The effect of ascorbate supplementation on oxidative stress in the streptozotocin diabetic rat

Free Radical Biology & Medicine, Vol. 13, pp. 41-46, 1992 Printed in the USA. All rights reserved.

0891-5849/92 $5.00 + .00 Copyright © 1992 Pergamon th~ss Ltd.

Original Contribution THE

EFFECT OF ASCORBATE SUPPLEMENTATION ON OXIDATIVE STRESS IN THE STREPTOZOTOCIN DIABETIC RAT

IAN S. YOUNG, JOHN J. TORNEY, and ELISABETH R. TRIMBLE Department of Clinical Biochemistry, Queen's University of Belfast, Northern Ireland (Received 9 December 1991; Accepted 11 February 1992) Abstract--An increase in oxidative stress may contribute to the development of diabetic complications. The key aqueousphase chain-breaking antioxidant ascorbate is known to be deficient in diabetes, and we have therefore investigated the effects of ascorbate supplementation on oxidative stress in the streptozotocin diabetic rat. Markers of lipid peroxidation (malondialdehyde [MDA] and diene conjugates) were increased in plasma and erythrocytes of untreated diabetic animals, and levels of the antioxidants ascorbate and retinol were reduced. Plasma tocopherol was unchanged. Insulin treatment normalized MDA and ascorbate levels, although ascorbate metabolism remained disturbed, as indicated by increased levels of dehydroascorbate. High-dose ascorbate supplementation in the absence of insulin treatment restored plasma ascorbate to normal and increased plasma retinol and tocopherol levels. However, MDA and diene conjugate levels remained unchanged, possibly as a result of increased iron availability. High-dose ascorbate supplementation should be approached with caution in diabetes, as ascorbate may exert both antioxidant and prooxidant effects in vivo. Keywords--Ascorbate, Dehydroascorbate, Tocopherol, Retinol, Malondialdehyde, Animal, Free radicals

Among the chain-breaking antioxidants which prevent the propagation of free-radical-induced chain reactions, ascorbate and tocopherol are particularly important. Tocopherol is a major lipid-phase antioxidant, and deficiency is associated with increased lipid peroxidation. Ascorbate may recycle tocopherol from the relatively stable tocopherol radical at the lipid interface ~ and also functions as a key aqueous-phase antioxidant in its own right. 12 There are marked derangements of ascorbate metabolism in diabetes, with reduced levels of ascorbate and increased levels of the oxidation product dehydroascorbate.13 In view of these disturbances and the particularly important role of ascorbate as an antioxidant, ascorbate treatment appears a logical approach to reduce oxidative stress in diabetes. However, ascotbate may act as a prooxidant in certain settings,14 particulady when there is increased transition metal availability, as has been reported in diabetes. ~s,16 It is important to ensure that the antioxidant effects of ascorbate will predominate before advocating the indiscriminate use of this antioxidant in diabetes. The aim of the present study was, therefore, to assess the effect of ascorbate supplementation on free radical

INTRODUCTION

There is currently great interest in the potential contribution of increased oxidative stress to the development of complications in diabetes mellitus. ~ An increase in oxidative stress may occur due to either an increase in free radical production or a reduction in antioxidant defenses. Increased production of free radicals in diabetes may arise in a number of ways, including autooxidation of glucose and glycated proteins, 2 particularly in the presence of transition metals. 3 Diabetic monocytes also have an increased capacity to produce superoxide. 4 In addition, there are widespread disturbances of antioxidant defense systems, 5-7 suggesting that reduced resistance to freeradical-induced tissue damage may also occur. This is reflected by increased levels of biochemical markers of lipid peroxidation and free radical activity, particularly in the presence of microangiopathic complications, s-lo

Address correspondence to: Dr. I. S. Young, Department of Clinical Biochemistry, Institute of Clinical Science, Royal Victoria Hospital, Belfast BT12 6BA, Northern Ireland.

