CHANGES IN OXIDATIVE STATUS OF THE HEART IN RATS RECEIVING VITAMIN C SUPPLEMENTS

We studied the oxidative status of the heart in rats receiving by two doses of vitamin C over a period of four weeks. The activities of copper zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD), and catalase, hydrogen peroxide concentration, the level of lipid peroxidation, and total vitamin C content were determined in the heart of the experimental animals, as well as the concentration of vitamin C in their serum. Our results indicate that, apart from the ability of rats to synthesize vitamin C, supplementation leads to additional antioxidative protection.


INTRODUCTION
During respiration, a small portion of oxygen molecules is transformed into superoxide radicals (O 2 •-) through mitochondrial complexes I and III (Tu r r e n s , 1997).Hydrogen peroxide, produced by O 2 •-dismutation, can be further converted to more reactive oxygen species (ROS), such as the hydroxyl radical.Thus, the level of respiration is an important factor for the susceptibility of tissues to oxidative stress.
The heart is an organ with high oxygen consumption, and increasing evidence suggests that ROS play a major role in the pathophysiology of heart diseases (K e i t h et al., 1998).As a consequence, antioxidative protection may be of great importance for its function.Vitamin C (L-ascorbic acid, ascorbate) is a hydrosoluble vitamin with confirmed antioxidative properties.Unlike humans, rats have the ability to synthesize it endogenously, and one can expect that in this way they satisfy normal daily needs for this nutrient.However, it is possible that additional vitamin C intake may improve antioxidative defense in these animals.Thus, our aim was to study the influence of vitamin C on the antioxidative status of rats receiving supplements of it for four weeks.We determined the activities of copper zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD), and catalase, hydrogen peroxide concentration, the level of lipid peroxidation and total vitamin C content in the heart of experimental animals, as well as the concentration of vitamin C in their serum.

Design of experiment
Male rats of the Wistar strain (Rattus norvegicus) weighing 200±45 g were used for the experiments.The animals were acclimated to 22±1°C and maintained under conditions of 12-h periods of light and dark, with free access to tap water and commercial rat food.
The rats were divided into three groups, each consisting of six animals.The first group was formed of control rats, which drank tap water.The second and the third groups consisted of animals receiving low (0.75 mg of ascorbic acid/kg body weight daily) or high (25 mg of ascorbic acid/kg body weight daily) doses of vitamin C dissolved in tap water.performed on a group of six animals over four weeks, the average daily consumption of water in rats was 240±5 ml per kilogram of body weight.Both doses of vitamin C were therefore dissolved in an adequate volume of tap water every day and administered to the appropriate group of animals.This supplementation was constant throughout four weeks.

Sample preparation
Animals were killed by decapitation with a Harvard guillotine without anesthesia, as recommended by the local ethical committee.After decapitation, the heart was removed and blood was collected.
The left half of the heart was excised and divided into two equal portions.One portion was homogenized in 25 mM phosphate buffered saline (PBS) (pH 7.0) and centrifuged at 9000g in a semi-preparative Sorvall Super T21 centrifuge for 20 min at 4°C.The supernatants were used for determination of catalase, CuZnSOD, and MnSOD activities, as well as for measurement of the H 2 O 2 concentration and total content of lipid hydroperoxides (the level of lipid peroxidation).The other portion of the heart and blood serum were used for determination of vitamin C content by using a similar procedure, except that 6% trichloroacetic acid (TCA) was used instead of PBS.

Total superoxide dismutase activity was determined in PBS samples by the adrenaline method of M i s r a and F r i d o v i c h (1972) using potassium cyanide as a selective CuZnSOD inhibitor for differential calculation of MnSOD activity (We i s i g e r and F r i d o v i c h , 1973).
Catalase activity was measured in PBS samples by the method of B e u t l e r (1982).and calculated against hydrogen peroxide standard curve absorption values.The concentration of lipid hydroperoxides was measured by FOX-2 (j i a n g et al., 1991), with the level of lipid peroxidation in samples expressed as a percent of the lipid peroxidation level in control group of animals.
Total vitamin C content was determined in TCA samples by the method of R o e and K u e t h e r (1943) against vitamin C standard curve absorption values.

