State of the antioxidant protection system of rat liver in ischemia and reperfusion

Purpose: determination of the state of the antioxidant protection system of the cytosolic fraction and suspension of rat liver mitochondria after experimental ischemia and reperfusion. Materials and methods: the study was conducted using white mature rats, divided into 3 groups: the control group (n = 15); The 2nd group of animals (n = 15), from which the liver was taken after 15 minutes of liver ischemia; the 3rd group of rats (n = 15), from which the liver was taken after a 15-minute reperfusion period, followed by a 15-minute ischemic period. Mitochondrial suspension and cytosolic fraction were isolated from liver tissue. Results: the obtained research results showed the presence of certain pathobiochemical changes in the suspension of mitochondria and the cytosolic fraction after ischemia or reperfusion. In the mitochondrial suspension during the reperfusion period it was found an adaptive increase in the activity of glutathione peroxidase by 39% and glutathione reductase by 61%. In the cytosolic fraction, it was the most remarkable increase of the total antioxidant capacity by 38% already during ischemia and a progressive decrease in the level of reduced glutathione form by 26% in ischemic and 55% in reperfusion period. The change in the state of the antioxidant system occurred against the background of an increase in the number of products of oxidative modifications of biomolecules by 40% during ischemia and 2.2 times after reperfusion. Conclusion: The results indicate the need to develop not only a mitochondria-oriented correction of oxidative disorders, but also active support for the components of the cytosol, which provide the main accumulation of free radical damage products and their subsequent removal from the cell, which is essential for survival.

Distribution of introduced human mitochondrial DNA in early stage mouse embryos

Objective. The aim of study was the analysis of human mitochondrial DNA (mtDNA) distribution among murine blastomeres in the embryos developing after an injection of human mitochondria suspension at the stage of one or two cells is presented. Material and methods. Mice CBA/C57Black from Rappolovo aged three weeks were used. Zygotes were obtained upon hormonal stimulation of animals and mated with males. 310 pL of mitochondrial suspension from HepG2 cells was injected into a zygote or one blastomere of a two-cell embryo. Zygotes or two-cell embryos cultured in M3 medium drops covered with mineral oil in Petri dishes. Upon reaching the two-, four- or eight-cell stage the cultured embryos were separated into blastomeres. The latter were lysed and the total DNA was isolated. Human mtDNA was detected by PCR using species-specific primers. Results. The development of 2848 mouse embryos was monitored. In 520 embryos that achieved the stage of 2, 4, 8 in proper time the presence of human mtDNA was assayed in each blastomere. Along with murine mtDNA all embryos contained human mitochondrial genome, which is an evidence of artificially modelled heteroplasmy. Not every blastomere of transmitochondrial embryos contained foreign (human) mtDNA. Mathematical elaboration evidenced an uneven distribution of human mtDNA in cytoplasm within the time elapsed between the injection of human mitochondria and the subsequent splitting of the embryo. Conclusion. The results obtained confirm our previous notion of the presence of 1011 segregation units of human mtDNA in the total amount of mitochondria (about 5 ∙ 102) injected into an embryo.

High levels of mitochondrial heteroplasmy modify the development of ovine - bovine interspecies nuclear transferred embryos

To investigate the effect of mitochondrial heteroplasmy on embryo development, cloned embryos produced using bovine oocytes as the recipient cytoplasm and ovine granulosa cells as the donor nuclei were complemented with 2 pL mitochondrial suspension isolated from ovine (BOOMT embryos) or bovine (BOBMT embryos) granulosa cells; cloned embryos without mitochondrial injection served as the control group (BO embryos). Reverse transcription–quantitative polymerase chain reaction (RT-qPCR) and sodium bisulfite genomic sequencing were used to analyse mRNA and methylation levels of pluripotency genes (OCT4, SOX2) and mitochondrial genes (TFAM, POLRMT) in the early developmental stages of cloned embryos. The number of mitochondrial DNA copies in 2 pL ovine-derived and bovine-derived mitochondrial suspensions was 960 ± 110 and 1000 ± 120, respectively. The blastocyst formation rates were similar in BOBMT and BO embryos (P > 0.05), but significantly higher than in BOOMT embryos (P < 0.01). Expression of OCT4 and SOX2, as detected by RT-qPCR, decreased significantly in BOOMT embryos (P < 0.05), whereas the expression of TFAM and POLRMT increased significantly, compared with expression in BOOMT and BO embryos (P < 0.05). In addition, methylation levels of OCT4 and SOX2 were significantly greater (P < 0.05), whereas those of TFAM and POLRMT were significantly lower (P < 0.01), in BOOMT embryos compared with BOBMT and BO embryos. Together, the results of the present study suggest that the degree of mitochondrial heteroplasmy may affect embryonic development.

Mechanism of the acceleration of CO2 production from pyruvate in liver mitochondria by HCO3-

To investigate the mechanism by which HCO3- accelerates pyruvate metabolism in guinea pig liver mitochondria, we measured continuously, at pH 7.4 and 37 degrees C, 13C16O2 production from [1-13C]pyruvate by mass spectrometry and NADH concentration by fluorescence and analyzed total malate, citrate, and beta-hydroxybutyrate produced by standard biochemical methods. When [1-13C]pyruvate is added to the mitochondrial suspension, 13C16O2 concentration rises steeply in the first seconds and then slows to a steady lower rate. Carbonic anhydrase (CA) eliminates this initial phase, which shows that decarboxylation of pyruvate produces CO2, not HCO3-, and it does this more rapidly than it can equilibrate without CA. HCO3- (25 mM) increased 13C16O2 production, O2 consumption and total malate and citrate production and decreased NADH concentration and total beta-hydroxybutyrate production. After obtaining the total amount of 13C16O2, malate, citrate, and beta-hydroxybutyrate produced, we calculated that the addition of 25 mM HCO3- to the suspension medium increased the amount of pyruvate decarboxylated by pyruvate dehydrogenase (PDH) 16% and increased the amount carboxylated by pyruvate carboxylase 300%. This supports our initial proposal that HCO3- accelerates the pyruvate carboxylation, which in turn consumes ATP directly and NADH and acetyl CoA secondarily, all of which increase PDH activity. However, we found no acceleration of pyruvate decarboxylation by 0.5 and 1 microM free Ca2+ concentration, unless the mitochondria were uncoupled and ATP was added.

