Measurement Of Dehydrogenase Activity Research Articles

Suitability of dehydrogenase activity assay as an index of soil biological activity

Dehydrogenase activity was studied in typical soils of the Northwest German Lower Plains in order to test some criticisms raised by nannipieri et al. (1990) about methodology. We found that in the same soils with the sampe crop, dehydrogenase activity varies significantly. In the field dehydrogenase activity was dependent on the soil type and not on the cropping system. There is no adequate explanation of these findings. Dehydrogenase activity is a parameter of soil microbiological activity and is influenced by many factors. Either purely accidental the results are or dehydrogenase activity is affected by unknown ecological interactions and by the composition of soil microflora. We conclude that without simultaneous investigations of other microbiological parameters (microbial biomass, ATP levels, enzyme activities, etc.) the measurement of dehydrogenase activity creates confusion and may impede valid ecological comparisons of soils.

Direct cytotoxicity of ethylcholine mustard aziridinium in cerebral microvascular endothelial cells.

The choline analogue ethylcholine mustard aziridinium (AF64A) is a potent and irreversible inhibitor of choline uptake in brain synaptosomes and is used as a neurotoxin to produce animal models of cholinergic hypofunction. However, previous studies have shown that intraocular administration of AF64A in rats not only reduced the number of cholinergic neurons in the retina, but also induced ultrastructural alterations in the microvasculature. The purpose of this study was to investigate whether AF64A has a direct cytotoxic effect on endothelial cells. As revealed by the measurement of lactate dehydrogenase activity in the culture medium, AF64A produced similar concentration-dependent cellular damage in cultures of bovine cerebral endothelial cells and in the human cholinergic neuroblastoma cell line SK-N-MC, but not in bovine cerebral smooth muscle cells. The toxic effect of AF64A correlated well with the affinity of the choline transport system detected in each cell type. The effect of the toxin on endothelial cells was mediated by its interaction with the endothelial cell choline carrier, as demonstrated by the following observations: (a) AF64A inhibited [3H]choline uptake in a concentration-dependent manner in both cultured and freshly isolated cerebral endothelial cells, and (b) the addition of choline or hemicholinium-3 to the culture medium prevented the AF64A-induced toxicity in endothelial cell cultures.

Is serum creatine kinase:aspartate aminotransferase ratio useful for diagnosing acute myocardial infarction in elderly patients?

To evaluate the usefulness of the serum creatine kinase: aspartate aminotransferase (CK:AST) ratio in differentiating between myocardial and non-myocardial increases in serum creatine kinase activity in the elderly. A three month prospective study of all patients admitted to an acute geriatric unit who were clinically assessed and investigated with electrocardiograms and measurement of CK, AST, and lactate dehydrogenase (LDH) activities on the first three days of admission. Excluding those with liver disease and alcohol misuse, patients with increased CK activity were then classified into four groups depending on whether they had fallen or had an acute myocardial infarction (AMI), or both. 270 patients were evaluated. CK activity was raised in 86 (31%) patients on any of the first three days of admission. Of these, 31 had fallen, 19 had an AMI, and five had both fallen and sustained an AMI. The CK:AST ratio, on all days, was higher (p < 0.05) in those who had fallen. On the second and third days, the CK:AST ratio was higher (p < 0.01) in those patients who did not have an AMI. The overlap, however, between these groups was large. These results did not allow discrimination to be made between myocardial and skeletal sources for increased CK activity. The CK:AST ratio is, therefore, of limited use when applied to the diagnosis of AMI in elderly patients. Clinical evaluation rather than the pattern of enzyme change is more likely to determine the cause of increased CK activity.

Calcium‐Dependent and ‐Independent Acetylcholine Release from Electric Organ Synaptosomes by Pardaxin: Evidence of a Biphasic Action of an Excitatory Neurotoxin

The effect of pardaxin, a new excitatory neurotoxin, on neurotransmitter release was tested using purely cholinergic synaptosomes of Torpedo marmorata electric organ. Pardaxin elicited the release of acetylcholine with a biphasic dose dependency. At low concentrations (up to 3 x 10(-7) M), the release was calcium-dependent and synaptosomal structure was well preserved as revealed by electron microscopy and measurements of occluded lactate dehydrogenase activity. At concentrations from 3 x 10(-7) M to 10(-5) M, the pardaxin-induced release of acetylcholine was independent of extracellular calcium, and occluded synaptosomal lactate dehydrogenase activity was lowered, indicating a synaptosomal membrane perturbation. Electron microscopy of 10(-6) M pardaxin-treated synaptosomes revealed nerve terminals depleted of synaptic vesicles and containing cisternae. At higher toxin concentrations (> or = 10(-5) M), there were striking effects on synaptosomal morphology and occluded lactate dehydrogenase activity, suggesting a membrane lytic effect. We conclude that, at low concentrations, this neurotoxin is a promising tool to investigate calcium-dependent mechanisms of neurotransmitter release in the nervous system.