41

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I.S. YOUNG ~'l a/.

activity and antioxidant defenses in an animal model of diabetes, the streptozotocin diabetic rat, in which increased oxidative stress is known to occur.~7

MATERIALS AND METHODS Male Hooded-Lister rats of 150-200 g weight were used. Diabetes was induced using a single intravenous injection of 50 mg/kg streptozotocin (Sigma Chemical Company, Poole, Dorset) in citrate buffer (pH 4.5) and was confirmed after 1 week by the presence of a plasma glucose > 15 mmol/L. Plasma glucose levels in untreated animals remained at this level for the duration of the study. Animals were individually housed in metabolic cages and given free access to standard chow and water for the duration of the study. Animals were divided into four groups, each containing eight animals: Group 1: Untreated diabetes Group 2: Ascorbate-treated diabetes. Ascorbate was added at a concentration of I g/L to drinking water and made up freshly on a daily basis. Group 3: Insulin-treated diabetes. Insulin treatment was started I week after streptozotocin injection. Animals were given a single daily injection of human ultratard insulin, with additional actrapid if required. The dose was adjusted to keep the daily urine output at < 5 mk/d. Group 4: Nondiabetic controls. After 6 weeks, animals were killed under ether anaesthesia by decapitation, and blood and organs were collected for biochemical analyses. Erythrocytes were prepared for estimation of membrane lipid peroxidation as described by Jain et al. 1° In the final week of the experiment, a 24-h urine collection was taken into 10% metaphosphoric acid in darkened bottles for estimation of urinary ascorbate and dehydroascorbate excretion, as described below. Ethylenediaminetetraacetic acid (EDTA) plasma was used for the measurement of malondialdehyde (MDA), a marker of lipid peroxidation, by a specific high-performance liquid chromatography (HPLC) technique.18 MDA in the erythrocyte preparation was measured using a similar technique. Diene conjugates were measured after extraction into chloroform/methanol by second derivative spectroscopy, as described by Corongiu and Milia. 19Vitamin E (as ~-tocopherol) and retinol (vitamin A) were also measured by HPLC using the method of Catignani et al. 2° Tocopherol is lipid bound in plasma, and levels were therefore corrected for plasma cholesterol. 21 Plasma ascorbate and dehydroascorbate were stabilized by the addition of an equal volume of 10% metaphosphoric acid to

plasma within 30 min before measurement by H P L ( using the method of Speek et al. 22 Cholesterol levels were measured on a Beckman (Beckman Instruments, Inc., Brea, CA) CX5 multichannel analyzer, and plasma glucose was measured using a glucose oxidase method. Serum iron was estimated using the ferrozine method on a Cobas Bio. (Hoffmann-l.a Roche, Welwyn Garden City, U.K.) analyzer. Hepatic iron stores were estimated by atomic absorption spectroscopy and expressed as/~g/g hepatic tissue. Groups were compared using analysis of" variance with Duncan's post hoc comparison of means to assess differences between individual groups. Results are given as mean _+ standard error unless otherwise indicated.

RESULTS Weight gain was greater in control and insulintreated animals than in ascorbate-treated or untreated (control 83.0 + 3.1 g, insulin treated 77.4 _+ 6.4, ascorbate treated 10.0 _+ 8.1, untreated 9.4 + 6.4, p < .01). The ascorbate-treated animals had a mean intake of 116 _+ 0.2 mg ascorbate/d in drinking water. There was no difference in plasma glucose at the time of death in ascorbate-treated and untreated diabetic animals, with significantly lower glucose levels in control and insulin-treated groups than in untreated or ascotbate-treated (control 9.2 +_ 0.2 mmol/L, insulin treated 12.6 + 0.6, ascorbate treated 39.7 _+ 1.6, untreated 39.5 + 1.4, p < .01). MDA, a marker of lipid peroxidation, was increased in both plasma and erythrocyte membranes in untreated diabetes (Figs. IA and 1B). Insulin treatment restored MDA to normal, while ascorbate supplementation had no overall effect. A similar increase in conjugated dienes was observed in plasma of untreated animals; this was abolished by insulin treatment, but ascorbate supplementation was without effect (Fig. 1C). Plasma ascorbate (AA) was significantly reduced in untreated diabetes (Fig. 2A), while dehydroascorbate (DHAA) levels remained unchanged (Fig. 2B): however, this did not give rise to a significant change in the DHAA/AA ratio (Fig. 2C). Urinary excretion of both ascorbate and DHAA was increased in untreated animals (Figs. 3A and 3B), and there was an increase in the urinary D H A A / A A ratio (Fig. 3C). Ascorbate supplementation restored AA and DHAA to normal (Fig. 2) at the expense of increased urinary excretion (Fig. 3). Insulin treatment restored AA to normal, but the presence of elevated DHAA levels and an in-

