Pretreatment of the ischemic myocardium with verapamil protects against mitochondrial respiratory depression observed during ischemic arrest as well as during reperfusion. Since ischemic mitochondrial function appears not to be altered further by reperfusion, the purpose of this study is to identify a biochemical event affecting mitochondria that is specifically associated with reperfusion injury. It has been proposed that increased cellular Ca2+ influx and oxygen toxicity may result from reintroduction of coronary flow. Increased cytosolic Ca2+ is transmitted to the mitochondria with subsequent activation of Ca2+-dependent events, including phospholipase A2. Net production of lysophospholipids (and loss of total diacylphospholipids from the mitochondria) will proceed when reacylation mechanisms are inhibited. Since acyl-CoA:lysophospholipid acyltransferase is a sulfhydryl-sensitive enzyme and since increased activity of glutathione peroxidase shifts the levels of the mitochondrial sulfhydryl buffer, glutathione, towards oxidation, levels of glutathione and its oxidation state were measured during reperfusion in the absence or presence of verapamil pretreatment. Ischemia lowers total glutathione and reduces the redox ratio (reduced glutathione: oxidized glutathione) by 85%. Reperfusion partially returns the redox ratio to control by causing oxidized glutathione to disappear from the matrix. Verapamil maintains both the concentration and the redox potential of glutathione at control levels. Concomitant with alterations in reduced glutathione:oxidized glutathione is a decrease in ischemic mitochondrial phospholipid content. During reperfusion, phosphatidylethanolamine and its major constituent fatty acids (C 18:0 and C 20:4) are specifically lost from the mitochondrial membrane. Accompanying the significant loss of arachidonic acid during reperfusion is the decreased content of 11-OH, 12-OH, and 15-OH arachidonate. These lipid peroxidation products are not increased in ischemia. It is proposed that oxidation of matrix glutathione to glutathione disulfide during ischemia results in formation of glutathione-protein mixed disulfides and inhibition of sulfhydryl-sensitive proteins, including acyl-CoA lysophosphatide acyltransferase. Thus, metabolic events occurring within the ischemic period set the stage for prolonged dysfunction during reperfusion.

SUMMARY A radioimmunoassay system for determining content of glutamate dehydrogenase (GDH) in human leukocytes was established and studied in 14 patients with spinocerebellar ataxia or atypical Parkinsonism. The protein content of leukocyte GDH was decreased in four patients and the reduction in the protein content was proportional to that in the enzyme activity. The ratio of GDH activity to protein content was invariable in healthy controls, diseased controls and patients with reduced GDH activity. These results suggested that at least a portion of the partial GDH deficiency was due to the decreased level of the enzyme protein.Spinocerebellar degeneration is a heterogeneous disease complex, and the majority of the diseases have not been characterised biochemically. Recently, several authors have reported the partial deficiency in glutamate dehydrogenase (GDH; EC 1.4.1.3) activity in leukocytes, fibroblasts, platelets and muscles from patients with nondominant or dominant form of olivopontocerebellar atrophy (OPCA).' -These findings suggested that systemic abnormality of glutamate metabolism might be related to a subgroup of OPCA. However, it remains unclear whether or not GDH deficiency is aetiologically significant, and little is known about the mechanism by which GDH activity is reduced.In this study, we established a radioimmunoassay for determination of GDH protein content in human leukocytes and studied patients with spinocerebellar ataxia. Materials and methodsFourteen patients with spinocerebellar degeneration or atypical Parkinsonism and eight age-matched, healthy controls were studied. Four patients who showed significantly lower level of GDH activity than controls are presented below. No members with similar diseases were known in the families of each case. Decreased glutamate dehydrogenase protein in spinocerebellar degenerationEnzyme assay of leukocyte GDH Leukocytes were separated with 6% dextran from heparinised venous blood, and were disrupted by freeze and thaw cycles. These cells were subjected to enzyme assay and radioimmunoassay. GDH activities were measured fluorometrically by a modification of the method described by Plaitakis et al.2 Aliquots of the homogenate were incubated at 47 5'C for 60 minutes. The activity determined after incubation was considered to represent "heat-stable" GDH. This value was subtracted from the total activity to obtain the activity of "heat-labile" GDH.Radioimmunoassay of leukocyte GDH protein GDH proteins purified from bovine liver, rat liver and bacteria (Proteus species) were purchased from Sigma USA. Rabbits were subcutaneously injected with 1 mg of bovine liver GDH emulsified in Freund's complete adjuvant at 2-week intervals and were bled 7 days after the third injection. The specificity and cross-reactivity of the antiserum was tested by immunoblot analysis with protein extracts from rat cerebellum and liver, and by ELISA using GDH purified from bovine liver, rat liver and bacteria as antigens. Crossreactivity of the antiserum with human GDH was evidenced ...

The thiobarbituric acid (TBA) reaction method established by Yagi to determine the lipid peroxide level in serum and animal tissues was applied to isolated mitochondria with some modifications. The TBA reaction was carried out in acetic acid (pH 2.0) in the presence of 0.7 °o sodium dodecyl sulfate at 951CC for 45 min. Chloroform was used to remove the lipid and protein from the reaction mixture. An aqueous layer was used for spectrophotometric measurement at 532.5 nm to determine the TBA reaction product with lipid peroxide. To evaluate the TBA assay system, the lipid peroxide content of mitochondrial suspensions exposed to ultraviolet light was examined. The lipid peroxide formed in mitochondria was proportional to the dose of ultraviolet energy. The lipid peroxide of methyl linoleate exposed to ultraviolet light was determined both with the TBA reaction and with the hydroperoxide reaction. The linear increase in TBA values following UV exposure was similar to the increase of hydroperoxide values determined iodometrically. The TBA assay was shown to be a good predictor of lipid peroxide formed in mitochondria. This assay system is useful for lipid peroxide determination in various tissues: epidermis, Ehrlich ascites tumor cells and B-16 melanoma culture cells.

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SummaryMale Wistar rats received intraperitoneal injections of adriamycin (4mg/kg body weight/day) and/or riboflavin-butyrate (20mg/kg body weight/day) for 6 consecutive days. Cardiac mitochondria were then prepared for our present experiment. The combined use of riboflavin-butyrate with adriamycin was evaluated for reduction of lipid peroxide formation in rat cardiac mitochondria. In order to find the mechanism of the effect of riboflavin-butyrate, the glutathione peroxidase glutathione reductase system was examined. Adriamycin reduced the glutathione reductase activity in rat cardiac mitochondria, but did not affect the glutathione peroxidase activity. The mitochondrial content of flavin adenine dinucleotide, a prosthetic group of glutathione reductase, was greatly reduced and apoprotein of glutathione reductase also de creased. The administration of riboflavin-butyrate with adriamycin in creased flavin adenine dinucleotide and glutathione reductase activity. These results suggest that exogenous administration of riboflavin-butyrate is capable of reducing lipid peroxide by both enzymatic detoxification through glutathione reductase and non-enzymatic detoxification due to direct reaction with lipid peroxide.