Standard thermal trauma of 3rda - 3rdb degree, of 15-20 per cent of body surface was induced on white male Wistar rats narcotized with thiopental The changes of the concentration of thioharhituric acid (TBA)- reactive products, the activity of some antioxidant enzymes such as superoxide dismutase (SOD), glucose-6-phosphate dehydrogenase (G-6-PD), and catalase as well as the percentage of haemolysis were investigated during the early post-burn period (24, 48, and 72 hours after burning). It was demonstrated that erythrocyte haemolysis increased along with activation of lipid peroxidation (LPO) after thermic injury. Alpha-tocopherol treatment in a dose of 20 mg/kg body mass reduced the elevated levels of TBA-reactive products and enhanced erythrocytic antioxidant defence and resistance. It could be clarified that LPO activation played an important role in the haemolysis and that alpha-tocopherol stabilized erythrocyte membrane after burns

Bekyarova, G.

Kozarev, I.

Yankova, T.

English

Varna Medical University Press: Journals

1 Scripta Scientifica Medica, vol. 2H (1993), pp 62-66 Copyright О Medical University, Varna A L P H A - T O C O P H E R O L S T A B I L I Z E S E R Y T H R O C Y T E M E M B R A N E DURING T H E E A R L Y S T A G E A F T E R T H E R M A L T R A U M A G. Bekyarova, I . Kozarev, T, Yankova* Department of Pathological Physiology, ^Department of Chemistry and Biochemistry, Medical University, Varna Standard thermal trauma of 3rda - 3rdb degree, of 15-20 per cent of body surface was induced on white male Wistar rats narcotized with thiopental The changes of the concentration of thioharhituric acid (TBA)- reactive products, the activity of some antioxidant enzymes such as superoxide dismutase (SOD), glucose-6-phosphate dehydrogenase (G-6-PD), and catalase as well as the percentage of haemolysis were investigated during the early post-burn period (24, 48, and 72 hours after burning). It was demonstrated that erythrocyte haemolysis increased along with activation of lipid peroxidation (LPO) after thermic injury. Alpha-tocopherol treatment in a dose of 20 mg/kg body mass reduced the elevated levels of'TBA-reactive products and enhanced erythrocytic antioxidant defence and resistance. It could be clarified that LPO activation played an important role in the haemolysis and that alpha-tocopherol stabilized erythrocyte membrane after burns. Key-words: Thermic injury, lipid peroxidation, alpha-tocopherol. erythrocytes, antioxidant enzymes, rats INTRODUCTION Changes in structural and functional properties of erythrocytes play an important role for microcirculatory disturbances after thermal trauma (4, 13). Red blood cells are transformed into exynocytes which are inclined to aggregation and haemolysis (3). These Address for correspondence: Dr. G. Bekyarova, Dept. of t'"thological Physiology, Medical I hiiversity, v::rna 55 Marin Drinov St. BG 9002 Varna, BULG.4RJA changes characterize the prelytic state of erythrocytes (5). Lipid peroxidation (LPO) is an important factor for destabilization of erythrocyte membrane (2). Disorders in functions and integrity of red blood cells are associated with high plasma concentration of lipoperoxides and low erythrocytic levels of alpha-tocopherol (12). Data about alterations of the activity of antioxidant enzymes such as superoxide dismutase (SOD), catalase. and glucose-6-phosphate dehydrogenase (G-6-PD) after burn injury are scarce and contradictory Alpha-tocopherol Stabilizes Erythrocyte Membrane. (10). Alpha-tocopherol has protective effect on erythrocytes in haemolytic anaemia (7). The purpose of the present study is to investigate the effect of alpha-tocopherol on the resistance of erythrocyte membrane during the early stage after experimental thermal trauma. MATERIAL AND METHODS Male Wistar rats weighing 222 ± 54 g (mean ± SD) were divided into three groups: 1. Controls (n = 51); 2. Burned (n = 62). and 3. Burned but treated with alpha-tocopherol (n= 54). They were fed a laboratory chew ad libitum. All bums were made under thiopental narcosis (in a dose of 30 mg.kg"1 body mass) on the back of animals by using an apparatus for heat convection on 15-20 per cent of the total body surface at 3rc*a until 3rc*b degree. Alpha-tocopherol (Serva, Germany) was intraperitoneal^ applied in a dose of 20 mg. kg" 1 body mass immediately after thermal trauma as well as at the 241*1 and 48 t h hour after bums. Blood samples for analysis were taken from the jugular vein at the 24 t h . 