Biomedical subjects
Publications and source records attributed to J F Koster.
The levels of iron, copper and ferritin were measured in seminal plasma from young healthy students and infertile men with severe teratospermia. No significant differences were found between them. The iron might be available for lipid peroxidation unless it is bound in some way. The role of ferritin in O2- formation is discussed.
In a comparative screening study of chelators intended for clinical use eleven iron chelators have been tested for their ability to mobilize (59Fe) iron from 59Fe-labelled ferritin and from hepatocytes of rats labelled with 59Fe-transferrin. The toxic effects of the chelators were also studied using microsomal lipid peroxidation induced by Fe3+/ADP and NADPH. From these tests it was shown that 1,2-dimethyl 3-hydroxypyrid-4-one (L1) and mimosine were the most effective iron chelators in iron mobilization and did not catalyse lipid peroxidation. In conclusion it can be stated that besides to investigate the iron binding capacity of new chelators also their ability to catalyse lipid peroxidation has to be ruled out.
The effects of the lipid-peroxidation product 4-hydroxynonenal on the formation of fluorescent chromolipids from microsomes, mitochondria and phospholipids were studied. Incubation of freshly prepared rat liver microsomes or mitochondria with 4-hydroxynonenal results in a slow formation of a fluorophore with an excitation maximum at 360 nm and an emission maximum at 430 nm. The rate and extent of the development of the 430 nm fluorescence can be significantly enhanced by ADP-iron (Fe3+). With microsomes, yet not with mitochondria. NADPH has a catalytic effect similar to that of ADP-iron. Fluorescent chromolipids with maximum excitation and emission at 360/430 nm are also formed during the NADPH-linked ADP-iron-stimulated lipid peroxidation. Phosphatidylethanolamine and phosphatidylserine react with 4-hydroxynonenal revealing a fluorophore with the same spectral characteristics as that obtained in the microsomal and mitochondrial system. The findings suggest that the fluorescent chromolipids formed by lipid peroxidation are not derived from malonaldehyde, but are formed from 4-hydroxynonenal or similar reactive aldehydes via a NADPH and/or ADP-iron-catalysed reaction with phosphatidylethanolamine and phosphatidylserine contained in the membrane.
A flow cytometric method to determine cellular GSH contents has been developed. This method is fast and simple and enables the determination of GSH contents in intact cells. Results obtained with the new method correlate well with the results obtained by a specific biochemical assay for GSH (r = 0.9984; n = 7). The method has been used to determine GSH recovery rates in cultured fibroblasts from healthy subjects and from patients with Werner's syndrome, Spielmeyer-Vogt syndrome and Fanconi's anemia. No obvious differences in GSH recovery rates were observed. GSH recovery rates were also not affected after in vitro ageing. Experiments with cells deficient in GSH synthetase revealed that the observed GSH recovery is exclusively due to de novo synthesis.
Xanthine oxidase is able to mobilize iron from ferritin. This mobilization can be blocked by 70% by superoxide dismutase, indicating that part of its action is mediated by superoxide (O2-). Uric acid induced the release of ferritin iron at concentrations normally found in serum. The O2(-)-independent mobilization of ferritin iron by xanthine oxidase cannot be attributed to uric acid, because uricase did not influence the O2(-)-independent part and acetaldehyde, a substrate for xanthine oxidase, also revealed an O2(-)-independent part, although no uric acid was produced. Presumably the amount of uric acid produced by xanthine oxidase and xanthine is insufficient to release a measurable amount of iron from ferritin. The liberation of iron from ferritin by xanthine oxidase has important consequences in ischaemia and inflammation. In these circumstances xanthine oxidase, formed from xanthine dehydrogenase, will stimulate the formation of a non-protein-bound iron pool, and the O2(-)-produced by xanthine oxidase, or granulocytes, will be converted by 'free' iron into much more highly toxic oxygen species such as hydroxyl radicals (OH.), exacerbating the tissue damage.
Isolated rat hepatocytes were incubated in the absence or presence of glucagon and the activity of fructose-1,6-bisphosphatase was measured in cell extracts. After glucagon treatment the Vmax was increased (20-50%) whereas the Km remained unchanged. The stimulation was complete at 5 min after addition of glucagon. The glucagon concentration needed for maximal stimulation was 10(-9) M. After gel filtration the fructose-1,6-bisphosphatase activity in extracts of glucagon-treated cells was lowered to the control level. The effect of glucagon could not be completely mimicked by dibutyryl cAMP. The data indicate that in addition to the possible regulatory role of enzyme phosphorylation, a positive effector is involved in the stimulation of fructose-1,6-bisphosphatase activity by glucagon.
In the process of lipid peroxidation of microsomes induced either by oxygen radicals generated by xanthine oxidase or by NADPH, ferritin is able to donate the necessary iron. The amount of ferritin necessary to catalyze the process of lipid peroxidation is in the physiological range. In contrast to the finding with phospholipid liposomes, catalase hardly stimulates the lipid peroxidation of microsomes.
