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Biomedical subjects

H de Groot

Publications and source records attributed to H de Groot.

At least 127 records · Page 7Linked to original sources
PubMed1989

Hypoxia, reactive oxygen, and cell injury.

Hypoxia usually decreases the formation of reactive oxygen species by oxidases and by autoxidation of components of cellular electron transfer pathways and of quinoid compounds such as menadione. In the case of menadione reactive oxygen species are liberated to a significant extent only at non-physiologically high oxygen partial pressures (PO2). At physiological and hypoxic PO2 values electron shuttling of menadione in the mitochondrial respiratory chain predominates. In contrast, lipid peroxidation induced by halogenated alkanes, such as carbon tetrachloride, in liver leads to an increase in the formation of reactive oxygen and thus in cell injury under hypoxic conditions. Reactive oxygen species may also be generated during reoxygenation of a previously hypoxic tissue. Based on experiments with isolated hepatocytes a three-zone-model of liver injury due to hypoxia and reoxygenation is presented; 1) a zone where the cells die by hypoxia; 2) a zone where the cells are destroyed upon reoxygenation, presumably mediated by an increase in the cellular ATP content; and 3) a zone where cell injury occurs upon reoxygenation, mediated by reactive oxygen species possibly liberated by xanthine oxidase.

Animals↗
PubMed1989

Hypoxic liver cell death: critical Po2 and dependence of viability on glycolysis.

Viability of isolated hepatocytes was not significantly dependent on oxygen at oxygen partial pressures (Po2) from 70 to 0.3 mmHg. The critical Po2 for induction of hypoxic cell death was close to 0.1 mmHg and was distinct from the value at which mitochondrial function becomes impaired (2-5 mmHg). Hypoxic damage in hepatocytes from fasted rats occurred within 1 h but was delayed by the addition of fructose, which increased rates of lactate formation from about 1 to 12 nmol.10(6) cells-1.min-1. Hepatocytes from fed rats maintained viability for almost 180 min of anaerobic incubation and then rapidly became damaged. Addition of fructose prevented hypoxic cell damage also in these hepatocytes. Rates of lactate formation were 11-15 nmol.10(6) cells-1.min-1 and were increased two- to threefold by fructose. The rapid initial degradation of glycogen and the release of glucose were delayed with fructose, which also could have contributed to sustained glycolytic rates. Further, under conditions in which lactate production was high, e.g., in the fed state, there was also a significantly better preservation of cellular ATP levels.

Adenosine Triphosphate↗
PubMed1989

Hypoxia-reoxygenation injury and the generation of reactive oxygen in isolated hepatocytes.

Reoxygenation following hypoxia enhanced loss of viability of isolated hepatocytes compared to cells maintained under hypoxic conditions. Cell damage due to reoxygenation was not dependent on the conversion of xanthine dehydrogenase to xanthine oxidase which occurred at a time when almost all the hepatocytes had lost their viability. The effect of reoxygenation was critically linked to the duration of the hypoxic period. During the hypoxic period degradation of endogenous glycogen may have provided sufficient substrate for glycolysis to contribute to maintenance of cell integrity by preservation of adenine nucleotides.

Aerobiosis↗
PubMed1988

Reoxygenation injury in isolated hepatocytes: cell death precedes conversion of xanthine dehydrogenase to xanthine oxidase.

Reoxygenation of isolated hepatocytes from fed rats after 3 h of anaerobic incubation led to a significantly enhanced loss of cell viability. No evidence for the participation of reactive oxygen species generated by xanthine oxidase in this reoxygenation injury was found. Conversion of xanthine dehydrogenase to xanthine oxidase occurred at a time when almost all of the hepatocytes had lost their viability. Furthermore, xanthine dehydrogenase was first released from the severely injured cells and then converted to the oxidase form. The results suggest that in the intact organ participation of reactive oxygen species, generated by xanthine oxidase, in reoxygenation injury may only occur when, upon reoxygenation, hypoxic cell injury in part of the tissue has progressed to such an extent that there is a significant conversion of xanthine dehydrogenase to xanthine oxidase.

Anaerobiosis↗
PubMed1988

Dependence of mitochondrial and cytosolic adenine nucleotides on oxygen partial pressure in isolated hepatocytes. Application of a new rapid high pressure filtration technique for fractionation.

