The Journal of Nutrition Vol. 127 No. 10 October 1997, pp. 2060-2064
Copyright ©1997 by the American Society for Nutritional Sciences

Interactions between Vitamin C and Vitamin E Are Observed in Tissues of Inherently Scorbutic Rats^1,2

Kyoko Tanaka, Tomoko Hashimoto, Sadako Tokumaru^*, Hiroshi Iguchi, and and Shosuke Kojo³

Department of Food Science and Nutrition, Nara Women's University, Nara 630 Japan; ^* Joetsu University of Education, Joetsu, Niigata 943 Japan; and  Department of Public Health, Faculty of Medicine, Kyoto University, Kyoto 606 Japan

ABSTRACT

INTRODUCTION

MATERIALS AND METHODS

RESULTS AND DISCUSSION

FOOTNOTES

LITERATURE CITED

ABSTRACT

To investigate in vivo interactions between antioxidant vitamins C and E, sparing effects of vitamin C on vitamin E as well as those of vitamin E on vitamin C were evaluated using inherently scorbutic [Osteogenic Disorder Shionogi (ODS)] rats. Rats were divided into four groups (control, vitamin E-deficient, vitamin C-deficient and simultaneously vitamins C and E-deficient). The levels of vitamins C and E in tissues were determined at 0, 14 and 21 d of deficiency. On d 14, the vitamin E concentration in plasma, liver, brain and lung of the vitamin C-deficient group was significantly lower than that of the control, in agreement with the literature concerning the sparing of vitamin E by ascorbate. The vitamin E concentration of the vitamin C-deficient group also was significantly lower in plasma, heart, liver, lung and kidney than that of the control group on d 21. On the basis of two-way ANOVA, significant interactions between vitamins C and E were observed on d 21 for vitamin E concentration in these tissues. The ascorbate level in plasma, heart, liver, muscle and kidney of the vitamin E-deficient group was significantly lower than that of the corresponding control group on d 21. Significant interactions between vitamins C and E were observed on d 21 for vitamin C concentration in these tissues. These results suggest a sparing effect of vitamin E on vitamin C, an effect that was observed for the first time in this study. These results suggest that the interaction between vitamins C and E exists in vivo and that the extent of the interaction depends on the tissue. Thiobarbituric acid reactive substances (TBARS) in plasma and liver of the vitamin C-deficient rats were significantly higher than those of the control and the vitamin E-deficient groups on d 21, suggesting that the deficiency of vitamin C caused a larger increase in oxidative stress than the deficiency of vitamin E. TBARS of the liver in rats deficient in both vitamins C and E were significantly higher than those in all other groups, suggesting an additive effect of the deficiencies of vitamins C and E on hepatic TBARS. These data suggest that in vivo, vitamins E and C interact, and each can exert sparing effects in the absence of the other.

INTRODUCTION

The regeneration of vitamin E from tocopheryl radical by vitamin C via the donation of a hydrogen atom has been well characterized by in vitro studies (Mukai et al. 1991, Packer et al. 1979). However, the nature of the interaction between these vitamins in vivo is still controversial (Burton et al. 1990). This interaction has been explored in experiments that measured change in animal models fed diets containing various levels of these vitamins (Chen 1981, Chen and Thacker 1986 and 1987, Chen et al. 1980, Ginter et al. 1984, Hruba et al. 1982, Igarashi et al. 1991). The method for determination of vitamin C in these studies was based on the widely used colorimetric method developed over half a century ago (Roe and Kuether 1943). Recently, we reported that the specificity of the conventional method was very low on the basis of the observation that this method gave a value three times as high as the true level of vitamin C in rat plasma as determined by a new specific method involving chemical derivatization and HPLC (Kishida et al. 1992). With the use of the new method, we reported (Tokumaru et al. 1996) that the decreasing profile of vitamin C during its deficiency depended on the nature of tissues in inherently scorbutic (ODS)⁴ rats (Mizushima et al. 1984), which were demonstrated to be a good model for the study of vitamin C.

In this paper, we studied the nature of the interaction between vitamins C and E by evaluating the change in tissue levels of these vitamins caused by depletion of vitamin C, vitamin E, or both in ODS rats by using specific and sensitive assays to measure these vitamins (Buttriss and Diplock 1984, Kishida et al. 1992).

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MATERIALS AND METHODS

Materials. Dehydro-L-ascorbic acid bis(2,4-dinitrophenyl)hydrazone was prepared according to the literature (Kishida et al. 1992). All other chemicals were purchased from Wako Pure Chemical (Osaka, Japan) and were of analytical grade.