43

Ascorbate in diabetes

treatment produced a more substantial increase in retinol levels, although these remained below normal. It is well recognized that oral ascorbate may increase iron absorption by maintaining iron in the Fe’+ state and that ascorbate and iron may interact to facilitate free radical production.r4 We therefore assessed iron status in the four groups of animals. Plasma iron was significantly increased in the ascorbate-treated animals in comparison with the other three groups (control 22.4 f 0.9 pmol/L, insulin treated 22.8 + 1.4, untreated 23.0 f 1.8, vs. ascorbate treated 28.0 + 1.O, p < .05). In addition, hepatic iron stores, a good

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creased DHAA/AA ratio (Figs. 2B and 2C) indicated the presence of a persistent abnormality in ascorbate metabolism. This was confirmed by increased urinary excretion of DHAA and an increased urinary DHAA/ AA ratio (Figs. 3B and 3C). Tocopherol levels remained unchanged in untreated diabetes and were not affected by insulin treatment. However, ascorbate supplementation significantly increased plasma tocopherol (Fig. 4). Retinol levels were significantly reduced in untreated diabetes (Fig. 5). Ascorbate supplementation resulted in a small increase in plasma retinol, whereas insulin

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44

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Fig. 4. Plasma vitamin E (alpha tocopherol) in diabetic and control rats. Results given as mean ,~.ith standard error. *p < .05 compared to all other groups.

plasma is exposed to a free-radical-generating source, lipid peroxidation does not occur until all of the ascorbate present has been oxidized, t2 In addition, aqueous ascorbate can recycle lipid-phase tocopherol in vitro when the latter is oxidized, l~ Consequently, the observed deficiency of ascorbate in plasma and cells from diabetic subjects may be of considerable significance, and it has been suggested that ascorbate supplementation may be of therapeutic benefit in diabetes as a result of its capacity to reduce oxidative stress. In this study, we have investigated the effect of high-dose ascorbate supplementation in the streptozotocin diabetic rat. We used a highly specific HPLC technique to confirm the presence of increased levels of MDA in the plasma and erythrocytes of untreated animals. Previous reports of increased levels of thiobarbituric acid reactive substances in plasma and erythrocytes of diabetic patients 24 and animal models 25 have used less specific spectrophotometric or fluorimetric techniques, which are more prone to in-

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marker of total body iron status, were higher in ascotbate-treated animals than in any of the other groups (control 296.5 _+ 20.8 ug/g liver, insulin treated 377.9 +_ 21.5, untreated 374.3 + 19.2, vs. ascorbate treated 403.7 +_ 9.5, p < .05). However, hepatic iron was also higher in untreated and insulin-treated animals in comparison to the control group (p < .05).

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DISCUSSION

The importance of ascorbate as an antioxidant has been increasingly recognized in recent years. 23 When

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Fig. 5. Plasma vitamin A in diabetic and control rats. Results given as mean with standard error. Each group differs from all others by p < .05.

