Acta Ph?jszol Scand 1991, 142, 527-528

ADONIS

000167729100152Y

Oxidative stress in skeletal muscle atrophied by immobi Iizat ion H. KONDO, M. M I U R A and Y. I T O K A W A Department of Hygiene, Faculty of Medicine, Kyoto University, Kyoto, 606, Japan Oxidative stress is known to cause free radical reactions such as lipid peroxidation, which have a role in damaging biological structures and cellular functions. Exhaustive exercise has been shown to increase levels of lipid peroxidation in skeletal muscle, which is generally thought to be related to the pathogenesis of exercise myopathy (Davies et al. 1982). However there has been few reports on the level of lipid peroxide in atrophied muscle. I n this study we report the increased oxidative stress in skeletal muscle atrophied by immobilization. I n addition, we shed light on the effect of Vitamin E -an antioxidant on the muscular atrophy to clarify whether such oxidative stress played a role in the onset of muscle proteolysis or not. Fifteen male Wistar rats (14 weeks old) were used; 5 rats for lipid peroxidation, and 10 rats for the effect of Vitamin E. The latter 10 rats were divided into two groups of 5 rats and were injected intraperitoneally either placebo or Vitamin E, once daily for 6 days before immobilization and every other day during immobilization. Vitamin E was given in the form of alpha-tocopherol solubilized in 10% polyoxyethylene hydrogenerated caster oil/lO yo propylene glycol Received 13 March 1991, accepted 1 May 1991. Key words: atrophy, glutathione, immobilization, lipid peroxidation, oxidative stress, skeletal muscle, Vitamin E. Correspondence : Hisao Kondo. Department of Hygiene, Faculty of Medicine, Kyoto University, Konoe-cho, Sakyo-ku, Kyoto, 606, Japan.

buffered by sodium citrate (Eisai Co. Ltd, Tokyo), in a dose of 30mg kg-'. Under anaesthesia the ankle joint of one hind limb was immobilized in the extended position using a Kirschner wire. After 12 days' immobilization soleus muscles were collected from both hind limbs, the contralateral one being used as a control. The sample was assayed immediately after collection. Homogenization was done under Ar gas flow to lessen the oxidative-reductive change. Thiobarbituric acid-reactive substance (TBARS) was assayed by fluorimetric method of Ohkawa et al. (1979) with a slight modification; i.e. addition of 0.0125 vol. of ethanolic 2% butylated hydroxytoluene. Total glutathione (total GSH) and glutathione disulfide (GSSG) were assayed by the method of Anderson (1985). T h e data were expressed as meanf SE and analqsed statistically by t-test or paired t-test. In atrophied muscles the levels of TBARS and GSSG were significantly higher, and that of total G S H was significantly lower compared with those in control muscles (Table 1). The increase of TBARS level strongly suggests acceleration of lipid peroxidation. It is also generally accepted that under conditions of increased oxidative stress to cells, levels of G S H are usually reduced and levels of GSSG are increased (Lew et al. 1985). Thus, the changes of TBARS, total GSH and GSSG in the present study prove the increased level of oxidative stress in the atrophied muscle. As far as we know, this is the first report on the oxidative stress in atrophied muscle induced by immobilization. On the other hand, Gilbert

Table 1. The tissue levels of TBA reactive substance (TBARS), glutathione disulfide (GSSG), and total glutathione (total GSH) in the skeletal muscle atrophied by 12 days immobilization Control

Atrophy

- ~ --

Weight (wet, mg)

159f8 ( 100%)

TBARS (nmol MDA g ') GSSG (nmol GSH g-') Total G S H (nmol G S H g-') GSSG (total GSH)

28.6f 1.7 l5.6f 1.6 2462 105 0.0063 f0.0005

74 f 4" (46.5% of Control) 45.2 f2.4" 22.2 f 1.9" 1606 78" 0.0138 f 0.0010*

Values are mean fSE ( n = 5). "Significant difference (P< 0.01) from control by paired t-test.

527

H . Kondo et al.

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T a b l e 2. Effect of Vitamin F. injection on the muscular atrophy Vitamin E

Placebo Control .

~

~

~

~~~

~-

~

_~

.4trophy

I)

Control

-4trophy

.

68 & 3% 54.7 k 1.0 ( 100 O O ) 24.7k0.5 41.0+ 1.4% 151 + 6

Weight (wet, mg) Rate of atroph!- ( O 0 ) TBARS (nmol MD.4 g

_.._

l58i7 84*3* 46.7 f 1.91 (85.3°~0of placebo) 26.0k0.1 33.0+ l.l*y

Values are mean k SE ( n = 5 ) . * Significant difference ( P < 0.01) from control by paired t-test; difference ( P < 0.01) from placebo and placebo-atrophy respectively by t-test.

(1982) has reported thc possibilities of enz!-me regulation by thiol/disulfide eschange and modulation of the thioi/disulfide ratio in vivo to serve as a ‘third messenger ’ in response to c.43lP levels. In our study the ratio of GSSG to total GSH in atrophied muscle increased more than two times, which is thought to be one ofthe causes of the metabolic change in atrophied muscles. In the Vitamin E group the TBARS level in atrophied muscle decreased significantly as compared with that in placebo group (Table 2), which show that Vitamin E injected intraperitoneallj- served effectively as an antioxidant to lighten the oxidative stress in atrophied muscle. In the Vitamin E group the rate of atroph!- was less than that in placebo group by about 15 Oi, (Table 2), which suggests that muscular atrophy proceeded mildy with lower osidatire stress. dlthough it is uncertain whether it played a direct or indirect role, this indicates that oxidative stress accelerated muscle proteolysis in the course of atrophy. We are grateful to Dr H. Iguchi (Kpoto University)

t1 significant

for his helpful advice and Dr S. M. Ahmed (our laboratory) for assistance in the preparation of the manuscript.

REFERENCES ANDERSON, M.E. 1985. Determination of glutathione and glutathione disulfide in biological samples. Meth Enz,ymol 113, 548-552 DAVIES, K.J.A., QUINTANILHA, A.T., BRROKS, G.A. & PACKERL. 1982. Free radicals and tissue damage produced by exercise. Biochem Bioph,ys Res Commun 107, 1198-1205 GILBERT,H.F. 1982. Biological disulfides : T h e third messenger?. 3. B i d Chem 257, 12086-12091 LEW,H., PYKE,S. & QUINTANILHA, A. 1985. Changes in the glutathione status of plasma, liver and muscle following exhaustive exercise in rats. FEBS Lett 185, 262-266 OHKAWA, H., OHISHI,N. & YAGI,K. 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem. 95, 351-358