Microbial Activity and Performance of an Anaerobic Reactor Combining a Filter and a Sludge Bed

Microbial activity and performance of anaerobic upflow sludge bed filter(AUBF) were investigated using two types of AUBF: 1/2 packed (AUBF-1/2) and 1/7 packed (AUBF-1/7) with media, and a fully packed anaerobic filter (AF). Each reactor was loaded with 1 to 3 kg COD/m3-day of distillery wastewater using naked barley as raw material. Adenosine triphosphate and dehydrogenase activity measurements were applied to measure the microbial activity in the reactors. AUBF presented comparable treatment efficiencies, while the microbial activity in AUBF is equal to or higher than in AF. Upper packing and lower sludge bed were very efficient to retain the biomass and reduce the amount of packings needed without sacrificing the performance. However, AUBF resulted in less process stability at shock loading than AF, and operation of AUBF needs more careful control to maintain the stability.

A new, simple assay for long-chain acyl-CoA dehydrogenase in cultured skin fibroblasts using stable isotopes and GC-MS

In this paper, we present a new method for measurement of long-chain acyl-CoA dehydrogenase (LCAD) activities in cultured skin fibroblasts. The method is based upon gas chromatographic/mass spectrometric determination of 3-OH-hexadecanoic acid formed during incubation of fibroblasts in a medium containing palmitoyl-CoA and crotonase, to convert the enoyl-CoA ester produced into the 3-hydroxyacyl-CoA ester. The validity of the method demonstrated by the finding of a full deficiency of LCAD in fibroblasts from three patients with an established deficiency of LCAD.

Optimization of the Cascade System of Activated Sludge by Means of the Metabolic Activity Measurements of Microorganisms

A system composed of four sections of the same size and a final clarifier has been investigated. Wastewater from the production of fodder yeast of average COD of 10220 mg/l has been purified. The investigations have shown that the processes which occurred in the first section of the cascade played a dominant role in the removal of the organic impurities. An analysis of the dynamics of the processes in the particular sections of the cascade has shown, however, that the substrate removed in the first section was metabolized only in the next ones, in the oxygen processes, whereas the measurements of dehydrogenase activity have proved that after the termination of the intensive biological oxygen processes, the microorganisms still metabolize the adsorbed substrate in a process which does not consume large quantities of oxygen. The metabolic activity measurements permitted the readjustment of the sewage detention time in the particular sections of the cascade to the observed phasing of the biochemical transformations. In the cascade system studied the optimum aeration time was 96 hours, and the substrate loading - about 0.84 gCOD/g MLSS.d. Such parameters made possible a complete metabolizing of the substrate by microorganisms while preserving favourable biochemical and physical features.

Measurement of acyl-CoA dehydrogenase activity in cultured skin fibroblasts and blood platelets.

The measurement of acyl-CoA dehydrogenase activity is an essential part of the investigation of patients with suspected defects of fatty acid oxidation, and recently the organometallic oxidant ferricenium hexafluorophosphate has been introduced as an electron acceptor for these assays. However, we show that when medium-chain acyl-CoA dehydrogenase activity was measured in cultured skin fibroblasts and platelets from patients with proven defects of this enzyme, there was considerable residual enzyme activity when this electron acceptor was used. The ferricenium assay is not as specific as the anaerobic ETF-linked assay in the biochemical diagnosis of medium-chain acyl-CoA dehydrogenase deficiency in fibroblasts, and therefore is of limited clinical applicability in its present form.