By using a new rapid high pressure filtration technique, mitochondrial and cytosolic ATP and ADP contents were determined in isolated hepatocytes at different oxygen partial pressures. At 670 mmHg, subcellular adenine nucleotide contents and ATP/ADP ratios were comparable with values obtained with the digitonin fractionation technique. However at lower oxygen partial pressure ADP appears to be rephosphorylated during digitonin fractionation whereas with high pressure filtration fractionation rephosphorylation of ADP is avoided due to shorter fractionation times. Cytosolic and mitochondrial ATP/ADP ratios decrease if oxygen partial pressure is lowered. However the absolute values of ATP/ADP ratios depend critically on the incubation conditions. Thus incubation of hepatocytes in an oxystat system, where oxygen partial pressure is maintained constant by infusing oxygen-saturated medium and the hepatocyte suspension is continuously stirred, yields much higher subcellular and overall ATP/ADP ratios than incubation in Erlenmeyer flasks gassed with different gas mixtures and shaken in a water bath. This is ascribed to limited diffusion of oxygen from the medium into the cell if the suspension is not mixed thoroughly by stirring. The strong dependence of subcellular ATP/ADP ratios on incubation conditions indicates that oxygen may be one rate-controlling factor for oxidative phosphorylation in the intact cell.

Adenine Nucleotides↗
PubMed1988

Monoclonal antibodies to the major feline allergen Fel d I. I. Serologic and biologic activity of affinity-purified Fel d I and of Fel d I-depleted extract.

Monoclonal antibodies were raised against a major feline allergen, Fel d I. The specificity of the antibodies for Fel d I was demonstrated by a modified crossed immunoelectrophoretic procedure. These antibodies were used in affinity purification and depletion. Fed d I was eluted from the affinity matrix at a low pH. It was tested in serologic and biologic assays. Results of both the RAST and the histamine release test with affinity-purified Fel d I confirmed that it is a very potent allergen for the majority of patients allergic to cats. In the extract depleted with the monoclonal antibody, the ratio Fel d I/cat albumin was reduced by a factor of 20. With this depleted extract, 18 of 25 patients showed a reduction greater than 50% of the bound anti-IgE in the RAST compared with the RAST with crude cat extract. In the histamine release test with cells from two patients, the activity of the depleted extract was reduced by factors of 30 and 40, respectively. Depletion of Fel d I by polyclonal antibodies resulted in a reduction of the ratio Fel d I/cat albumin by a factor of 330. In the RAST, a reduction of the IgE response greater than 50% was found in 23 of 25 patients; in 12 patients the reduction was greater than 90%. In the histamine release test, the polyclonally depleted extract was 200 to 300 times less potent than the crude cat-dander extract. These results indicate that a very large proportion of the allergenic activity of cat-dander extract is caused by a single molecule: Fel d I.

Allergens↗
PubMed1988

Discrepancies between the skin test and IgE antibody assays: study of histamine release, complement activation in vitro, and occurrence of allergen-specific IgG.

Intracutaneous skin tests (STs) and RAST with the common allergens, grass pollen, house dust mite, and cat dander, were performed on 660 adult patients. In 117 patients (18%), we found 140 discordances (7%) in a total number of 1980 ST and RAST combinations. In agreement with studies in the literature, greater than 80% of the discordances consisted of positive skin reactions without detectable allergen-specific IgE antibodies in serum. The percentages of discordant results were similar for the three allergens. Reproducibility of both the RAST and the ST was evaluated in the discordant group. Repetition of the routine RAST procedure elicited results similar to those in the first test in 81% (105/130). A second ST elicited identical results in 89% (47/53). In addition to the routine IgE antibody assay, sera of patients with a positive ST but without detectable IgE antibodies were tested in two other RAST systems: (1) a RAST with allergen extracts from the same production batch as the ST reagents, and (2) the Pharmacia RAST. In spite of having a clearly positive ST, sera from 68 (80%) of 85 patients remained completely negative in all three RAST systems. Histamine release (HR) in vitro from washed leukocytes was studied in 35 patients with a reproducible positive ST and negative RAST results with serum. Interpretation of this test was possible in 28 patients. In 82% (23/28) of these patients, clearly detectable HR was found with the relevant allergen extract. A role of IgE in the skin reactions and HR tests was confirmed by positive RAST results with IgE that was affinity purified from serum of seven of these patients. Allergen-specific IgG4 antibodies are unlikely to be implicated, since no antibodies against grass pollen and house dust mite were detectable in sera of these patients. Only 18% of the patients with an unexplained skin reaction with cat dander have detectable IgG4 antibodies, but these antibodies were found in a similar frequency in a nonallergic, ST negative control group. Low total IgG responses precluded false negative RAST results caused by competition of IgG antibodies with IgE antibodies. There were no significant differences in the degree of complement activation in vitro by house-dust extracts between healthy control subjects, nonallergic patients, and patients with unexplained skin reactivity. It is concluded that a high proportion of the positive skin reactions with common inhalant allergens, which are not accompanied by a positive RAST, are probably caused by IgE antibodies that are not detectable in serum with any of the RAST procedures.(ABSTRACT TRUNCATED AT 400 WORDS)

