Parameters of the redox status of the synaptosomal glutathione system and the functional state of the synaptosomal membrane of rat brain were investigated after activation of free radical processes in vitro. A decrease in the content of reduced glutathione along with a simultaneous increase in that of its oxidized form and inhibition of Na+,K+-ATPase and acetylcholine esterase were observed for the model of oxidative stress. These parameters correlated with changes in the total phospholipids: cholesterol ratio and in the balance of thiol- and disulphide groups of proteins of synaptosomal membranes. Preincubation of synaptosomes with 0.5 mM sodium pantothenate prevented the negative effects of the Fenton reaction, but had no effect in the presence of the buthionine-sulfoximine inhibitor of glutathione biosynthesis. Our results provide evidence for the regulatory effect of the glutathione redox status on the structural and functional state of synaptosomal membranes, and for its possible modulation by the CoA biosynthetic precursor.

We have previously observed (summarized in BioFactors 17 (2003) 61) that pantothenic acid, pantothenol and other derivatives that are precursors of CoA protect cells and whole organs against peroxidative damage by increasing the content of cell glutathione. The present investigation was aimed to elucidate the mechanism of this increase in human lymphoblastoic (Jurkat) cells. It showed that incubation of the cells with pantothenic acid or pantothenol increased mainly the content of free glutathione, with little effect on protein-bound glutathione. Buthionine sulfoximine, an inhibitor of glutathione synthesis, prevented this increase. Increase of the content of free glutathione, as produced by pantothenic acid or pantothenol, was largely prevented by respiratory chain inhibitor rotenone, inhibitor of mitochondrial ATP synthesis oligomycin and uncoupler of oxidative phosphorylation of carbonyl cyanide 3-chlorophenylhydrazone. These treatments also decreased the cellular content of ATP. Preincubation with pantothenic acid or pantothenol also increased cell respiration with pyruvate as the exogenous substrate. Although no significant increase of total cell CoA content could be found, it is concluded that the increase of the glutathione level was due to increased production of ATP that was, in turn, a result of the increased content of mitochondrial CoA.

Development of Academician R. V. Chagovets' ideas of the regulatory role of vitamins and their derivatives in thiol-containing compound metabolism and antioxidant system formation as well as in studying non-coenzymatic functions of B-vitamins and vitamin-binding proteins had a considerable effect on the almost 40-year studies on pantothenic acid metabolism and biochemical functions by scholars at the vitaminologic school in Grodno. The concept concerning the intracellular structure of the pantothenate coenzyme form, CoA, pool (content and ratio of CoA-SH, acetyl-CoA, short-chain and long-chain acyls-CoA, coenzyme disulfide forms and CoA-S-S-proteins) was substantiated as an important metabolic regulatory factor (including glutathione system redox potential), with changes being a principal mechanism of pantothenate derivative vitamin and pharmacotherapeutic activity implementation. The effect of the latter is mediated through the systems of CoA biosynthesis and phosphopantetheine proteins, changed CoA-S-S-protein levels, which in turn maintain the intracellular level of CoA-SH as well as cytosolic and mitochondrial transport of its vitamin-containing precursors. A universal CoA biosynthetic function was revealed in prevention of lipid peroxidation initiation and oxidative stress development.

Pantothenic acid belongs to vitamin B group (it is usually termed vitamin B 5) and is the building stone of coenzyme A (Formula 1). It is not synthesised by animal tissues, but is produced by intestinal bacteria and is ubiquitous in food products. Therefore, pantothenic acid deficiency in animal and human subjects is not known, except for experimental conditions. Nevertheless beneficial effects of pantothenic acid and its reduced derivative, pantothenol, have been observed in radiation injury [1– 4] and wound healing, especially in tissues exposed to atmospheric oxygen, like skin [5–10], lung epithelium [11] and eye cornea [12,13]. Protection by pantothenic acid against heart and liver injury caused by ischemia and oxidative stress has also been reported [14–20]. Thus, beneficial effects of pantothenic acid and pantothenol have been reported under conditions when damage by oxygen free radicals might be suspected. This report will briefly describe research carried out in the authors’ laboratories on the protective action of pantothenic acid and its derivatives on mammalian cells subjected to various kinds of oxidative stress. It will also present the mechanism underlying these effects that involves glutathione and, possibly, other cellular thiols.