Neutrophil Aldose Reductase Activity and its Association with Established Diabetic Microvascular Complications

Direct investigation of the polyol pathway is rarely possible in studies of human diabetes. A spectrophotometric assay has been developed for the measurement of aldose reductase and sorbitol dehydrogenase activity in the neutrophil. Neutrophil aldose reductase activity was increased in patients with Type 1 diabetes with complications (median 40 (interquartile range 28-48) u, where 1 unit of enzyme activity = nmol NADPH min-1 10(8)-cells-1) compared with those without complications (20 (16-36) u, p less than 0.01) and normal control subjects (20 (8-36) u, p less than 0.01). In Type 2 diabetes, patients with complications also had higher aldose reductase activity (40 (28-52) u) than those without complications (24 (16-36) u, p less than 0.01). There were no differences between patients without complications and normal control subjects. Sorbitol dehydrogenase activity was decreased in diabetic patients (p less than 0.02) but not significantly different between diabetic patients with and without complications.

Biochemical studies carried out on different groups ofCandida parapsilosis andCandida rhagii strains by comparing some cellular and mitochondrial activities

A comparative biochemical study was performed on some strains of Candida rhagii and on strains belonging to different subgroups of Candida parapsilosis. Measurements of alcohol dehydrogenase activity, resistance to drugs and occurrence of an alternative pathway enabled us to confirm the classification between several subgroups within the C. parapsilosis species.

Effects of hydrogen sulfide exposure on surface properties of lung surfactant

Hydrogen sulfide is an irritant and chemical asphyxiant gas that exerts its primary toxic effects on the respiratory and neurological systems. Exposure to hydrogen sulfide above a threshold value of 200-300 ppm is characterized by the sudden onset of hemorrhagic pulmonary edema. The purpose of this study was to determine whether this response is associated with changes in the surface properties of pulmonary surfactant. Bronchoalveolar lavage fluid was retrieved from the lungs of Fischer 344 rats exposed to two concentrations of hydrogen sulfide or fresh air for 4 h. Surface tension-lowering properties were assayed using a captive bubble surface tensiometer. Lung injury was assessed by histopathology and measurements of total protein and lactate dehydrogenase activity in the lavagate. Marked abnormalities in surfactant activity were demonstrated in the lavagates from rats exposed to the highest concentration (300 ppm) of hydrogen sulfide. These involved the properties of adsorption to the air-water interface and surface tension lowering under quasi-static interfacial compression. Exposure to 200 ppm hydrogen sulfide had no effect on minimum surface tension despite a significant increase in protein and lactate dehydrogenase in the lavagate. This would suggest a threshold-type response for the inhibition of surfactant activity by hydrogen sulfide. In vitro studies using normal rat surfactant showed that the abnormalities in surfactant activity were due to inhibitors in the edema fluid and not to a direct effect of sulfide on surfactant. The pathophysiological consequences of increased alveolar surface tension after hydrogen sulfide exposure may need to be considered in the clinical setting.

Microbial metabolic activities in soils of old-field communities following eleven years of nutrient enrichment

Effects of 11 years (1978–1988) of nutrient enrichment (fertilizer or sludge) on microbial metabolic activity in soil samples collected from contrasting types of old-field communities were studied during September 1989. During the 1989 growing season, subplots were manipulated by tilling and/or liming to evaluate mechanisms of ecosystem recovery or were left undisturbed. Metabolic activities of soil microorganisms were determined by measuring dehydrogenase activity within soil samples collected from these subplots. The amounts of 2,3,5-triphenyltetrazolium formazan formed during incubation by the reduction of 2,3,5-triphenyltetrazolium chloride were used to evaluate dehydrogenase activity. Plots that had received long-term applications of sludge or fertilizer had significantly lower rates of microbial activity ( P<0·05) than did control plots. Fertilizer and sludge plots treated with lime had significantly higher microbial metabolic activity ( P<0·05) than those not receiving lime. Whereas liming stimulated microbial activity to near control levels, tilling had no significant treatment effect.

Quisqualate‐induced excitotoxic death of glial cells: Transient vulnerability of cultured astrocytes

The cytotoxic effect of quisqualate, a potent excitatory amino acid (EAA), was examined in purified astrocyte cultures obtained from neonatal rat cerebral cortex and grown in a serum-free, defined medium (G5). Excitotoxicity was evaluated by phase contrast microscopy and quantified by the measurement of lactic dehydrogenase (LDH) activity released from the damaged cells into the culture medium. Quisqualate evoked not only osmotic swelling, but also cytotoxic cell death of cultured astrocytes. The vulnerability of cells was restricted to a period between day 4 and 8 in subculture, with a maximal sensitivity on day 7. The present findings indicate that not only neurons, but also glial cells may exhibit excitotoxic vulnerability. The vulnerability to quisqualate appears to be a transient phenomenon, at least in cultured astrocytes.