Adolescent↗
PubMed1988

Hypoxic toxicity of misonidazole in a glucose-6-phosphate dehydrogenase deficient mutant Chinese hamster ovary cell line.

The metabolic activation of misonidazole (MISO) and its effects on the hexose monophosphate pathway (HMP) and on cell viability were studied in hypoxic mutant Chinese hamster ovary (CHO) cells deficient in glucose-6-phosphate dehydrogenase and their parent wildtype cells. The metabolic activation of MISO was similar in both cell lines as indicated by the binding of 14C-MISO to the acid-insoluble fraction of these cells; it was decreased by the absence of glucose. In the wildtype CHO cells, MISO caused a significant stimulation of the activity of the HMP while in the mutant CHO cells no HMP activity was measurable, even in the presence of MISO. In both cell lines clonogenicity began to decline after 2 hr and trypan blue exclusion after 4 hr of hypoxic incubation. The effect of MISO on both parameters of cell viability was somewhat more pronounced in the wildtype CHO cells. This difference became especially significant at the longer incubation times. The results indicate that reducing equivalents for the metabolic activation of MISO are provided not only by the HMP but that pathways other than the HMP, such as glycolysis or pathways starting from mitochondrial tricarboxylates, are of similar or even greater importance in this respect.

Animals↗
PubMed1988

Partial purification of rat liver microsomal glucose-6-phosphatase on hydroxylapatite.

Glucose-6-phosphatase was effectively solubilized from rat liver-microsomal membrane by the nonionic detergent Renex 690 in the presence of 0.6M sodium chloride. Subsequent separation on hydroxylapatite proved to be a successful and rapid initial step towards the purification of this enzyme. Glucose-6-phosphatase appeared in the colourless void volume with a yield of about 40-50%. The specific activity in the pooled void volume was 3-4 U/mg protein representing an enrichment of 30- to 40-fold. The best final specific activity obtained in an enriched fraction was 6.7 U/mg protein. Analysis of the pooled glucose-6-phosphatase-enriched fraction by SDS electrophoresis revealed 2 dominant protein bands with the apparent molecular mass of 17 and 18.5 kDa and few weak protein bands in the range of 21 to 42 kDa.

Animals↗
PubMed1987

Distinct temporal relation among oxygen uptake, malondialdehyde formation, and low-level chemiluminescence during microsomal lipid peroxidation.

An oxystat system was employed in conjunction with a single-photon counting apparatus for simultaneous monitoring of oxygen uptake, oxidative decomposition of membrane lipids, and occurrence of electronically excited species during microsomal lipid peroxidation. During NADPH/ADP-iron-promoted lipid peroxidation at a steady state oxygen partial pressure (pO2) of 30 mm Hg, complex time relationships among oxygen uptake, malondialdehyde (MDA) formation, and low-level chemiluminescence were observed. While the first two parameters occurred nearly simultaneously, low-level chemiluminescence occurred with a significant delay. A decrease of the steady state pO2 to 3 mm Hg led to significant increases of the lag phases of all three parameters and a further enhancement of the time displacement of low-level chemiluminescence in relation to oxygen uptake and MDA formation. At a pO2 of 0.5 mm Hg, the lowest pO2 maintained during this study, no low-level chemiluminescence was observed while oxygen uptake and MDA formation were still detected. In contrast, during NADPH/CCl4-promoted lipid peroxidation at a pO2 of 0.5 mm Hg a sudden drastic rise of low-level chemiluminescence accompanying oxygen uptake and MDA formation was observed. At pO2 between 0.5 and 3 mm Hg all three parameters occurred nearly concomitantly during the entire incubation. At pO2 levels above 3 mm Hg all three parameters showed principally the same behavior. However, the respective maxima of low-level chemiluminescence were reached with some delay. The present observations support the assumption that the decomposition of membrane lipid peroxyl radicals to MDA and the formation of electronically excited species proceed via different pathways. The time displacement between oxygen uptake and MDA formation, on the one hand, and low-level chemiluminescence, on the other hand, depends on the type of initiating radical system and on the steady state pO2 level. It is suggested that the differences are due to distinct subsets (chemical or spatial) of secondary peroxyl radicals in the membrane.

