Yukimasa Matsuda

Detection of sarin hydrolysis products from sarin-like organophosphorus agent-exposed human erythrocytes

A sarin-like organophosphorus agent, [bis(isopropyl methyl)phosphonate; BIMP], was synthesized. This agent has the same phosphonate group as sarin and also has the same anti-acetylcholinesterase activity potency as sarin. The ID50 and LD50 values of BIMP in mice after intravenous injection were 3.9 nM and 0.8 mg/kg, respectively. The AChE activities of their red blood cells and brains were dose-dependently reduced by intravenous BIMP. After preparation of experimental BIMP-exposed human red blood cells, BIMP-bound acetylcholinesterase (AChE) was solubilized from erythrocyte membranes, purified by immunoaffinity chromatography, digested with trypsin, and the sarin hydrolysis products bound to AChE were released by alkaline phosphatase digestion. The digested sarin hydrolysis products were subjected to trimethylsilyl (TMS) derivatization and detected by gas chromatography-mass spectrometry. Isopropyl methylphosphonic- and methylphosphonic acids, which are the sarin hydrolysis products, were detected in experimental BIMP-exposed human red blood cells. This new method, which enables sarins hydrolysis products to be detected in BIMP-exposed erythrocytes, is a useful tool for studying sarin-poisoning victims.

Formation of Keto and Hydroxy Compounds of Linoleic Acid in Submitochondrial Particles of Bovine Heart

To observe lipid peroxidation of additive-free submitochondrial particles, we incubated submitochondrial particles in the absence of exogenous irons and t-butyl hydroperoxide. After the incubation, the phospholipids were hydrolyzed by phopholipase A2, and the fatty acid constituents were analyzed by high-performance liquid chromatography, gas chromatography-mass spectrometry, and liquid chromatography-mass spectrometry. Contrary to a commonly accepted theory, lipid peroxidation in the submitochondrial particles did not need the addition of NADH. In the phospholipid constituent fatty acids of the oxidized submitochondrial particles, derivatives of hydroperoxides of linoleic acid such as keto, hydroxy, trihydroxy, and hydroxyepoxy compounds were generated. Lipid peroxidation in the submitochondrial particles was not inhibited by the addition of catalase, superoxide dismutase, hydroxyl radical scavengers, or ethylenediaminetetraacetic acid, but was inhibited by the addition of KCN, antimycin-A, NADH, ubiquinol, deferoxamine mesylate, ascorbic acid, and alpha-tocopherol. The cardiolipin-cytochrome c lipid peroxidation system could mimic the lipid peroxidation of the submitochondrial particles, in terms of linoleic acid products and the inhibitory patterns of radical scavengers and electron transfer chain inhibitors. Thus, lipid peroxidation in the submitochondrial particles seems to be due to phospholipid-hemoprotein lipid peroxidation systems such as the cardiolipin-cytochrome c system.

Does the sequence of onset of rigor mortis depend on the proportion of muscle fibre types and on intra-muscular glycogen content?

Abstract We examined the postmortem changes in the levels of ATP, glycogen and lactic acid in two masticatory muscles and three leg muscles of rats. The proportion of fibre types of the muscles was determined with NIH image software. The ATP levels in the white muscles did not decrease up to 1 h after death, and the ATP levels 1 and 2 h after death in the white muscles were higher than those in the red muscles with a single exception. The glycogen level at death and 1 h after death and the lactic acid level 1 h after death in masticatory muscles were lower than in the leg muscles. It is possible that the differences in the proportion of muscle fibre types and in glycogen level in muscles influences the postmortem change in ATP and lactic acid, which would accelerate or retard rigor mortis of the muscles.