Parallel development of excitotoxic vulnerability to N-methyl- l-aspartate and kainate in dispersed cultures of the rat cerebral cortex

The development of excitotoxic cell death caused by l-glutamate, N-methyl- d-aspartate , quinolinate and kainate was examined in dispersed primary cultures of the rat cerebral cortex. Cell death was evaluated by phase-contrast microscopy and quantified by the measurement of lactic dehydrogenase activity in the culture medium. Cells obtained from embryonic cerebral cortex on days 16–18 of pregnancy, and maintained in a serum-supplemented medium, started to respond to glutamate, N-methyl- d-aspartate quinolinate and kainate by cell death on day 7 in vitro. The sensitivity to the neurotoxins increased rapidly, and in a similar fashion, during the second week and remained unchanged up to day 21. Our findings indicate that, unlike the cerebral cortex in situ, the sensitivity of cultured cortical cells to the cytotoxicity mediated by N-methyl- d-aspartate and kainate receptors develops in a nearly parallel fashion.

Regional changes in creatine kinase and myocyte size in hypertensive and nonhypertensive cardiac hypertrophy.

Several intracellular enzymes have been shown to have altered total activity or isoenzyme composition in cardiac hypertrophy. This study tests the hypothesis that the accumulation of the fetal-type (BB + MB) creatine kinase (CK) isoenzymes in hypertrophied adult myocardium is related to an increase in blood pressure. Consideration was made for the location, size, and hemodynamic load of the myocytes. By using the two-kidney, one-clip (2K1C) rat model of renal hypertension with and without hydralazine treatment, CK (total and isoenzyme), lactate dehydrogenase, and citrate synthase activities and myocyte size were measured. An increased heart weight/body weight ratio occurred in both untreated 2K1C rats (4.15 +/- 0.09) and hydralazine-treated 2K1C rats (4.12 +/- 0.13) as compared with control rats (3.25 +/- 0.10). Blood pressure was high only in untreated 2K1C rats (196 +/- 9 mm Hg), as compared with hydralazine-treated 2K1C rats (142 +/- 6 mm Hg) and control rats (135 +/- 3 mm Hg). Myocytes were isolated from five ventricular regions: left ventricular epicardial and endocardial free wall, left and right halves of the interventricular septum, and right ventricular free wall. Regional differences in normal and hypertrophied myocardium were demonstrated for morphological and biochemical parameters, with the greatest changes occurring in left ventricular endocardium. The shift in CK isoenzyme expression toward accumulating more BB + MB was greater in "hypertensive hypertrophy" (untreated 2K1C rats) than in "nonhypertensive hypertrophy" (hydralazine-treated 2K1C rats). Calculations incorporating isolated myocyte volume showed that the cellular content of total CK remained the same during the hypertrophic process, accounting for a decrease in the tissue activity. Measurement of lactate dehydrogenase and citrate synthase activities suggests that hypertrophied myocardium has relatively higher glycolytic capacity and that this effect is exacerbated in the presence of high blood pressure. We conclude that increased blood pressure is more closely linked to the fetal CK isoenzyme shift than is hypertrophy alone.

Measurement of α-ketoglutarate dehydrogenase activity in tissue extracts and human platelets using reversed-phase high-performance liquid chromatography

A new method for the determination of the activity of α-ketoglutarate dehydrogenase complex (KGDHC) in mouse brain and liver mitochondria and in human platelets using reversed-phase high-performance liquid chromatography is described. This method is based on the quantification of succinyl-CoA formed in the reaction catalyzed by KGDHC. Succinyl-CoA was separated using a YMC-Pack C 8 column employing isocratic elution and detected spectrophotometrically at 254 nm. The detection limit of succinyl-CoA was 0.05 nmol. Succinyl-CoA in the supernatant of the assay mixture was stable for several hours at 4°C and for a week when stored at −20°C. The KGDHC assay showed good linearity with time and added protein, and all tissues demonstrated an absolute requirement for added α-ketoglutarate, nicotinamide dinucleotide, and coenzyme A and partial or no requirement for thiamine pyrophosphate, magnesium chloride, and dithiothreitol. The specific activities in liver and brain mitochondria and platelet homogenates determined by the present method were 19.2 ± 09, 18.1 ± 2.8, and 2.6 ± 0.3 nmol/min/mg protein, respectively. In human platelets, the present method gives higher specific activity and lower blank values than a prior method using 14CO 2 and may be useful in the diagnosis of KGDHC deficiency. This method is simple, rapid, and can be readily employed for the determination of KGDHC activity in various animal tissues and human platelets.