Animal and diets. Guidelines of the Prime Minister's Office of Japan (# 6 of 27 March 1980) for the care and use of laboratory animals were followed. The homozygous male ODS rats (od/od), 5 wk old, were purchased from Clea Japan (Tokyo, Japan). The rats were housed in a room with a temperature of 24 ± 2°C and a 12-h light:dark cycle. Rats were permitted free access to food and water. For the first week, all rats were supplied with the synthetic basal diet prepared by Funahashi Farm (Chiba, Japan) according to AIN 76 (American Institute of Nutrition 1977) and were offered ion-exchanged water containing 1 g vitamin C/L, an amount sufficient to maintain normal growth (Mizushima et al. 1984). After the week of acclimation, rats were divided into four groups [the control, vitamin C-deficient (designated as C), vitamin E-deficient (designated as E) and simultaneouly vitamins C and E-deficient (defined as C,E) groups]. The number of rats in each group was 4 or 5. The diet of the E group was prepared by Funahashi Farm with stripped corn oil (5 g/100 g) as the fat. The control group received vitamin C (1 g/L) in drinking water and the synthetic basal diet, which contained stripped corn oil (also 5 g/100 g) and all-rac--tocophrol (50 mg/kg). The C group was offered the synthetic basal diet and vitamin C-free water. The C,E group was offered vitamin C-free water and the vitamin E-free diet as described above.

Analytical methods. On the indicated day, each rat was killed as follows (1 animal per day) and all determinations were made on the day of killing. Rats were anesthetized with diethyl ether and killed by collecting the blood from the inferior vena cava using a syringe containing sodium heparin as an anticoagulant. After perfusion of ice-cooled saline through the portal vein, organs were removed. The excised tissue was homogenized in 5 volumes of 10 mmol/L PBS (pH 7.2) under cooling in an ice bath. All determinations were made in duplicate. The determination of vitamin C was as described (Kishida et al. 1992, Tokumaru et al. 1996). The level of -tocopherol was determined by a method of Buttriss and Diplock (1984). The conditions for the use of HPLC and the fluorescence detector (Shimadzu RF-535, Kyoto, Japan) were reported previously (Kishida et al. 1993, Tokumaru et al. 1997). Thiobarbituric acid reactive substances (TBARS) were measured as described (Buege and Aust 1978) and expressed as nanomole equivalents of malondialdehyde (MDA) per gram of tissue.

Protein concentrations were determined according to the method of Lowry et al. (1951) with bovine serum albumin as the standard.

Data were expressed as means ± SD and analyzed by ANOVA using StatView software (Abacus Concepts, Berkeley, CA). Group means of all experimental groups were compared using two-way ANOVA to examine the interaction among dietary treatments. Differences between group means were analyzed using Fisher's protected least significant difference test (PLSD). Differences were considered significant at P < 0.05.

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RESULTS AND DISCUSSION

Change of body weight of ODS rats. Body weight of the control rats increased steadily as described in the literature (Kimura et al. 1992, Tokumaru et al. 1996). The change in body weight of the E group did not differ from that of the control group, thus showing that vitamin E deficiency for 3 wk did not affect body weight in agreement with the report of Tokumaru et al. (1997). Body weights of the C and the C,E groups also increased steadily but began decreasing on d 14, consistent with the literature (Kimura et al. 1992, Tokumaru et al. 1996). On d 14, body weights of the C and C,E groups were 168.2 ± 10.5 and 166.5 ± 10.4 g, respectively; these were significantly lower than the control (178.3 ± 11.9 g) and the E (185.0 ± 3.9 g) groups. On d 21, body weight of the C and C,E rats was 158.36 ± 12.85 and 162.54 ± 13.46 g, respectively; again, these were significantly lower than the weights of the control (216.08 ± 5.59 g) and the E (209.74 ± 13.28 g) groups.

Change in the level of vitamin E in tissues. The -tocopherol concentration in plasma of the C group on d 21 was significantly lower than that of the control group (Fig. 1). Other tocopherols (,  and ) were not detected in this experiment. Similar results were obtained in the heart, lung, liver and kidney on d 21 and in plasma, brain, lung and liver on d 14 (Fig. 1). Significant interactions between these vitamins on the concentration of -tocophrol were observed on d 21 in plasma, heart, lung, liver and kidney. These results demonstrated that vitamin C spared vitamin E in vivo and supported the presence of in vivo interactions, including direct reaction of the tocopheryl radical with ascorbic acid as shown by in vitro studies (Mukai et al. 1991, Packer et al. 1979). This was consistent with the literature (Hruba et al. 1982) reporting that chronic vitamin C deficiency decreased the concentration of -tocopherol in the liver and lung of guinea pigs but was not consistent with a report (Igarashi et al. 1991) that the tocopherol level in tissues of ODS rats fed the least vitamin C was the highest. This discrepancy may be partly due to the different doses of vitamin C applied.

