Elevated lipid peroxidation and vitamin e-quinone levels in heart ventricles of streptozotocin-treated diabetic rats

Free Radical Biology & Medicine,Vol. 18, No. 2, pp. 337-341, 1995 Copyright © 1995 Elsevier Science Lid Printed in the USA. All rights reserved 0891-5849/95 $9.50 + .00

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Brief Communication ELEVATED LIPID PEROXIDATION AND VITAMIN E-QUINONE IN HEART VENTRICLES OF STREPTOZOTOCIN-TREATED DIABETIC RATS

LEVELS

SUSHIL K. JAIN and STEVEN N. LEVINE Departments of Pediatrics and Medicine, Louisiana State University School of Medicine, Shreveport, LA, USA

(Received 18 March 1994; Accepted 20 May 1994) Abstract---Diabetic patients develop cardiomyopathy characterized mainly by left ventricular contractile dysfunction and congestive heart failure. This study has investigated the effects of diabetes and insulin treatment on lipid peroxidation, vitamin E, and vitamin E-quinone levels in the heart ventricles of rats made diabetic by streptozotocin treatment. Controls were injected with buffer alone; a subgroup of diabetic rats were injected daily with insulin for 2 months. Membrane lipid peroxidation was measured by determining the thiobarbituric acid (TBA)-reactivity. Vitamin E and vitamin E-quinone were measured by using the high pressure liquid chromatography. There was a significant (p < 0.02) increase in the vitamin E-quinone in the heart ventricles of diabetic rats (0.33 _+ 0.05 pg/mg phospholipid) compared with control rats (0.19 ___ 0,02). This increase was prevented in insulin-treated diabetic rats (0.20 +_ 0.03). Vitamin E levels were higher (14.15 +_ 1.17 /zg/mg phospholipid) in diabetic rats compared to control rats (9.93 _+ 1.29 (p < 0.03). However, insulin treatment to diabetic rats did not cause any change in vitamin E levels (11.75 ± 1.02) compared with diabetic rats. TBA reactivity was higher in the heart ventricles of diabetic rats ( 1.09 _+ 0.11 nmole/mg phospholipid) compared with controls (0.78 _+ 0.08, p < 0.04). Insulin treatment to diabetic rats prevented the increase in the lipid peroxidation (0.79 +_ 0.07); there were no statistically significant differences in TBAreactivity levels in heart ventricles of insulin-treated diabetic and control rats. This study documents accumulation of vitamin E-quinone and lipid peroxidation products in heart ventricles in diabetic rats, which may have a role in the altered contractile property of the heart ventricles in diabetes. Keywords--Vitamin E, Vitamin E-quinone, Diabetes, Heart, Free radicals

INTRODUCTION

and vitamin E-quinone levels in heart ventricles of streptozotocin-treated diabetic and control rats.

Diabetic patients often develop cardiomyopathy characterized mainly by left ventricular contractile dysfunction and congestive heart failure, t The diabetic cardiomyopathy can be reproduced in experimental animals made diabetic with alloxan or streptozotocin treatments J-4 It is also known that oxygen radical metabolites can inhibit prostacyclin synthesis and cause impairment in vascular relaxation mechanisms and contractile dysfunction.5-~2 Recent studies in a cell-free system, red blood cells and endothelial cells in vitro, ~3-t5 and in lens, retina, kidney, liver, platelets, and red blood cells in diabetic animals and patients t6-28 have suggested that elevated glucose levels can generate oxygen radicals and cause membrane lipid peroxidation. This study has investigated the effects of diabetes and insulin treatment on lipid peroxidation, vitamin E,

MATERIALS AND METHODS

Female Sprague-Dawley rats weighing 180-200 g were divided into three groups: control (C), diabetic (D), and insulin-treated Diabetic (D + I). There was no specific reason for using female rats. Rats in groups D and D + I were made diabetic by a single intravenous injection of 55 mg/kg streptozotocin dissolved in citrate buffer (pH 4.5). Control rats were injected only with buffer. Two days after treatment, tail vein blood glucose was measured in all animals. Any streptozotocin-treated rat having a blood glucose < 250 mg/dl was excluded from further study. Rats in group D + I were injected subcutaneously daily with 1.4 units protamine zinc insulin/100 g body weight. All animals were provided with food and water ad lib. Sixteen hours before sacrifice, food was withdrawn, but the animals were allowed free access to water.

Address correspondence to: Sushil K. Jain, Department of Pediatrics, LSU Medical Center, Shreveport, LA 71130, USA. 337

338

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Fig. 1. TBA reactivity of heart ventricles from control (C), diabetic (D), and insulin-treated diabetic (D + 1) groups. TBA reactivity was higher in the heart ventricles of diabetic rats compared with controls. Insulin treatment of diabetic rats (D + I) prevented the increase in heart lipid peroxidation.

At the time of sacrifice, rats were weighed and anesthetized with 40 mg/kg sodium pentobarbital IP. Blood from the heart was collected into EDTA-tubes and heart resected. The ventricles were dissected free from the atria and vascular tissues, blotted dry, and weighed. The ventricular tissue of each animal was placed in 25 ml of ice cold 10 mM Tris maleate (pH 6.8) and homogenized with a Sorvall Omnimixer (set at 90% for 1 min) followed by 10 strokes with a Teflon pestle. The homogenate was processed for measurements of lipid peroxidation, vitamin E and vitamin E-quinone, and lipid extraction.