Iron mobilized from ferritin is able to convert superoxide and hydrogen peroxide, which are produced in large amounts in rheumatoid arthritis (RA), to the extremely toxic hydroxyl radical. We have found that synovial fluid ferritin is increased significantly in RA patients compared with levels in controls. The high synovial fluid:serum ferritin ratio is compatible with the hypothesis that synovial fluid ferritin is derived from the synovial membrane. We found no difference in ferritin concentrations in the synovial membranes of RA patients compared with those of controls. Quantitative data on the amount of iron bound to ferritin showed that the level was 2.9 times higher in RA synovial membranes than in those of controls. Moreover, RA synovial fluid contained considerable amounts of iron bound to ferritin. Calculation of the iron saturation of ferritin revealed that RA synovial membranes contained a mean of 2,210 moles of iron per mole of ferritin: a significant elevation when compared with the mean value of 1,500 moles found in the synovial membranes of the controls. The decreased saturation of ferritin in RA synovial fluid, compared with that in the synovial membrane, could be caused by an uncompensated release of iron from ferritin, which has been induced by superoxide that is produced by stimulated granulocytes. The results demonstrate that in the joints of RA patients, sufficient ferritin loaded with iron is available to stimulate oxygen free radical damage.
It is not known which message and mechanism triggers the cell to mobilize iron from ferritin. In this paper we present the results of incubation experiments with 59Fe-labelled hepatocytes. Anemic serum gives a significant higher rate of iron mobilization than normal serum. The involvement of apo-transferrin is ruled out because it did not increase iron mobilization. Citrate increased iron mobilization which is not the result of an increase in NADH/NAD+-ratio because addition of ethanol did not stimulate iron mobilization. Desferrioxamine is used clinically in iron overloaded patients and it is known that iron removal is a very slow process. Although desferrioxamine can mobilize iron from ferritin in hepatocytes, a considerable amount remains inside the cell as a low molecular weight fraction. This fraction represents chelator bound iron and is slowly released into the circulation.
We detected no difference in the reduced glutathione content of erythrocytes obtained from patients with rheumatoid arthritis (RA) and controls. The stability of glutathione to oxidative stress (cumene hydroperoxide) was also the same. Although measured in the erythrocyte, our results indicate that changes in intracellular reduced glutathione are not involved in the aetiology of RA. Serum from patients with RA had a significantly reduced (p less than 0.01) sulphydryl (SH) concentration (415 +/- 89 (SD) mumol/l) compared with controls (583 +/- 74 mumol/l). This was also valid if the SH groups were expressed per gram of protein. Serum and synovial fluid from RA patients contained similar levels of SH groups (mumol/g protein).
The superoxide (O2-) production of stimulated polymorphonuclear leucocytes is increased in patients with rheumatoid arthritis and osteoarthritis compared with controls. Treatment of these different groups with pharmacological amounts of the non-steroidal anti-inflammatory drug piroxicam in vivo resulted in a decrease of about 25% in O2- secretion by isolated granulocytes. In vitro experiments showed that piroxicam inhibits O2- production of granulocytes by interference with the stimulation of the NADPH-oxidase. Piroxicam caused diminished O2- production of membrane fragments if it was present during the stimulation of the NADPH-oxidase of the intact cells. During the actual O2- production of the stimulated membrane fragments piroxicam had no effect. It is concluded that piroxicam is able to inhibit granulocyte O2- production by blocking the activation of NADPH-oxidase, which results in diminished tissue destruction by oxygen free radicals in inflammatory diseases.
1) The effect of 4-hydroxynonenal and lipid peroxidation on the activities of glucose-6-phosphatase and palmitoyl CoA hydrolase were studied. 2) 4-Hydroxynonenal inactivates glucose-6-phosphatase but has no effect on palmitoyl-CoA hydrolase. These effects are similar with those observed during lipid peroxidation of microsomes. 3) The inhibition of glucose-6-phosphatase by 4-hydroxynonenal can be prevented by glutathione but not by vitamin E. The inactivation of glucose-6-phosphatase during lipid peroxidation is prevented by glutathione and delayed by vitamin E. 4) The formation of 4-hydroxynonenal during lipid peroxidation was followed in relation to the inactivation of glucose-6-phosphatase. At 50% inactivation of glucose-6-phosphatase the 4-hydroxynonenal concentration was 1.5 microM. To obtain 50% inactivation of glucose-6-phosphatase by added 4-hydroxynonenal a concentration of 150 microM or 300 microM was needed with a preincubation time of 30 and 60 min, respectively. 5) It is concluded that the glucose-6-phosphatase inactivation during lipid peroxidation can be due to the formation of 4-hydroxynonenal. The formed 4-hydroxynonenal which inactivates glucose-6-phosphatase is located in the membrane. If this mechanism is valid it implies that a functional SH group of glucose-6-phosphatase is layered in the membrane. However, an inactivation of glucose-6-phosphatase by desintegration of the membrane by lipid peroxidation cannot be ruled out.