Statistical analysis
The data are expressed as means ± SEM.Oneway ANOVA was used for multiple range comparison, with posterior testing for determination of significant differences among groups.The minimal level of probability of significance was set at P < 0.05.

RESULTS
As presented on Figs. 1 and 2, both doses of vitamin C decreased the activities of heart CuZnSOD and catalase, while the activity of MnSOD and H 2 O 2 concentration remained unchanged.Vitamin C supplementation increased the total content of ascorbate in the serum, but did not affect its concentration in the heart (Fig. 3).As presented on the same figure, the level of the heart lipid peroxidation was decreased.Our experiments showed that vitamin C supplementation lowered the activity of heart CuZnSOD, but did not affect MnSOD activity (Fig. 1).According to these results, ascorbate exerts different effects in different cell compartments of the heart.It seems that in cytoplasm, where CuZnSOD is located, vitamin C plays an antioxidative role.However, this is not the case with mitochondria which are MnSOD containing parts of cell.
Mitochondria are considered to be the major source of superoxide in eukaryotic cells because they consume most of the oxygen used by the cell.From this point of view, the unchanged MnSOD activity may imply that mitochondrial antioxidative defense has an intrinsic capacity sufficient to cope with oxidative stress under physiological conditions.Moreover, the presence of CuZnSOD in the intermembranous space of mitochondria has been confirmed (O k a d o -M a t s u m o t o and F r i d o v i c h , 2001).Thus, any O 2 •-entering the mitochondria would be scavenged by mitochondrial CuZnSOD.
In the cytoplasm of cardiomyocytes, vitamin C exerts an antioxidant role.It has become evident that oxidative stress in the heart may involve ROS production by various highly specialized enzymes, not only respiratory ones (F i n k e l , 1999).There are several potential sources of O 2 •-in the cytoplasm of heart cells, for example xanthine oxidase (B e r r y and H a r e , 2004) or NADPH oxidases (C a v e et al., 2005).Probably acting through the removal of superoxide radicals, ascorbate reduces activity of CuZnSOD, thus preventing further ROS generation and lowering the level of heart lipid peroxidation (Fig. 3).
The function of SODs is catalytic dismutation of O 2 •-with hydrogen peroxide as a final product.Thus, decreased CuZnSOD activity may imply reduced H 2 O 2 production, followed by consequent catalase activity decrease.Our data show that vitamin C supplementation decreased activity of catalase in the heart, while the concentration of hydrogen peroxide remained unchanged (Fig. 2).The purpose of this could be to keep of the H 2 O 2 content at the optimal, rather than the minimal level (C l e m e n t and P e r v a i z , 2001), since it acts as one of the most important redox regulators in cell (T h a n n i c k a l and F a n b u r g , 2000).Thus, it seems that maintaining the hydrogen peroxide concentration at the basal level in the heart is not linked with the activity of SODs, but with some other, probably cytoplasm-derived sources of H 2 O 2 (S h a h and C h a n n o n , 2004).
It is interesting that vitamin C supplementation increases ascorbate concentration in the blood, but not in the heart (Fig. 3).This phenomenon has no adequate explanation, inasmuch as the concentration of vitamin C in the heart is among the lowest in the organism.It is possible that the heart's ascorbate content must be kept at a low level in order to maintain an appropriate ROS concentration.If that is the case, the extensive ascorbate turnover with blood could be responsible for vitamin C homeostasis in the heart.
In conclusion, the presented results demonstrate that vitamin C supplementation exerts additional antioxidative protection in the heart of rats, apart from their ability to synthesize vitamin C.

Промене оксидативног статуса срца
Both lipid hydroperoxides and H 2 O 2 were determined in PBS samples by the ferrous ion oxidation (FOX) assay (Wo l f , 1994).There are two versions of FOX assay, FOX-1 and FOX-2 (B a n e r j e e et al., 2002).The concentration of hydrogen peroxide was measured by FOX-1 (G a y and G e b i c k i , 2000)

Fig. 1 .
Fig. 1.Influence of low (0.75 mg/kg b.w. per day) and high (25 mg/kg b.w. per day) doses of vitamin C on activity of CuZnSOD (left) and MnSOD (right) in the rat heart.Statistically significant differences (P < 0.05) in relation to the control are marked with an asterisk above the columns, while statistically significant differences between the two doses of the vitamin C are marked with an asterisk inside the columns.