedure described here and in previous papers yielded preparations enriched in certain neuronal structures and a fraction in which 'heavy' free mitochondria was concentrated. To characterize these preparations the activities of two transmitter enzymes (CHAT and glutamate decarboxylase, GAD) and 6 mitochondrial enzymes (succinate dehydrogenase (SDH), glutamate dehydrogenase (GDH), monoamine oxidase, citrate synthase, fumarase and GABA-aminotransferase) were determined. The distribution of the transmitter enzymes was clearly different in the preparations containing various neuronal structures. The GAD:ChAT RSA ratio was 2.4 for the glomerulus particles, 1.3 for the molecular layer fragments, 0.6 for the myelinated axon segments, and 0.2 for the 'cholinergic' synaptosomes. The mitochondrial enzyme profile of the preparations comprising mainly neuronal structures differed markedly from that of the 'free' mitochondrial fraction. Notably the latter was greatly enriched in GDH (RSA 5.6), whereas the SDH:GDH RSA ratio was relatively high in the former preparations. Nevertheless there were notable differences in the enzyme profile of the fractions of predominantly neuronal origin indicating that the enzyme composition of mitochondria of neuronal processes is not uniform.

A radiochemical method for the determination of transketolase activity in erythrocyte hemolysates.

A radiochemical method for the determination of transketolase activity is reported. It is based on incubation of erythrocyte hemolysates with radioactive ribose 5-phosphate followed by isolation of sedoheptulose 7-phosphate on anion-exchange columns. The experimental conditions are discussed and the presence of phosphatase activity in the incubation mixture is demonstrated. The proposed method is compared with a current colorimetric method. Reference values are given for the transketolase activity and the thiamine pyrophosphate effect.

Short-chain fatty acid synthesis in brain. Subcellular localization and changes during development.

Acetyl-CoA synthase (EC 6.2.1.1), Propionyl-CoA synthase (EC 6.2.1.-) and butyryl-CoA synthase (EC 6.2.1.2) were measured in subcellular fractions prepared by primary and density-gradient fractionation from adult rat brain by a method resulting in recoveries close to 100%. Most of the activity of the three enzymes was recovered in the crude mitochondrial fraction. On subfractionation of this crude mitochondrial fraction with continuous sucrose density gradients, most of the activity of the three enzymes was found at a higher density than NAD+-isocitrate dehydrogenase and at about the same density as glutamate dehydrogenase, confirming earlier reported data for acetyl-CoA synthase. The finding that propionyl-CoA synthase and butyryl-CoA synthase had about the same distribution in the gradients as acetyl-CoA synthase adds support to the hypothesis that mitochondria involved in the metabolism of these short-chain fatty acids (all three of which have been shown to result in a rapid and high labelling of glutamine in vivo) form a distinct subpopulation of the total mitochondrial population. The three synthase activities were found to differ from each other in their rate of change and their subcellular localization during rat brain development. This, in combination with the observation that in gradients of adult brain preparations the three activities did not completely overlap, suggests that the three synthase activities are not present in the same proportion to each other in the same subpopulation (s) of mitochondria in the brain.

Subcellular localization of gamma-aminobutyrate transaminase and glutamate dehydrogenase in adult rat brain. Evidence for at least two small glutamate compartments in brain.

The subcellular localizations of gamma-aminobutyrate transaminase (EC 2.6.1.19) and glutamate dehydrogenase (EC 1.4.1.2) in brain tissue of adult rats were compared with each other and with those of NAD+-isocitrate dehydrogenase (EC 1.1.41) and monoamine oxidase (EC 1.4.3.4; kynuramine as substrate). Crude mitochondrial fractions from brain tissue were centrifuged in continuous sucrose density gradients. gamma-Aminobutyrate transaminase and glutamate dehydrogenase were always found at a higher density than NAD+-isocitrate dehydrogenase and monoamine oxidase. When centrifuged for 1 h at 53 000gav., there was a slight difference between the distribution profiles of glutamate dehydrogenase and gamma-aminobutyrate transaminase. This difference was larger when the centrifugation time was only 15 min. It is concluded that there are subpopulations of brain mitochondria with differing proportions of gamma-aminobutyrate transaminase and glutamate dehydrogenase. The results are discussed in relation to evidence obtained with labelled precursors in vivo that there are at least two small glutamate compartments in adult brain.