Several theories were formulated to explain the process of aging. Now they often appeared out of date. The free radical theory is a rather recent one that is based on the production of free radicals during normal physiological respiration. A lot of data indicate their role in the progress of aging. Clearly also other factors play a role in this process: the toxicity of free radicals may for instance depend on the availability of free iron. So on the one hand we need oxygen for existence and free radicals may destroy infectious bacteria; on the other hand oxygen appears to be toxic and may be supposed to underly the process of aging.
Hexachlorobenzene (HCB) induces a porphyria characterized by a diminished activity of the enzyme uroporphyrinogen decarboxylase (URO-D), presumably due to inactivation by reactive metabolites of HCB. We studied the effect of iron on HCB porphyria in female rats, to determine whether the iron dependent process of lipid peroxidation was involved in the pathogenesis of porphyria. We showed that malondialdehyde formation is increased in rat liver tissue of porphyric rats and that high molecular weight proteins due to cross-linking are formed. We also showed that the induction of porphyria by HCB is dependent on the presence of iron. Our findings suggest that lipid peroxidation is involved in the toxicity of HCB and that the aggravating effects of iron on HCB are mediated by lipid peroxidation.
In the isolated, perfused rat heart, lipid peroxidation, induced by cumene hydroperoxide (Cum OOH), is accompanied by the release of malondialdehyde (MDA). Using a modified perfusion technique resulting in the separate collection of coronary and interstitial effluent, it can be shown that upon Cum OOH (0.5 mM) perfusion there is an immediate release of MDA in the coronary effluent and a delayed release in the interstitial fluid, indicating the susceptibility and coronary vascular tissue towards free radical-induced lipid peroxidation. Perfusion with Cum OOH leads to an initial increase of the coronary flow and a depressed contractility followed by a cardiac arrest concomitantly with the onset of MDA release in the interstitial fluid. Finally, during prolonged perfusion the coronary flow diminishes and contracture of the heart muscle ('stone heart') develops. These phenomena resemble those occurring during the 'calcium paradox'. Although the contractility diminishes immediately after the perfusion with Cum OOH the tissue ATP level and energy charge (formula; see text) remain constant. From the moment of cardiac arrest the ATP and creatine phosphate levels gradually decrease and the energy charge drops simultaneously with the appearance of MDA in the interstitial fluid. In contrast to the calcium paradox there is no simultaneous increase in the myocardial AMP level. Various mitochondrial enzymes (cytochrome c oxidase, monoamine oxidase, carnitinepalmitoyltransferase I and palmitoyl CoA synthetase) were tested and not affected by Cum OOH perfusion. During the development of contracture after 20 min of Cum OOH perfusion massive contraction band necrosis of cardiac tissue occurs. However, overall protein release is lower when compared with the protein release during the calcium paradox.(ABSTRACT TRUNCATED AT 250 WORDS)
The control of recessively inherited inborn errors of metabolism may benefit from quantitative biochemical screening assays enabling the identification of heterozygous individuals. Based on the principle of partial enzyme deficiency in heterozygotes, an attempt was made to identify heterozygous animals in a Lapland dog family with canine glycogen storage disease type II (acid alpha-glucosidase deficiency). Acid alpha-glucosidase activity was determined in peripheral blood leucocyte extracts of 12 related Lapland dogs, two of which were obligate heterozygotes. The use of an antiserum against acid alpha-glucosidase was necessary to increase the specificity of the assay. Twice the obligate heterozygous enzyme level was assumed to indicate the homozygous normal level. Five dogs were designated as presumptive heterozygotes, and five as presumptive normal homozygotes. The results in two dogs were inconclusive. The information obtained in this preliminary investigation may be helpful in the control of the disease in the Lapland dog breed.
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A recently described canine model (Lapland dog) of glycogen storage disease type II (GSD II, Pompe disease, acid alpha-glucosidase deficiency) was identified with several biochemical genetic methods. Complementation studies in which fibroblasts from a GSD II dog were fused with fibroblasts derived from control dogs and from human patients with different clinical forms of the disease did not lead to restoration of acid alpha-glucosidase activity in the heterokaryon cell populations. These results indicate that acid alpha-glucosidase deficiency is the primary defect in canine GSD II and that there is a close genetic parallelism with human GSD II. Immunotitration analysis of the residual acid alpha-glucosidase activity in the canine GSD II fibroblasts and liver demonstrated that this residual activity was not due to acid alpha-glucosidase enzyme, in which respect canine GSD II was similar to the infantile form of the human disease. Double immunodiffusion studies showed the presence of catalytically inactive acid alpha-glucosidase enzyme protein in canine GSD II. This is consistent with a structural gene mutation. It is concluded that canine GSD II in the Lapland dog is a homologous model of the infantile form of human GSD II, a conclusion in concordance with clinical and pathological studies.