Adenosine Diphosphate↗
PubMed1987

Dependence of hepatic gluconeogenesis on PO2: inhibitory effects of halothane.

The dependence of gluconeogenesis and O2 uptake on PO2 in isolated rat hepatocytes is presented. Maintenance of steady-state PO2 was achieved with an oxystat system (Biochem. J. 236: 765-769, 1986). O2 uptake showed a half-maximal (K0.5) value of 0.5 Torr PO2, whereas the glucose synthesis rate was half-maximal at 1.2 Torr PO2. Halothane at concentrations greater than 1 mM exerted a parallel inhibition of O2 uptake and glucose synthesis at all PO2 levels studied. In contrast, at halothane concentrations less than 1 mM, inhibition of glucose synthesis occurred only at less than 20 Torr PO2. At these low concentrations, halothane was without significant effects on cellular O2 uptake. In isolated mitochondria, inhibition of O2 uptake was already half-maximal at a halothane concentration of 0.5 mM. In this subcellular system the inhibitory effect of halothane was independent of PO2. These results demonstrate that the critical PO2 at which cellular O2 utilization begins to decrease and the PO2 at which glucose synthesis begins to decrease are comparable; both PO2 levels are approximately 5 Torr. The metabolic zonation of the liver lobule is discussed in view of the results presented.

Animals↗
PubMed1986

A computer-supported oxystat system maintaining steady-state O2 partial pressures and simultaneously monitoring O2 uptake in biological systems.

A feedback-controlled oxystat system is described maintaining steady-state O2 partial pressures (pO2) between 0.01 mmHg (14 nM-O2) and 150 mmHg (210 microM-O2) and simultaneously monitoring O2 uptake at rates between 0.1 and 120 microM-O2 X min-1 in suspensions of cells, in subcellular fractions and in solutions of enzymes. At pO2 values between 0.2 and 150 mmHg (0.28 and 210 microM-O2) a polarographic O2 sensor was used, and below a pO2 of 0.2 mmHg (0.28 microM-O2) the O2-dependent luminescence of the photobacterium Vibrio fischeri was utilized to monitor the actual pO2. At a selected pO2, O2 supply is maintained by injecting appropriate amounts of O2-saturated aqueous medium into the reaction chamber by using a motor-driven burette. The oxystat system is under control of a computer that reads the O2 sensors, interacts with the motor-driven burette, calculates the O2 uptake from the amounts of O2-saturated medium added, collects data from further measuring devices and provides the documentation of the results during incubation.

Computers↗
PubMed1986

Alterations of the microsomal glucose-6-phosphatase system evoked by ferrous iron- and haloalkane free-radical-mediated lipid peroxidation.