Lack of excitotoxic cell death in serum-free cultures of rat cerebral cortex

The excitotoxicity of glutamate,N-methyl- d-aspartate (NMDA), quisqualate and kainate were compared in rat cerebro-cortical cell cultures grown in different media. Excitotoxic cell death was examined by phase contrast microscopy and by the measurement of lactic dehydrogenase (LDH) activity released in the culture medium. Cells grown for 14 days in a serum-free, defined (N2) medium were resistant, while sister cultures maintained in the presence of serum responded to the excitotoxins by a significant degree of cell death. Cells cultured in serum-free medium for 7 days, and fed by a serum-containing one for an additional week exhibited vulnerability to the excitotoxins. It is concluded that the development of excitotoxicity in rat cortical cultures depends on one or more serum constituents.

The circadian rhythm of intra-acinar profiles of alcohol dehydrogenase activity in rat liver: a microquantitative study

Using microquantitative measurements of alcohol dehydrogenase activity in microdissected samples of liver tissue along the sinusoidal length, the intra-acinar distribution profiles were studied in seven groups of female rats at different times during 24 h with a light phase from 6:30 h to 18:30 h. The mean values of alcohol dehydrogenase activity showed a circadian rhythm with a minimum at 13.30 h and a maximum at 17.30 h (p less than 0.001). However, the intra-acinar gradients remained almost unchanged, indicating that increase and decrease in enzyme activity takes place simultaneously in all parts of the liver acinus. This observation, together with data from the literature, suggests that the circadian rhythm of alcohol dehydrogenase activity reflects variations in different liver cell constituents, rather than enzyme protein synthesis or proteolysis.

Radioisotopic measurement of femtomolar amounts of NAD(P)H in the assay of enzymatic activity at a single cell level

A radioisotopic method for the assay of reduced or oxidized pyridine nucleotides, based on the interconversion of 2-[U- 14C]ketoglutarate or 2-keto[3,4- 3H]glutarate and labelled l-glutamate in the reaction catalyzed by glutamate dehydrogenase, was applied to the measurement of lactate dehydrogenase activity in rat pancreatic islet homogenates. Using the tritiated tracer, the limit of sensitivity of the procedure for NAD(P)H assay was close to 1.0 fmol/sample, and lactate dehydrogenase activity could be measured in as little as 0.0005 islet/sample i.e., at a single cell level. This radioisotopic procedure, which can be used for the assay of various metabolites and enzymic activities, thus provides a tool for investigating the heterogeneity in metabolic behaviour of individual cells.

Glutamate dehydrogenase activities in microdissected rat nephron segments: effects of acid-base loading

A method for measurement of glutamate dehydrogenase (GDH) activity in single renal tubules was employed to determine the distribution and regulation of GDH in tubule segments. Fresh microdissected tubules from collagenase-treated kidneys were permeabilized by hyposmotic shock and freezing. The rate of conversion of alpha-ketoglutarate, NH4+, and NADH to glutamate and NAD was measured at 37 degrees C fluorometrically. Very high activities were found in proximal tubule segments (150-210 pmol.min-1.mm tubule length-1), intermediate values (40-90 pmol.min-1.mm-1) in distal convoluted tubules, cortical thick ascending limbs, connecting tubules, medullary thick ascending limbs, and lower values (5-30 pmol.min-1.mm-1) in cortical collecting ducts, inner medullary collecting ducts, outer medullary collecting ducts, outer medullary thin limbs, and inner medullary thin limbs. To determine the effects of acid-base loading on GDH activity, 0.28 M NH4Cl (acid) or 0.28 M NaHCO3 (alkali) was added to the animals' drinking water for 7 days. Acid intake by the rats increased GDH activity in S1 and S2 proximal tubules by threefold, with no effect in other segments, including S3 proximal tubules. Alkali intake decreased GDH activity in the S3 proximal tubule by 40%, with no effect in other segments. We conclude that GDH activities are highest in proximal tubule segments and are regulated only in proximal tubule segments. Thus the results are consistent with the view that the proximal tubule is the chief site of the regulated production of ammonium in the kidney.