Continuous measurement of 13C16O2 production from [13C]pyruvate by intact liver mitochondria: effect of HCO3-

We have measured continuously the production of mass 45 CO2(13C16O2) from 13C-labeled pyruvate in a guinea pig liver mitochondrial suspension and simultaneously the O2 consumption at 37 degrees C and pH 7.4. The reactions took place in a closed 3-ml volume, stirred, thermoregulated chamber separated from the ion source of a mass spectrometer by a gas-permeable membrane that permitted recording the mass peaks of any gas dissolved in the reaction mixture with a response time as fast as 3 s. If the pyruvate was labeled on C-2, no 13C16O2 was formed, even after 1 h, indicating that C-2 and C-3 were not metabolized in the citric acid cycle. We found that production of 13C16O2 was five times greater in the presence of 25 mM HCO3- than in its absence. A probable mechanism of this CO2/HCO3- effect is carboxylation of pyruvate to oxaloacetate, which would react with acetyl CoA to form citrate and with NADH to form malate, thus removing two major inhibitors of pyruvate dehydrogenase. We conclude that CO2/HCO3- has a potent and hitherto unappreciated regulatory effect on liver pyruvate dehydrogenase.

Astragalus Membranaceus and Polygonum Multijlorum Protect Rat Heart Mitochondria Against Lipid Peroxidation

We isolated rat heart mitochondria and induced lipid peroxidation with ADP and FeSO4.Oxygen consumption and MDA formation were measured for quantitating the amount of lipid peroxidation. Using these methods, we screened the water extracts of 14 Chinese medicinal herbs for their effect on lipid peroxidation. It was found that Astragalus membranaceus inhibited 42.1 ± 3.4% of oxygen consumption and 39.8 ± 3.2% of MDA production at concentration of 2 mg dried herb/ml mitochondrial suspension. At the same concentration, Polygonum multijlorum inhibited 52.1 ± 7.3% of oxygen consumption and 50.9 ± 5.3% of MDA production. Other herbs did not inhibit lipid peroxidation to 50% of control at concentration up to 6 mg dried herb/ml mitochondrial suspension. Purification and identification of the active component(s) in Astragalus membranaceus and Polygonum multijlorum as well as their clinical application await further studies.

The reversible Ca2+-induced permeabilization of rat liver mitochondria

Rat liver mitochondria became permeabilized to sucrose according to an apparent first-order process after accumulating 35 nmol of Ca2+/mg of protein in the presence of 2.5 mM-Pi, but not in its absence. A fraction (24-32%) of the internal space remains sucrose-inaccessible. The rate constant for permeabilization to sucrose decreases slightly when the pH is decreased from 7.5 to 6.5, whereas the rate of inner-membrane potential (delta psi) dissipation is markedly increased, which indicates that H+ permeation precedes sucrose permeation. Permeabilization does not release mitochondrial proteins. [14C]Sucrose appears to enter permeabilized mitochondria instantaneously. Chelation of Ca2+ with EGTA restores delta psi and entraps sucrose in the matrix space. With 20 mM-sucrose at the instant of resealing, about 21 nmol of sucrose/mg of protein becomes entrapped. The amount of sucrose entrapped is proportional to the degree of permeabilization. Entrapped sucrose is not removed by dilution of the mitochondrial suspension. Resealed mitochondria washed three times retain about 74% of the entrapped sucrose. In the presence of Ruthenium Red and Ca2+ buffers permeabilized mitochondria reseal only partially with free [Ca2+] greater than 3 microM. [14C]Sucrose enters partially resealed mitochondria continuously with time, despite maintenance of delta psi, in accordance with continued interconversion of permeable and impermeable forms. Kinetic analyses of [14C]sucrose entry indicate two Ca2+-sensitive reactions in permeabilization. This conclusion is supported by the biphasic time courses of resealing and repolarization of permeabilized mitochondria and the acute dependence of the rapid repolarization on the free [Ca2+]. A hypothetical model of permeabilization and resealing is suggested and the potential of the procedure for matrix entrapment of substances is discussed.

Assay of succinate dehydrogenase activity by the tetrazolium method: evaluation of an improved technique in skeletal muscle fractions.

An improved spectrophotometric method for measuring succinate dehydrogenase (EC 1.3.99.1) activity with the use of 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride (INT) is described. The procedure has been evaluated in mitochondrial fractions and homogenates of frog skeletal muscle. For mitochondrial suspensions, extraction of formazan with alcohol was found to be superior to extraction with ethyl acetate. For homogenates, complete extraction of formazan required sequential treatment with alcohol and ethyl acetate; the generally employed procedure of extracting once with ethyl acetate alone led to serious underestimation of the amount of formazan in the tissue. Observations of mitochondrial suspension incubated with various concentrations of INT led to the selection of 0.8 mM INT for optimal results. Higher concentrations, although commonly used, can exert undesirable inhibitory effects on succinate dehydrogenase activity, especially at low concentrations of mitochondria and after longer periods of incubation. The problem of instability of succinate dehydrogenase was solved by the addition of buffer at pH 7.5.