48 t h , and 7 2 n d hour after ernial injury. LPO in erythrocytes was estimated by е concentration of thiobarbituric acid (ТВ A) reactive products. They were spectrophotometrically determined at wave length of 532 nm with extinction coefficient of l,56.10- 5.M- 1.cm- 1 (11). Erythrocytic SOD activity was measured by the method of M i s r a and F r i d о v i с h (1972), G-6-PD one was assessed by Boehringer's (Germany) test reactives, and catalase one was estimated after A s a t i a n i 1 s method (1969). J a g e r ' s method (1968) е was used to detemiine erythrocyte haemolysis. Results were presented as the means ± SEM. Statistical significance was estimated by S t u d e n t - F i s h e r ' s t-test. RESULTS The concentrations of TBA-reactive products increase during the interval studied by 38, 74, and 80 per cent (p < 0,001), respectively, in comparison with nmol/g Hb В 60H о • о Q. 4 0 -Q) > О со 2 2 0 -< 1. Control 2. Burned 3. Burned + a - T 0 24 48 72 Hours Post burn period Fig. 1. Changes of TBA-reactive product level after bir::s and alpha-tocopherol treatment U/mg Hb 7 -6-5 -Q О О 4 -3 -2 -1 -0 ***IVj " 0 0 0 1 + + + & , -cO.001 + + P j - 0 . 0 1 1. Control 2. Burned 3. Burned + a - T —T— 0 24 48 72 Hours Post burn period Fig. 2. Changes of erythrocytic SOD activity after burns and alpha-tocopherol treatment • — i —  63 1 G. Bekyarova, I . Kozarev, T. Yankova their initial values (Fig. 1). The content of these products of LPO decreases significantly by 22 per cent at the 24 t h hour and by 38 per cent at the 72n c* hour, respectively. SOD activity reduces most significantly by 45 per cent at the 48* hour after thermic trauma (Fig. 2). It enhances statistically significantly at the 24* and 4 8 * hour and normalizes at the 72n <* hour after burning and alpha-tocopherol treatment. There is no significant reduction of the catalase index during the early stage after bum injury as well as after alpha-tocopherol treatment (Fig. 3). G-6-PD activity enhances by 68 and 78 per cent at the 4 8 t h and 7 2 n d hour after burns, respectively, when compared with that in control animals. The activity of this enzyme decreases 48 hours after alpha-tocopherol treatment and remains significantly elevated as compared with that of controls (Fig. 4). Haemolysis of erythrocytes increases by 400 per cent at the 24* hour after burns. This percentage decreases, however, significantly after alpha-tocopherol treatment by 26 per cent at the 24* hour and by 38 per cent at the 7 2 n d hour after thermic trauma (Fig. 5). DISCUSSION Our results demonstrate that there is a LPO activation manifested by elevated blood concentration of TBA-reactive products. Other authors also report high plasma lipid peroxide levels in the early stage after thermal trauma (10). Activity of antioxidant enzymes piotecting erythrocytes against free-radial damage changes to a different extent and dynamics. 64 rel. units x ш ~o _c (1) со J? 03 2 -m 1 -c0 О 1. Control 2. Burned 3. Burned + a - T —|— 72 0 24 48 Post burn period Fig. 3. Changes of catalase index after burns and alpha-tocopherol treatment mll/mg Hb 1CH Hours Q O L C D 6 6 -4 -2 - 1. Control 2. Burned 3. Burned + a -* * * R . - 0 . 0 0 1 * * P,, < 0 . 0 0 1 — I — 0 24 48 72 Post burn period Fig. 4. Changes of erythrocytic G-6-PD activity after burns and alpha-tocopherol treatment Hours CO CO _>» о Е ш X 1. Control 2. Burned 3. Burned + a - T R, , <:O.OOl + « 0 01 0 24 48 72 Post burn period Fig. 5. Changes of erythrocytic haemolysis after burns and alpha-tocopherol treatment Alpha-tocopherol Stabilizes Erythrocyte Membrane. SOD activity reduces after burns. It could be due to oxidation of SH-groups in the active centre of the enzyme by oxygen species. It has been rtJported that hydrogen peroxide inhibits SOD activity (6). However, we are not able to indicate the concrete reasons for the absent essential changes in the activity of this enzyme. Erythrocytic G-6-PD activity enhances after bum injury. Our data suggest the assumption that this enzyme of the pentose-phosphate cycle supports a high level of reduced glutathione to protect against free-radical damage probably by intensified production of NADH in erythrocytes. Alpha-tocopherol restricts the extent of haemolysis during the acute phase after burns. This fact could be due to its ability to react with free radicals as well as to improve the antioxidant defence of erythrocytes and protect them against oxidative injuries (8). CONCLUSION Based on these data the following coclusions can be drawn: 1. LPO activation plays a substantial role in erythrocytic haemolysis after bum trauma. 