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Fig. 1. Concentrations of vitamin E in tissues of inherently scorbutic rats fed control, vitamin C-deficient, vitamin E-deficient and simultaneously vitamins C and E-deficient diets. Values are means ± SD, n = 4 or 5. Different letters at each time point indicate significant differences among groups by Fisher's protected least significant difference test (P < 0.05).
[View Larger Version of this Image (33K GIF file)]

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The vitamin E concentrations in all tissues of the E and the C,E groups were significantly lower than those in the control and the C rats on d 14 as well as on d 21 (Fig. 1). No significant differences were observed between the E and the C,E groups perhaps because, except in brain, the effect of ascorbate was masked by the low tocopherol level caused by its deficiency (see below).

Change in the level of vitamin C in tissues. On d 14, the plasma vitamin C concentration of the control rats was significantly higher than that of the E group (Fig. 2). A similar difference was observed for heart and kidney on d 14 and for plasma, heart, liver, muscle and kidney on d 21. These results suggested that the lack of vitamin E accelerated the metabolism of vitamin C in these tissues. The sparing effect of vitamin E on vitamin C was observed for the first time in the present in vivo study. Because the direct regeneration of ascorbate from monodehydroascorbate and dehydroascorbate by tocopherol is unlikely, these observations suggest that the deficiency of vitamin E enhances radical reactions in the hydrophobic region, leading to the elevated oxidative stress in the aqueous phase of the cell to consume more vitamin C, a strong antioxidant in the aqueous phase (Frei et al. 1989).

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Fig. 2. Concentrations of vitamin C in tissues of inherently scorbutic rats fed control, vitamin C-deficient, vitamin E-deficient and simultaneously vitamins C and E-deficient diets. Values are means ± SD, n = 4 or 5. Different letters at each time point indicate significant differences among groups by Fisher's protected least significant difference test (P < 0.05).
[View Larger Version of this Image (32K GIF file)]

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In the C group, ascorbate level in all tissues decreased rapidly as shown in Figure 2, a result consistent with our previous report (Tokumaru et al. 1996). The vitamin C levels of the C and the C,E groups were significantly lower than those of the control and the E groups for all tissues on d 14 as well as d 21. In plasma, the vitamin C level on d 14 in these groups was under the detection limit. In plasma, heart, kidney and muscle, vitamin C levels of both the C and the C,E groups on 21 d were below the detection limit. In muscle, the concentration of vitamin C of the C,E group was significantly lower than that of the C group on 14 d, suggesting again a saving effect of vitamin E on the tissue ascorbate. Statistically significant interactions between these vitamins on the concentration of vitamin C were observed in plasma and muscle on d 14 and in plasma, heart, liver, kidney and muscle on d 21.

In the brain, where both vitamins C and E decreased most slowly during their deficiencies (Tokumaru et al. 1996 and 1997), the interaction of these vitamins was not observed (Figs. 1, 2). The sparing effect of vitamin E on vitamin C was not detected in the lung. These results suggested that the interaction of these vitamins depended on the nature of tissues studied.

TBARS concentrations. In plasma and liver, TBARS of the C and C,E groups were significantly higher than those of the control and the E group on d 21 (Table 1), suggesting that the oxidative stress caused by vitamin C deficiency was stronger than that resulting from vitamin E deficiency. The decrease of ascorbate during vitamin C deficiency may have been more extensive than that of vitamin E during its deficiency, although the relative contribution of these vitamin deficiencies in determining TBARS is not clear at present.

Table 1. Thiobarbituric acid reactive substances (TBARS) in plasma and liver of inherently scorbutic rats fed control, vitamin C-deficient, vitamin E-deficient and simultaneously vitamin C and E-deficient diets for 21 d1,2 [View Table]

In the liver, the TBARS level of the C,E group was significantly higher than those of all other groups, suggesting an additive effect of the deficiencies of both vitamins. A significant interaction between these vitamins on TBARS was observed only in liver (Table 1). In the brain, kidney, heart, lung and muscle, a significant difference in TBARS among the four groups was not observed (data not shown).

By using a specific and sensitive method (Kishida et al. 1992), the interaction between two antioxidant vitamins was investigated. A sparing effect of vitamin C on vitamin E was observed, which supported possible in vivo regeneration of vitamin E by ascorbate as suggested by in vitro studies (Mukai et al. 1991, Packer et al. 1979). On the other hand, a sparing effect of vitamin C by vitamin E was observed in the present in vivo experiments. This effect was not expected from in vitro studies because direct regeneration of vitamin C by tocopherols was unlikely. This observation suggested that the enhanced oxidative stress in the membrane region caused by vitamin E deficiency was transferred to the aqueous phase in the cell, resulting in a decrease of vitamin C, a strong hydrophilic antioxidant (Frei et al. 1989). Statistically significant interactions between vitamins C and E were observed in plasma, heart, lung, liver, kidney and muscle.

FOOTNOTES

Manuscript received 4 March 1997. Initial reviews completed 14 April 1997. Revision accepted 17 June 1997.

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