Formation of leukotoxin (9,10-epoxy-12-octadecenoic acid) during the autoxidation of phospholipids promoted by hemoproteins

Myoglobin (Mb) and cytochrome c (Cyt c) are known to promote lipid peroxidation when mixed with certain types of phospholipids. In the presence of phospholipids such as cardiolipin (CL), ferrous Mb and Cyt c were converted to ferric hemoproteins, and autoxidation of the phospholipids and the oxidation of free linoleic acid (LA) added to the reaction mixture were observed. When the reaction mixture comprising 0.01 mM Cyt c, 0.2 mM CL and 0.1 mM LA was incubated, 92.7% of LA was consumed, and the LA products included 2.49 microM 9,10-epoxy-12-octadecenoic acid (leukotoxin) and its isomer which are potent inhibitors of mitochondrial respiration and have toxic effects on cardiac function. Hemoglobin (Hb) could promote almost no lipid peroxidation in the presence of any kinds of phospholipids. The experiments using some scavengers of active oxygen species revealed that tocopherol and ascorbic acid could strongly reduced LA oxidation caused by Cyt c or Mb. As LTx production was also observed when LA was mixed with Fe2+, LTx may be a common product where non-enzymatic lipid peroxidation occurs.

Calcium is required for quasi-lipoxygenase activity of hemoproteins.

Bovine and guinea pig heart homogenates, porcine leukocyte homogenate, and human hemolysate were found to vigorously oxidize linoleic acid, with lipoxygenase-like activity, to its hydroperoxy, epoxy, hydroxy-epoxy, and keto compounds in the presence of calcium chloride. In the absence of calcium, the reaction was significantly reduced. Attempts to characterize this quasi-lipoxygenase activity revealed that calcium potentiated the quasi-lipoxygenase activities of hemoproteins (hemoglobin, myoglobin, myeloperoxidase, catalase, cytochrome c) and hemin at the physiological pH of 7.5. Lipid peroxidation by hemoproteins was inhibited by albumin and erythrocyte membranes in blood, as well as by a low concentration of calcium in cells. However, it seems possible that in extracellular fluid, which contains a high concentration of calcium and a low concentration of albumin, hemoprotein released from damaged cells could oxidize unsaturated fatty acids derived by phospholipase-A2 from phospholipids of damaged cellular membranes. In a model of quasi-lipoxygenase activation under such conditions, lipids of erythrocyte membranes were oxidized by hemoglobin in the presence of phospholipase-A2 and calcium. The effect of nitrogen oxide, paraquat, and bleomycin on oxidation by hemoproteins and hemin was also discussed.

Onset of rigor mortis is earlier in red muscle than in white muscle

Abstract Rigor mortis is thought to be related to falling ATP levels in muscles postmortem. We measured rigor mortis as tension determined isometrically in three rat leg muscles in liquid paraffin kept at 37 °C or 25 °C – two red muscles, red gastrocnemius (RG) and soleus (SO) and one white muscle, white gastrocnemius (WG). Onset, half and full rigor mortis occurred earlier in RG and SO than in WG both at 37 °C and at 25 °C even though RG and WG were portions of the same muscle. This suggests that rigor mortis directly reflects the postmortem intramuscular ATP level, which decreases more rapidly in red muscle than in white muscle after death. Rigor mortis was more retarded at 25 °C than at 37 °C in each type of muscle.

Postmortem changes in cytochrome c oxidase activity in various organs of the rat and in human heart.

Cytochrome c oxidase (COX), a mitochondrial enzyme, is inactivated by cyanide or carbon monoxide (CO) intoxication. To test whether cytochrome c has potential as an indicator of these toxins in cadavers, we measured COX activity in the main organs of the rat, and in the human heart, at various times after death. Each tissue sample or organ was homogenized and the COX activity in the mitochondrial fraction was measured using ferrous cytochrome c as the substrate. COX activity was significantly higher in rat brain, heart and kidney than in lung and liver from 0 to 4 days after death. The loss of COX activity was significantly slower in the brain and heart than in the lung, liver and kidney. Most importantly, COX activity correlated with the time-since-death for each of the rat organs we tested (r2=0.70-0.95), but for the human heart (r2=0.47). It may be possible that COX activity is likely to be a useful indicator of the time-since-death, and is worth pursuing as an indicator of the tissue cyanide and CO content.

A ‘keyhole lesion’ gunshot wound in an adipocere case

A corpse completely converted into adipocere and showing two adjacent bone defects--a typical gunshot entrance wound and a keyhole lesion--is reported. Postmortem changes, a comminuted fracture of the cranial base, and destruction of the bullets made it impossible to determine the direction of fire through the keyhole lesion. Gunshot wounds may show various atypical forms, including keyhole lesions, and especially in old corpses, the distinction between an entrance wound and an exit wound can be very difficult, even if a careful complete autopsy is performed.