An original experimental model for detecting organ-specific markers of predisposition to ethanol hepatotoxicity is proposed. A relationship between congenital activity of LPO processes in rat liver (before ethanol intoxication) and the type and severity of ethanol-induced damage to the liver was demonstrated using methods of mathematical modeling. It was proven that intact rats with genetically high MDA levels in the liver and more active systems of MDA generation in ascorbate- and NADPH-dependent reactions are prone to ethanol-induced damage to the liver.

Incubation of rat brain synaptosomal/mitochondrial fraction with tert-butylhydroperoxide resulted in accumulation of the lipid peroxidation product, conjugated dienes, damage of the synaptosomal membrane as evidenced by leakage of lactate dehydrogenase, and decrease of the total content of glutathione and of the GSH/GSSG ratio. This treatment also produced a considerable decrease of the ouabain-sensitive ATPase activity and a much smaller diminution of the activities of glutathione reductase and glutathione transferase. Preincubation of the synaptosomal/mitochondrial fraction with 0.5 or 1.0 mM L-methionine significantly protected against lipid peroxidation, membrane damage and changes in the glutathione system produced by low (1 mM) concentrations of tert-butylhydroperoxide and completely prevented inactivation of ouabain-sensitive ATPase, glutathione reductase and glutathione transferase by such treatment. The importance of L-methionine in antioxidant protection is discussed.

Human leukemic T lymphocytes (Jurkat cells) were induced to undergo apoptosis by brief irradiation with ultraviolet C light (254 nm). This was accompanied by accumulation of lipid peroxidation products in the form of conjugated dienes, a decrease of total glutathione content, and a shift of its redox state towards the oxidized form. Preincubation of the cells with 1 mM pantothenate resulted in a significant elevation of total glutathione content of the cells, reaching its maximum level, 160% of the control, after 3 h. Similar increase was observed after preincubation with 5 mM N-acetylcysteine, a known precursor of glutathione. Both pantothenic acid and N-acetylcysteine alleviated the ultraviolet-induced decrease of glutathione content, diminished lipid peroxidation, and partly protected the cells against apoptosis produced by ultraviolet irradiation.

BioFactorsVolume 11, Issue 1-2 p. 53-55 Extended abstract Pantothenic acid in maintaining thiol and immune homeostasis A. G. Moiseenok, A. G. Moiseenok Laboratory of Coenzymes, Institute of Biochemistry of the National Academy of Sciences, Department of Infectional Disease, Grodno State Medical Institute, Institute of Biochemistry, BLK 50, Grodno 230017, BelarusSearch for more papers by this authorV. I. Komar, V. I. Komar Laboratory of Coenzymes, Institute of Biochemistry of the National Academy of Sciences, Department of Infectional Disease, Grodno State Medical Institute, Institute of Biochemistry, BLK 50, Grodno 230017, BelarusSearch for more papers by this authorT. I. Khomich, T. I. Khomich Laboratory of Coenzymes, Institute of Biochemistry of the National Academy of Sciences, Department of Infectional Disease, Grodno State Medical Institute, Institute of Biochemistry, BLK 50, Grodno 230017, BelarusSearch for more papers by this authorN. P. Kanunnikova, N. P. Kanunnikova Laboratory of Coenzymes, Institute of Biochemistry of the National Academy of Sciences, Department of Infectional Disease, Grodno State Medical Institute, Institute of Biochemistry, BLK 50, Grodno 230017, BelarusSearch for more papers by this authorV. S. Slyshenkov, V. S. Slyshenkov Laboratory of Coenzymes, Institute of Biochemistry of the National Academy of Sciences, Department of Infectional Disease, Grodno State Medical Institute, Institute of Biochemistry, BLK 50, Grodno 230017, BelarusSearch for more papers by this author A. G. Moiseenok, A. G. Moiseenok Laboratory of Coenzymes, Institute of Biochemistry of the National Academy of Sciences, Department of Infectional Disease, Grodno State Medical Institute, Institute of Biochemistry, BLK 50, Grodno 230017, BelarusSearch for more papers by this authorV. I. Komar, V. I. Komar Laboratory of Coenzymes, Institute of Biochemistry of the National Academy of Sciences, Department of Infectional Disease, Grodno State Medical Institute, Institute of Biochemistry, BLK 50, Grodno 230017, BelarusSearch for more papers by this authorT. I. Khomich, T. I. Khomich Laboratory of Coenzymes, Institute of Biochemistry of the National Academy of Sciences, Department of Infectional Disease, Grodno State Medical Institute, Institute of Biochemistry, BLK 50, Grodno 230017, BelarusSearch for more papers by this authorN. P. Kanunnikova, N. P. Kanunnikova Laboratory of Coenzymes, Institute of Biochemistry of the National Academy of Sciences, Department of Infectional Disease, Grodno State Medical Institute, Institute of Biochemistry, BLK 50, Grodno 230017, BelarusSearch for more papers by this authorV. S. Slyshenkov, V. S. Slyshenkov Laboratory of Coenzymes, Institute of Biochemistry of the National Academy of Sciences, Department of Infectional Disease, Grodno State Medical Institute, Institute of Biochemistry, BLK 50, Grodno 230017, BelarusSearch for more papers by this author First published: 16 December 2008 https://doi.org/10.1002/biof.5520110115Citations: 8AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume11, Issue1-22000Pages 53-55 RelatedInformation