Alterations of catalytic activities of the microsomal glucose-6-phosphatase system were examined following either ferrous iron- or halothane (CF3CHBrCl) and carbon tetrachloride (CCl4) free-radical-mediated peroxidation of the microsomal membrane. Enzyme assays were performed in native and solubilized microsomes using either glucose 6-phosphate or mannose 6-phosphate as substrate. Lipid peroxidation was assessed by the amounts of malondialdehyde equivalents formed. Regardless of whether the experiments were performed in the presence of NADPH/Fe3+, NADPH/CF3CHBrCl, or NADPH/CCl4, with the onset of lipid peroxidation, mannose-6-phosphatase activity of the native microsomes increased immediately, while further alterations in catalytic activities were only detectable when lipid peroxidation had passed characteristic threshold values: above 2 nmol malondialdehyde/mg microsomal protein, glucose-6-phosphatase activity of the native microsomes was lost, and at 10 nmol malondialdehyde/mg microsomal protein, glucose-6-phosphatase and mannose-6-phosphatase activity of the solubilized microsomes started to decline. It is concluded that the latter alterations are due to an irreversible damage of the phosphohydrolase active site of the glucose-6-phosphatase system, while the changes observed at earlier stages of microsomal lipid peroxidation may also reflect alterations of the transporter components of the glucose-6-phosphatase system. Virtually no changes in the catalytic activities of the glucose-6-phosphatase system occurred under anaerobic conditions, indicating that CF3CHCl and CCl3 radicals are without direct damaging effect on the glucose-6-phosphatase system. Further, maximum effects of carbon tetrachloride and halothane on lipid peroxidation and enzyme activities were observed at an oxygen partial pressure (PO2) of 2 mmHg, providing additional evidence for the crucial role of low PO2 in the hepatotoxicity of both haloalkanes.

Animals↗
PubMed1985

Enzymic determination of inorganic phosphates, organic phosphates and phosphate-liberating enzymes by use of nucleoside phosphorylase-xanthine oxidase (dehydrogenase)-coupled reactions.

Coupled enzyme assays are described for measuring inorganic phosphates, organic phosphates and phosphate-liberating enzymes in biological material. The assays all determine Pi by its reaction with inosine, catalysed by nucleoside phosphorylase; this yields ribose 1-phosphate and hypoxanthine. The hypoxanthine is oxidized to uric acid by xanthine oxidase, and may be measured either by the absorbance of the uric acid, or by the formazan formed when a tetrazolium salt is used as the oxidant. The coupled enzyme assays are characterized by high sensitivity, quantitative utilization of phosphates and stoichiometric formation of the measurable products, measurement at pH 6.0-8.5, determination of phosphates within a single analytical step, and continuous measurement of phosphohydrolase activity in a corresponding rate assay. Examples include determinations of substrates such as Pi, PPi and AMP, and of enzymes such as 5'-nucleotidase, inorganic pyrophosphatase and glucose-6-phosphatase. Directions for further examples are given.

5'-Nucleotidase↗
PubMed1985

Loss of latent activity of liver microsomal membrane enzymes evoked by lipid peroxidation. Studies of nucleoside diphosphatase, glucose-6-phosphatase, and UDP glucuronyltransferase.

The effects of lipid peroxidation on latent microsomal enzyme activities were examined in NADPH-reduced microsomes from phenobarbital-pretreated male rats. Lipid peroxidation, stimulated by iron or carbon tetrachloride, was assayed as malondialdehyde formation. Independent of the stimulating agent of lipid peroxidation, latency of microsomal nucleoside diphosphatase activity remained unaffected up to microsomal peroxidation equivalent to the formation of about 12 nmol malondialdehyde/mg microsomal protein. However, above this threshold a close correlation was found between lipid peroxidation and loss of latent enzyme activity. The loss of latency evoked by lipid peroxidation was comparable to the loss of latency attainable by disrupting the microsomal membrane by detergent. Loss of latent enzyme activity produced by lipid peroxidation was also observed for microsomal glucose-6-phosphatase and UDPglucuronyltransferase. In contrast to nucleoside diphosphatase, however, both enzymes were inactivated by lipid peroxidation, as indicated by pronounced decreases of their activities in detergent-treated microsomes. According to the respective optimal oxygen partial pressure (po2) for lipid peroxidation, the iron-mediated effects on enzyme activities were maximal at a po2 of 80 mmHg and the one mediated by carbon tetrachloride at a po2 of 5 mmHg. Under anaerobic conditions no alterations of enzyme activities were detected. These results demonstrate that loss of microsomal latency only occurs when peroxidation of the microsomal membrane has reached a certain extent, and that beyond this threshold lipid peroxidation leads to severe disintegration of the microsomal membrane resulting in a loss of its selective permeability, a damage which should be of pathological consequences for the liver cell. Because of its resistance against lipid peroxidation nucleoside diphosphatase is a well-suited intrinsic microsomal parameter to estimate this effect of lipid peroxidation on the microsomal membrane.

Acid Anhydride Hydrolases↗