Nobuya Haramaki

Cytosolic and Mitochondrial Systems for NADH- and NADPH-dependent Reduction of α-Lipoic Acid

Abstract In cellular, tissue, and organismal systems, exogenously supplied α-lipoic acid (thioctic acid) has a variety of significant effects, including direct radical scavenging, redox modulation of cell metabolism, and potential to inhibit oxidatively-induced injury. Because reduction of lipoate to dihydrolipoate is a crucial step in many of these processes, we investigated mechanisms of its reduction. The mitochondrial NADH-dependent dihydrolipoamide dehydrogenase exhibits a marked preference for R(+)-lipoate, whereas NADPH-dependent glutathione reductase shows slightly greater activity toward the S(−)-lipoate stereoisomer. Rat liver mitochondria also reduced exogenous lipoic acid. The rate of reduction was stimulated by substrates which increased the NADH content of the mitochondria, and was inhibited by methoxyindole-2-carboxylic acid, a dihydrolipoamide dehydrogenase inhibitor. In rat liver cytosol, NADPH-dependent reduction was greater than NADH, and lipoate reduction was inhibited by glutathione disulfide. In rat heart, kidney, and brain whole cell-soluble fractions, NADH contributed more to reduction (70–90%) than NADPH, whereas with liver, NADH and NADPH were about equally active. An intact organ, the isolated perfused rat heart, reduced R-lipoate six to eight times more rapidly than S-lipoate, consistent with high mitochondrial dihydrolipoamide dehydrogenase activity and results with isolated cardiac mitochondria. On the other hand, erythrocytes, which lack mitochondria, somewhat more actively reduced S- than R-lipoate. These results demonstrate differing stereospecific reduction by intact cells and tissues. Thus, mechanisms of reduction of α-lipoate are highly tissue-specific and effects of exogenously supplied α-lipoate are determined by tissue glutathione reductase and dihydrolipoamide dehydrogenase activity.xa0Copyright

Effects of natural antioxidant ginkgo biloba extract (EGB 761) on myocardial ischemia-reperfusion injury.

Recently, it was reported that Ginkgo biloba extract (EGb 761), which is known to have antioxidant properties, also has antiarrhythmic effects on cardiac reperfusion-induced arrhythmias. In the present study, effects of EGb 761 on cardiac ischemia-reperfusion injury were investigated from the point of view of recovery of mechanical function as well as the endogenous antioxidant status of ascorbate. Isolated rat hearts were perfused using the Langendorff technique, and 40 min of global ischemia were followed by 20 min of reperfusion. EGb 761 improved cardiac mechanical recovery and suppressed the leakage of lactate dehydrogenase (LDH) during reperfusion. Furthermore, EGb 761 diminished the decrease of myocardial ascorbate content after 40 min of ischemia and 20 min of reperfusion. Interestingly, EGb 761 also suppressed the increase of dehydroascorbate. These results indicate that EGb 761 protects against cardiac ischemia-reperfusion injury and suggest that the protective effects of EGb 761 depend on its antioxidant properties.

Reduction and transport of lipoic acid by human erythrocytes.

Reduction of exogenous lipoic acid to dihydrolipoate is known to occur in several mammalian cells and tissues. Dihydrolipoate is a potent radical scavenger, and may provide significant antioxidant protection. Because lipoic acid appears in the bloodstream after oral administration, we have examined the reduction of exogenous lipoate by human erythrocytes. Normal human erythrocytes reduced lipoate to dihydrolipoate only in the presence of glucose; deoxyglucose did not substitute for glucose, indicating that the reduction of lipoate requires glucose metabolism. Furthermore, the reduction was shown to be NADPH dependent. Erythrocytes isolated from a human subject with a genetic deficiency of glucose-6-phosphate dehydrogenase (and, therefore, deficient in the formation of NADPH) did not reduce lipoate. Dehydroepiandrosterone, a specific inhibitor of glucose-6-phosphate dehydrogenase, inhibited lipoate reduction. Our findings imply that some of the reduction of exogenous lipoic acid is catalysed by glutathione reductase, a flavoprotein dehydrogenase; mitomycin C, an inhibitor of FAD-dependent reductases, inhibited lipoate reduction by erythrocytes, and glutathione reductase purified from human erythrocytes was observed to reduce lipoic acid in a cell-free system. We further explored these findings with erythrocyte ghosts and liposomes. Our results indicate that a transport system exists for alpha-lipoic acid and dihydrolipoate; resealed erythrocyte ghosts, containing trapped lipoamide dehydrogenase and pyridine nucleotides, reduced externally added lipoate. By contrast, liposomes prepared with enzyme and pyridine nucleotides did not catalyze reduction of lipoate. This work indicates that uptake of exogenous lipoate and reduction to dihydrolipoate by normal human erythrocytes may contribute to oxidant protection in the human bloodstream.