Rats were exposed to a total dose of 0.75 Gy of gamma radiation from a 60Co source, receiving three doses of 0.25 Gy at weekly intervals. During two days before each irradiation, the animals received daily intragastric doses of 26 mg pantothenol or 15 mg beta-carotene per kg body mass. The animals were killed after the third irradiation session, and their blood and livers were analyzed. As found previously (Slyshenkov, V.S., Omelyanchik, S.N., Moiseenok, A.G., Trebukhina, R.V. & Wojtczak, L. (1998) Free Radical Biol. Med. 24, 894-899), in livers of animals not supplied with either pantothenol or beta-carotene and killed one hour after the irradiation, a large accumulation of lipid peroxidation products, as conjugated dienes, ketotrienes and thiobarbituric acid-reactive substances, could be observed. The contents of CoA, pantothenic acid, total phospholipids, total glutathione and GSH/GSSG ratio were considerably decreased, whereas the NAD/NADH ratio was increased. All these effects were alleviated in animals supplied with beta-carotene and were completely abolished in animals supplied with pantothenol. In the present paper, we extended our observations of irradiation effects over a period of up to 7 days after the last irradiation session. We found that most of these changes, with the exception of GSH/GSSG ratio, disappeared spontaneously, whereas supplementation with beta-carotene shortened the time required for the normalization of biochemical parameters. In addition, we found that the activities of glutathione reductase, glutathione peroxidase, catalase and NADP-dependent malate (decarboxylating) dehydrogenase ('malic enzyme') in liver were also significantly decreased one hour after irradiation but returned to the normal level within 7 days. Little or no decrease in these activities, already 1 h after the irradiation, could be seen in animals supplemented with either beta-carotene or pantothenol. It is concluded that pantothenol is an excellent radioprotective agent against low-dose gamma radiation.

Rats were exposed to γ radiation from a 60Co source, receiving 0.25 Gy at weekly intervals. During 2 d before each irradiation, the animals received daily intragastric doses of 26 mg pantothenol or 15 mg β-carotene per kg body weight. One hour after the third irradiation session, the animals were killed and their livers were analyzed. In animals not supplied with pantothenol, the irradiation resulted in a significant decrease of total liver lipids and a 50% decrease in phospholipids. Liver cholesterol was decreased by about 20%. Irradiation produced lipid peroxidation as expressed by doubling of the amounts of conjugated dienes and ketone dienes and of thiobarbituric acid reactive compounds. The amount of CoA in liver was decreased by 24% and that of reduced glutathione by 40%. The NAD+/NADH ratio was increased by 60% and the activity of NADP-dependent malate dehydrogenase (decarboxylating) was decreased by 26%. The amount of pantothenic acid and its derivatives (expressed as pantolactone-generating compounds) in blood decreased by about 80%. In rats to which pantothenol was administered, the content of pantothenic acid in blood was tripled compared to nonirradiated (control) rats, and all the biochemical parameters measured in liver were the same as in nonirradiated animals.

