Analysis of reactive oxygen species generated by neutrophils using a chemiluminescence probe L-012.

Reactive oxygen species (ROS) play important roles in the defense mechanism against infection and in the pathogenesis of various diseases. Although chemical properties of ROS generated by leukocytes have been studied extensively, methods available for their analysis are not sufficiently sensitive. We found that 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4-(2H,3H)dione (L-012) reacted with various types of ROS generated by activated neutrophils in human blood and oral cavity, and from peritoneal cavity of the rat, and developed strong chemiluminescence (CHL). Under physiological conditions, opsonized zymosan-dependent CHL intensity of L-012 in human blood and rat peritoneal neutrophils was about 100 and 10 times higher than that of luminol and luciferin analog MCLA, respectively. Phorbol ester-activated CHL of oral neutrophils was also higher with L-012 than that with luminol and MCLA. The presence of either superoxide dismutase, catalase, uric acid, deferoxamine, or azide decreased CHL intensity of L-012 by 52, 57, 57, 63, and 91%, respectively. Kinetic analysis revealed that L-012 developed CHL predominantly by reacting with hydroxyl radical and hypochlorite. Thus, highly sensitive L-012 permits studies on ROS generation by complex biological systems, such as leukocytes, and on the role of ROS in the pathogenesis of various diseases.

Mevalonic acidemia: first case of Japan.

Mevalonic acidemia is a rare metabolic disorder due to mevalonate kinase deficiency which affects the biosynthesis of cholesterol and nonsterol isoprenes. We report the first case of Japan. The clinical course is characterized by intrauterine growth retardation, postnatal growth failure, intractable diarrhea, liver dysfunctions and death at three months of age. Dysmorphic features including triangular face, protrusion of forehead, hypertelorism, low set ears and micrognathism were noted. High mevalonic acid level was found by GC/MS.

Effects of 6-(10-hydroxydecyl)-2,3-dimethoxy-5-methyl-1,4-benzoquinone (idebenone) and related benzoquinones on porcine pancreas phospholipase A2 activity.

6-(10-Hydroxydecyl)-2,3-dimethoxy-5-methyl-1,4-benzoquinone (idebenone) has been found to have a membrane-stabilizing activity in studies using lysosomes. Using dimyristoyl L-alpha-phosphatidylcholine (DMPC) as a substrate, the effects of idebenone and related benzoquinones on phospholipid digestion by phospholipase A2 (PLA2) was investigated. Free myristic acid, released from DMPC upon PLA2 treatment, was anthrylmethylated with 9-anthryldiazomethane (ADAM) and determined by reversed-phase HPLC. Idebenone and a related benzoquinone, 2,3-dimethoxy-5-methyl-6-(10-morpholinodecyl)-1, 4-benzquinone hydrochloride (QS-10.Mor), inhibited the hydrolysis of the substrate with PLA2 in a dose-dependent manner. It is suggested that the effect of idebenone on PLA2, in addition to its antioxidant activity lipid peroxidation, can be attributed to membrane-stabilizing activity.

Effects of idebenone and related compounds on respiratory activities of brain mitochondria, and on lipid peroxidation of their membranes.

The oxidation of succinate and NADH in a ubiquinone-depleted canine brain mitochondrial preparation was restored by a low molecular weight benzoquinone, idebenone. Idebenone inhibited NADH(NADPH)/ADP-Fe3+-dependent lipid peroxidation in canine brain mitochondria and protected against the resulting inactivation of NADH-cytochrome c reductase activity. Idebenone did not affect the activities of succinate oxidase in canine and rat brain mitochondria but suppressed the oxygen consumption in NADH oxidation. This suppression might be related to the inhibition of lipid peroxidation. These results suggest that idebenone functions as an electron carrier in the respiratory chains of brain mitochondria and as an antioxidant against membrane damage caused by lipid peroxidation in brain mitochondria.

Effects of idebenone (CV-2619) and its metabolites on respiratory activity and lipid peroxidation in brain mitochondria from rats and dogs.

The effects of idebenone (CV-2619) and its metabolites on respiratory activity and lipid peroxidation in isolated brain mitochondria from rats and dogs were studied. CV-2619 was easily reduced by canine brain mitochondria in the presence of respiratory substrates. Reduced CV-2619 (2H-CV-2619) was rapidly oxidized through the cytochrome b chain, indicating that the compound functioned simply as an electron carrier of mitochondrial respiratory system. Both nicotinamide adenine dinucleotide (NADH)- and nicotinamide adenine dinucleotide phosphate (NADPH)-dependent lipid peroxidations were examined in canine brain mitochondria in the presence of adenosine diphosphate (ADP) and Fe3+. NADH-cytochrome c reductase activity was sensitive to NADPH-dependent lipid peroxidation. CV-2619 (10(-5)M) strongly inhibited both types of the lipid peroxidation reactions and protected the resultant inactivation of the NADH-cytochrome c reductase activity. Activities of succinate oxidase in rat and canine brain mitochondria were virtually unaffected by CV-2619 and its metabolites (10(-5)-10(-6) M). On the other hand, CV-2619 markedly suppressed the state 3 respiration in glutamate oxidation in a dose dependent manner without any effect on the state 4 respiration and the ADP/O ratio in intact rat brain mitochondria. The inhibitory effect of CV-2619 was also observed in NADH-cytochrome c reductase, but not in NADH-2,6-dichlorophenolindophenol (DCIP) and NADH-ubiquinone reductases in canine brain mitochondria. These facts and results of inhibitor analysis suggest that the action site of CV-2619 is NADH-linked complex I in the mitochondrial respiratory chain and is different from that of inhibitors of oxidative phosphorylation such as rotenone, oligomycin and 2,4-dinitrophenol. Finally, the above findings suggest that CV-2619 acts as an electron carrier in respiratory chains and functions as an antioxidant against membrane damage caused by lipid peroxidation in brain mitochondria. It appears likely that the inhibition of oxygen consumption caused by CV-2619 is related to the effect on non-respiratory systems such as lipid peroxidation which also consumes oxygen.