2. Alpha-tocopherol treatment stabilizes erythrocyte membrane during the early stage after thermal trauma. REFERENCES 1. А с а т и а н и , В. С. В: Ферментные методы анализа. Москва, 1969.- 2. В л а д и м и р о в , Ю. А. Патол. физиол. жсперим. тер., 1980, № 4, 17-19.- 3. З а е ц , Т. Л., В. А. Л а в р о в , А. И. М а р ч у к , И. М. Н о с о в а . Бюлл. жсперим. биол. мед., 1990, № 1, 27-30.- 4. Л а в р о в , В. А., Т. Л. 3 а е ц . Бюлл. жсперим. биол. мед., 1990, № 2, 43-50.- 5. Ч е р н и ц к и й , Е. Л., А. В. В о р о б е й . Структура и функция эритроцитарных мембран. Минск, 1981.- 6. B a r t o s z , G . , Т. T a n n e r t , R . F r i e d , W. L e y k o . Experientia, 34, 1978, 1464.- 7. В i е r i , J . , R. С о г а с h , V. H u 1 b е r d . New Engl. J. Med., 308, 1983, 1063-1074.- 8. D i p l o c k , A., J . L u c y . FEBS Lett., 29, 1973, 205-220.- 9. M i s r a , P . , J . F r i d o v i c h . J . Biol Chem., 274, 1972, 1370.- 10. N i s h i g a k i , I . , M. H a g i h a r a , M. H i r a m a t s u . Biochem. Med., 24, 1980, 185-189.- 11. P o r t e r , N . N . , Y . N o r t o n , I . R a m d a s . Biochim. Biophys. Acta, 441, 1976, 596-599.- 12. S a s a k i , J . , G . Gollam , С. B a x t e r . J . Burn Care RehabiL, 4, 1983, 251-253.- 13. Y u a n , Y . , F . Z h i у a n d , Z. Z h i h о l g . Burns, 14, 1988, 365-368. Das Alpha-Tocopherol stabilisiert die Erythrozytenmembran wahrend des friihen Stadiums nach einem thermischen Trauma G . Bekjarowa, I . Kosarev, T. Jankowa* Lehrstuhl fiir pathologische Physiologic *Lehrstnhl filr Chemie und Biochemie, Medizinische Universitat Varna Zusammenfassung: Ein standartisiertes thermisches Trauma (des Grades I l ia -5 Scriptn Scientifica Medica 65 G. Bekyarova, I . Kozarev, T. Yankova Illb, auf 15 - 20 % der Korperflache) wurde bei weiBen mannlichen Wistar Ratten, die mit Thiopental narkotisiert worden waren, verursacht. Wahrend des fruhen Stadiums nach der Verbrennung (an der 24., 48. und der 72. Stunde) wurden die Veranderungen in der Konzentration der reaktiven Produkte der Thiobarbitursaure (TRP) und die Aktivitat einiger antioxidativen Enzyme wie der Superoxiddismutase (SOD), Glukose-6-Phosphat-Dehydrogenase (G-6-PD) und Katalse untersucht. Dabei studierte man auch die prozentige Anderung der Hamolyse. Es wurde gezeigt. daB nach dem thermischen Trauma die Hamolyse der Erythrozyten parallel zu der Aktivierung der Lipidperoxidation (LPO) steigte. Die Behandlung mit Alpha-Tocopherol in einer Dosierung von 20 mg/kg Korpermasse reduzierte den erhohten Spiegel der T R R und vergroBerte den antioxidativen Schutz und die Resistenz der Erythrozyten. Es wurde deutlich, daB die Aktivierung der LPO eine wesentliche Rolle bei der Hamolyse der Erythrozyten spielte, und daB das Alpha-Tocopherol die Erythrozytenmembran nach der Verbrennung stabilisierte. Alpha-tocopherol stabilise la membrane cTerythrocytes dans un premier stade d'un trauma thermique G. Bekiarova, I . Kosarev, T. Iankova* Chair е de physiologic pathologique, *Chaire de chimie et de hiochimie, Universite de medecine a Varna Resume: Un trauma thermique standartise (Ilia - Illb degre, sur 15-20 % de la superficie corporelle) a ete provoque sur des rats blancs males Wistar, qui etaient assoupis sous narcose par Taction de thiopental. On a suivi les changements que le contenu des acides thiobarbiturs (TBA) a subi - les produits reactifs dans le sang, Tactivite de quelques enzymes antioxydantes dans les erythrocytes comme superoxyde dismutase (SOD), glucose-6-phosphate dehydrogenase (G-6-PD), catalase et hemolyse des erythrocytes pendant la periode aigue, apres le trauma thermique (24, 48 et 72 heures). Les donnees de Tanalyse montrent que apres un trauma thermique l'hemolyse des erythrocytes sfaugmente quand s'active la peroxydation lipide. Apres un traitement avec alpha-tocopherol (20 mg/kg de masse corporelle) le contenu de TBA-produits reactifs diminue, tandis que la protection antioxydante et la resistance des erythrocytes s'activent. On arrive a la conclusion que Tactivation de la peroxydation lipide joue un role important dans l'hemolyse des erythrocytes, tandis que Talpha-tocopherol stabilise la membrane d'erythrocytes lors d'un trauma thermique. 66 

ALPHA-TOCOPHEROL STABILIZES ERYTHROCYTE MEMBRANE DURING THE EARLY STAGE AFTER THERMAL TRAUMA

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ALPHA-TOCOPHEROL STABILIZES ERYTHROCYTE MEMBRANE DURING THE EARLY STAGE AFTER THERMAL TRAUMA

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