Iron coordination by catechol derivative antioxidants

Iron complexes of nitrocatechols with different substituent groups [1: -CH = CR2; 2: -CH2-CHR2; 3: -CH = CR(R)] were synthesized and their effects on iron-induced free radical reactions of biological importance investigated. Catechol and nitrocatechol derivatives effectively inhibited iron-induced lipid peroxide-dependent lipid peroxidation. In the Fenton-like reaction, iron-catechol generated hydroxyl radicals more strongly than did iron citrate, and iron-nitrocatechol derivative 2 generated a small amount of hydroxyl radicals. The iron complexes of derivatives 1 and 3 did not generate hydroxyl radicals. Iron-catechol had the highest ratio of reduction to oxidation rate constants and the second was iron-nitrocatechol 2, suggesting that iron chelated by nitrocatechols 1 and 3 may be most difficult to reduce. To elucidate the structure and physical properties of the iron complexes, UV/vis absorption spectroscopic, ESR and 1H NMR studies were performed in aqueous and DMSO solutions. In aqueous solution at pH 7.4, iron complexes of the nitrocatechol derivatives were high-spin tris(nitrocatecholato)ferrate(III) with a characteristic ligand-to-metal charge transfer absorbance (pi -> d pi). The lambda max of iron-nitrocatechol derivative 2 was shorter than those of iron-nitrocatechol derivatives 1 and 3, suggesting that the reduction potential of iron-nitrocatechol 2 is higher than that of iron-nitrocatechols 1 and 3. Nitrocatechol derivatives with a conjugation structure can sequester the chelated iron more effectively than catechol and the derivative without the conjugation against free radical generation by keeping the iron in the ferric state, probably because of the reduction potentials.

Role of ascorbate in protection by nitecapone against cardiac ischemia-reperfusion injury

The antioxidant properties of nitecapone, a catechol derivative and an inhibitor of catechol-O-methyltransferase, were reported recently. In the present study, the influence of nitecapone on isolated rat heart ischemia-reperfusion injury was investigated to elucidate its cardioprotective role. Nitecapone, administered in the perfusion buffer from the beginning of the pre-ischemic phase, significantly improved recovery of cardiac mechanical function, suppressed enzyme leakage in the coronary effluent, and minimized loss of ascorbate, compared with the control group. In rats fed a diet containing 4% ascorbate, myocardial ascorbate content in ascorbate-fed rats after ischemia-reperfusion was higher than that in control rats fed a normal diet without ischemia. However, supplemented rats did not show any beneficial effects on cardiac mechanical recovery or enzyme leakage, suggesting that maintenance of tissue ascorbate level is not the cause, but the result of the protective effects of nitecapone against cardiac ischemia-reperfusion injury. The iron-chelating effect of nitecapone was also tested. It was confirmed, using electron spin resonance, that 50 microM nitecapone chelates the same concentration of iron released from the heart into the coronary effluent. Hence, the iron-chelating ability of nitecapone may be responsible, at least in part, for its cardioprotective effects in ischemia-reperfusion injury.

32 – Biological Effects of the Fermentation Product of Carica papaya (Bio-Normalizer)

Publisher Summary Despite accumulating data on the beneficial effects of bionormalizer (BN), the biological mechanisms responsible for the therapeutic activity of BN are not well understood. This chapter evaluates its activity under various conditions to gain further insights into the biological mechanisms of BN action and new possibilities for therapeutic applications. BN is a functional health-food supplement sold in Japan, the United States, and in other countries. This product is made from Carica papaya Linn. and other plants by yeast fermentation under strict quality control. Although ingredients of the product are still unknown in detail, carbohydrate (90%), protein, amino acids, and vitamins have been detected as main substances by chemical analysis. BN has been proposed as a free-radical modulating agent. In biological studies, BN has been found to scavenge hydroxyl radicals in vitro, and in animal studies, it has been reported to protect the rat brain against oxidative damage caused by aging, iron treatment, or ischemia-reperfusion. From such reports, it has been proposed that beneficial effects of BN might be due to its free radical scavenging properties. However, it has also been reported to upregulate phorbol ester-induced and zymosan-induced superoxide production in rat-peritoneal macrophages, natural killer-cell activity, and the level of interferon. Such evidence suggests that BN also possesses the ability to modulate immune effector cells in addition to its direct free-radical scavenging activity.