Effects of Q metabolites and related compounds on mitochondrial succinate and NADH oxidase systems.

The effects of Q metabolites (Q acid-I, Q acid-II) and related compounds (dihydro Q acid-I, dehydro Q acid-II, QS-n, and their esters) on mitochondrial succinate and NADH oxidase systems were investigated. The activity restoring succinate oxidation in acetone-treated beef heart mitochondria was found to decrease with descending order of carbon number (n) of the side chain of the Q metabolites; activity was restored with Q acid-I (n = 7) to one-third as much as that with Q-7 and Q-10, but Q acid-II (n = 5) did not restore any activity. Of the related compounds with a carboxyalkyl group (QS-n), QS-16-QS-18 (n = 16-18) were found to be most active, and their activities were also correlated with n. The relationship between the restoration of activity and the partition coefficient was considered. NADH oxidation in pentane-treated beef heart submitochondrial particles could be restored with esters of low molecular weight quinones to the same extent as with Q-10, but not with the metabolites.

The effect of ubiquinone-7 and its metabolites on the immune response. IV. Chemical structure-adjuvant activity relationship of quinonyl derivatives on humoral immune response.

The effects of the emulsion of quinonyl acids (QS-n, ES-n, KS-n) and related compounds (QSA-n) in Freund's incomplete adjuvant on the humoral immune response to bacterial alpha-amylase were assayed, and their structure-adjuvant activity relationships were discussed. All the quinonyl acids tested (500 microgram/mouse) enhanced the humoral immune response two to seven times as much as that of the control group, five weeks after immunization. 3'-Methyl and 2', 3'-double bond in the carboxy side chains of ubiquinone metabolites (Q acid-I, -II) were not essential for the adjuvant activity. The conversion of methoxyls on the quinone ring into methyls, and that of benzoquinone into phenol also did not affect the activity, but the activity seemed to depend on the carbon number of the carboxy side chain, and the prominent adjuvant activity was observed in the carboxylates having the carboxyalkyl chain of five to seven carbons. High doses (1 or 5 mg/mouse) of ubiquinone-7 and -2 enhanced the humoral immune response two to three times as much as that of the control group, and quinonyl alcohols (QSA-n) enhanced that with low dose (500 microgram/mouse).

The effect of ubiquinone-7 and its metabolites on the immune response. II. Adjuvant activity on the circulating antibody production and the effect on the liver lysosomal membrane.

The effects of the fat-soluble vitamins (retinol, alpha-tocopherol, phylloquinone and ubiquinone) and their related compounds were investigated on the immune response in vivo. E acid-I, dihydro E acid-I, ES-6, K acid-I, Q acid-II and QS-3 act as adjuvants on the production of circulating antibody to bacterial alpha-amylase in mice when they are immunized with in the water phase of water-in-oil emulsion. These compounds, however, can not act as adjuvants in enhancing helper activity of carrier-primed T-cells using an adoptive transfer system in mice. It is also shown that these compounds have no adjuvant activity on the development of delayed type hypersensitivity to ABA-N-acetyltyrosine in guinea pigs. No definite correlation between the adjuvant activity of these compounds and their labilizing activity on rat-liver lysosomal membrane was found.

The effect of ubiquinone-7 and its metabolites on the immune response. III. The effect on the immune response to sheep erythrocytes and DNP-Lys-Ficoll in mice.

The effects of Q acid-11, Q-7 and vitamin A palmitate on humoral immune response to two different types of antigens were examined. Particulate T-dependent antigen (SRBC) and T-independent antigen (DNP-Lys-Ficoll) were used to study the mode of action on immune system. Sodium-salt of Q acid-II (Q acid-11 Na) in the form of saline solution or water-in-oil emulsion showed suppressive effect on the direct and indirect PFC responses when administered simultaneously with SRBC. The suppressive effect of Q acid-II Na, however, was not observed when Q acid-II Na was administered two days before immunization. When Q acid-II Na was administered one day after immunization, the suppressive effect of Q acid-II Na was diminished and the level of PFC response was nearly equal to that of the control group. On the other hand, Q acid-II Na, Q-7 and vitamin A palmitate markedly enhanced the immune response to DNP-Lys-Ficoll (T-independent antigen). From the comparison of these humoral immune response, their modes of action are discussed.