Irradiation of Ehrlich ascites tumor cells with ultraviolet light or exposure to the Fenton reaction results in lesions in the mitochondrial energy-coupling system. Formation of the membrane potential and its utilization for ATP synthesis are more affected than the respiratory chain. Preincubation of the cells with pantothenic acid or its derivatives which can serve as precursors of CoA largely protects against the damage of mitochondrial energetics by oxygen reactive species formed by UV light or the Fenton reaction. Incubation of Ehrlich ascites tumor cells with pantothenic acid increases their content of glutathione (most of which is present in the reduced form) by 40%. It is concluded that the protective effect of precursors of CoA against lesions of the mitochondrial energy-coupling system by oxygen reactive species is mainly due to removal of free radicals and peroxides by glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase.

Azido derivatives of long-chain fatty acids, 12-(4-azido-2-nitrophenylamino)dodecanoic acid (N3-NpNH-Lau) and 16-(4-azido-2-nitrophenylamino)hexadecanoic acid (N3-NpNH-Pam), were used to study the mechanism of the protonophoric function of long-chain fatty acids in mitochondrial membranes. N3-NpNH-Lau was found to increase resting-state respiration and decrease the membrane potential in a dose-dependent way in a manner similar to that of the natural fatty acid, myristate. Both effects of N3-NpNH-Lau as well as of the myristate were reversed or prevented by the inhibitor of the mitochondrial ADP/ATP carrier (AAC), carboxyatractyloside. This protective effect of carboxyatractyloside was well expressed in rat heart mitochondria and less so in mitochondria within digitonin-permeabilized Ehrlich ascites tumour cells. Photomodification of Ehrlich ascites tumour mitochondria by ultraviolet irradiation in the presence of N3-NpNH-Lau made them more resistant to the uncoupling effect of myristate, and photomodification of rat heart mitochondria resulted in a strong inhibition of AAC which could not be reversed by serum albumin. Photolabelling of rat heart mitochondria with tritiated N3-NpNH-Pam revealed around 10 labelled bands on SDS/polyacrylamide gel electrophoresis. Based on immunodetection with a specific antibody, one of them, corresponding to 30 kDa, was identified as AAC. Specific interaction of AAC with azido fatty acids was confirmed by a high radiolabelling of this band. The role of fatty acids in fine control of the efficiency of oxidative phosphorylation is discussed.

Preincubation of Ehrlich ascites tumour cells with millimolar concentrations of pantothenic acid, pantothenol or pantethine, but not with homopantothenic acid, at 22 degrees C or 32 degrees C, but not at 0 degrees C, makes the plasma membrane more resistant to the damaging effect of submillimolar concentrations of digitonin. It is proposed that this increased resistance is due to the increased rate of cholesterol biosynthesis. In fact, incorporation of [14C]acetate into cholesterol is by 45% increased in the cells preincubated with pantothenic acid; this probably reflects elevation of the content of CoA in such cells [Slyshenkov, V.S., Rakowska, M., Moiseenok, A.G. & Wojtczak, L. (1995) Free Radical Biol. Med. 19, 767-772].

Preincubation of Ehrlich ascites tumor cells at 22 or 32°C, but not at 0°C, with pantothenic acid, 4′-phosphopantothenic acid, pantothenol, or pantethine reduced lipid peroxidation (measured by production of thiobarbituric acid-reactive compounds) induced by the Fenton reaction (Fe2+ + H2O2) and partly protected the plasma membrane against the leakiness to cytoplasmic proteins produced by the same reagent. Pantothenic acid and its derivatives did not inhibit (Fe2+ + H2O2)-induced peroxidation of phospholipid multilamellar vesicles, thus indicating that their effect on the cells was not due to the scavenging mechanism. Homopantothenic acid and its 4′-phosphate ester (which are not precursors of CoA) neither protected Ehrlich ascites tumor cells against lipid peroxidation nor prevented plasma membrane leakiness under the same conditions. Incubation of the cells with pantothenic acid, 4′-phosphopantothenic acid, pantothenol, or pantethine significantly increased the amount of cellular CoA and potentiated incorporation of added palmitate into phospholipids and cholesterol esters. It is concluded that pantothenic acid and its related compounds protect the plasma membrane of Ehrlich ascites tumor cells against the damage by oxygen free radicals due to increasing cellular level of CoA. The latter compound may act by diminishing propagation of lipid peroxidation and promoting repair mechanisms, mainly the synthesis of phospholipids.