Ascorbate in diabetes

terferences. We also found that diene conjugates, which reflect an earlier stage in the lipid peroxidation process, were increased in diabetic animals. Diene conjugates are known to be elevated in diabetic patients with microangiopathy.9 The major conjugated diene in the plasma of human subjects is 18;2(9,1 1)-linoleic acid; 26 this may be measured by HPLC, though some controversy exists as to whether this specific diene arises as a result of enzyme activity or free radical damage to fatty acids. 27 For this reason, we have used double derivative spectroscopy to measure total dienes rather than a specific isomer of linoleic acid. The presence of increased oxidative stress in this animal diabetic model is further confirmed by the presence of reduced ascorbate levels and an increase in the ratio of DHAA/AA in urine. Retinol, a less important plasma antioxidant, is also substantially reduced in untreated animals. The finding of no change in tocopherol levels in untreated diabetic animals is consistent with the in vitro observation that ascorbate will preserve and recycle tocopherol in the presence of increased free radical production, even at low ascotbate levels. 28 In this study, insulin treatment was successful in reducing plasma MDA levels to normal. This may be due to a reduction in the generation of free radicals by glucose autooxidation or from glycated proteins. Insulin treatment also restored plasma ascorbate levels to normal, although the presence of increased plasma DHAA and an increased DHAA/AA ratio in both plasma and urine suggests a persistent underlying disturbance ofascorbate metabolism. Previous work has produced conflicting evidence with regard to changes in the DHAA/AA ratio in diabetes. ~3,29-3~ However, this may be partly due to difficulties in stabilizing ascorbate prior to assay and the use of chemical assay methods in earlier studies. Two studies using a more specific HPLC assay for both AA and DHAA have reported an increased DHAA/AA ratio in treated diabetes which is in keeping with o u r r e s u l t s . 13'32 With careful stabilization of samples and specific assay methods, the DHAA/AA ratio may be of use as a marker of oxidative stress in diabetes. The mean additional ascorbate intake of supplemented animals in this study was 1 16 mg/d, approximately three times the amount of ascorbate synthesized per day by the rat. This dose was chosen because it has previously been shown to have significant effects on collagen synthesis in the diabetic rat. 33 The ascorbate intake of the supplemented animals was sufficient to restore plasma levels of both AA and DHAA to normal. However, this was achieved at the

45

expense of an increase in urinary ascorbate excretion. Ascorbate supplementation was associated with increased plasma levels of both tocopherol and retinol, supporting the idea that ascorbate may be important in recycling oxidized forms of both of these vitamins. The observation that ascorbate supplementation fails to reduce the increased plasma levels of MDA and diene conjugates observed in untreated diabetes suggests that prooxidant effects of ascorbate may be significant in vivo. A number of previous studies in nondiabetic subjects and animal models have suggested that ascorbate supplementation will reduce lipid peroxidation. 34'35 However, at least one study has reported that high-dose ascorbate may produce an increase in tissue lipid peroxidation in nondiabetic animals. 36 We are not aware of any reports describing the effects of ascorbate supplementation on oxidative stress in diabetes. It is well recognized that ascorbate may interact with iron in vitro to generate increased free radical production.~4 Oral ascorbate can cause increased iron absorption, and ascorbate may be harmful in iron overload states. 37 Disordered iron metabolism and a beneficial effect of treatment with the iron chelator desferrioxamine have been reported in diabetic patients. ~5 Serum iron levels and hepatic iron stores were higher in the ascorbate-treated rats in this study than in any of the other groups, and interactions between iron and ascorbate may explain the failure to reduce MDA and diene levels. Further work is underway to explore this possibility. In view of the known adverse effects of ascorbate deficiency and the changes in ascorbate metabolism observed in this and other studies, it seems reasonable to propose that ascorbate supplementation might be of benefit in reducing oxidative stress in diabetes. Nonetheless, as previously suggested and confirmed by this study, the effects of ascorbate on free radical production are complex and the relative contribution of antioxidant and prooxidant effects may depend on a number of factors, including the dose of ascorbate administered, the route of administration, and transition metal status. We therefore believe that further work is required before widespread supplementation with ascorbate is recommended in diabetes. Acknowledgement--We would like to thank Dorothy McMaster for measuring ion levels.

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