Thiobarbituric acid (TBA ) reactivity Malonyldialdehyde (MDA), an end product of fatty acid peroxidation, reacts with TBA to form a colored complex that has maximum absorbance at 532 nm. 29 Measurement of M D A by TBA reactivity is the most widely used method for assessing lipid peroxidation. For this purpose, 0.2 ml of homogenate was suspended in 0.8 ml phosphate-buffered saline (8.1 g NaCI, 2.302 g Na2HPO4, and 0.194 g NaH2POdL, pH 7.4) and 0.025 ml of butylated hydroxytoluene ([BHT] 88 mg/ 10 ml absolute alcohol). Thirty percent trichloroacetic acid (0.5 ml) was then added. Tubes were vortexed and allowed to stand in ice for at least 2 h. Tubes were centrifuged at 2000 rpm for 15 min. One milliliter each of the supernatant was transferred to another tube. To this was added 0.075 ml 0.1 M EDTA and 0.25 ml of

1% TBA in 0.05 N NaOH. Tubes were mixed and kept in a boiling water bath for 15 rain. Absorbance was read at 532 and 600 nm after tubes were cooled to room temperature in a double-beam Lambda 3B Perkin-Elmer spectrophotometer. BHT, an antioxidant, was added to prevent M D A formation during the assay, which could result in falsely elevated TBA reactivity. The addition of BHT to standard M D A did not affect the color development with TBA. Absorbance at 600 nm was subtracted from absorbance at 532 nm. MDA values in nanomoles were determined with the extinction coefficient of MDA-TBA complex at 532 nm = 1.56 × 105 cm -j M - ' . TBA reactivity was expressed per amount of total phospholipid. Total phospholipid was determined in the lipid extract by quantitating phospholipid-phosphorus by the method of Fiske and S u b b a r o w 9 For this, lipid extract was dried in the Pyrex glass tubes and then digested with 10N H2SO 4 to liberate free inorganic phosphorus, which was measured colorimetrically. Inorganic phosphorus was multiplied by a factor of 25 to obtain the amount of total phospholipid.

Measurement of vitamin E and vitamin E-quinone Vitamin E (alpha-tocopherol) and vitamin E-quinone (alpha-tocopherol quinone) were measured by using the high pressure liquid chromatography (HPLC) method of Hatam and Kayden. 3~ Freshly obtained RBC were treated with pyrogallol. Vitamin E in pyrogallol extract was analyzed within 1 month of its storage at -70°C. Preliminary studies found no effect of such storage on vitamin E levels. Separation of alpha and gamma tocopherols was carried out using reverse phase C-18 column (Waters), 95% methanol solvent system, and Waters HPLC system attached with multiwavelength uv/vis detector set at 292 nm. In additional analyses of the sample extracts, absorbance of peaks were measured at 265 nm to determine the level of tocopherol quinone. Values are expressed per total phospholipid. Glycosylated hemoglobin (HbA0, an index of the mean blood glucose level, was measured using Glycaffinity columns (Iso-Lab Inc, OH). Blood glucose was measured using a glucose oxidase method (Boehringer Mannheim Diagnostics, Indianapolis, IN). Data were analyzed statistically using nonpaired Student's t-test with Sigma Plot 4.1 statistical software for IBM. RESULTS

Fasting blood glucose and HbA~ at the time of sacrifice were significantly higher in the diabetic group (393

Membrane oxidative damage in diabetic heart

339

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(12) (12) (ii) Fig. 2. Vitamin E levels in the heart of control (C), diabetic (D), and insulin-treated diabetic (D + I) rats. Vitamin E levels were higher in diabetic rats compared with controls. Insulin treatment of diabetic rats (D + I) did not cause any change in vitamin E levels compared with diabetic (D) rats.

_ 97 mg/dL, 10.3 ___ 1.3% mean + SD) compared with control rats (113 + 9, 3.4 _ 0.4, respectively). Increase in fasting glucose (126 ___ 34) and HbA~ (3.5 +_ 0.5) was blocked in diabetic rats treated with insulin. Figure 1 illustrates the TBA reactivity of heart ventricles from control, diabetic, and insulin-treated diabetic groups. TBA-reactivity was higher in the heart ventricles of diabetic rats (1.09 ___0.11 nmole/mg phospholipid) compared with controls (0.78 _+ 0.08, p < 0.04). Insulin treatment of diabetic rats prevented the increase in lipid peroxidation (0.79 ___0.07); there were no statistically significant differences in TBA-reactivity levels in heart ventricles of control and insulintreated diabetic groups. Figure 2 illustrates vitamin E levels in the heart ventricles of control, diabetic, and insulin-treated diabetic rats. Vitamin E level was higher (14.15 ± 1.17 /zg/mg phospholipid) in diabetic rats compared to controls (9.93 ___ 1.29, p < 0.03). However, insulin treatment of diabetic rats did not cause significant change in vitamin E (11.75 _ 1.02) levels compared with diabetic rats. Figure 3 illustrates levels of vitamin E-quinone in the heart ventricles of control, diabetic, and insulintreated diabetic rats. There was a significant increase in the vitamin E-quinone in heart ventricles of diabetic rats (0.33 +__0.05 #g/mg phospholipid, p < 0.02) compared with controls (0.19 +_ 0.02). This increase was prevented when rats were treated with insulin (0.20 ± 0.03) to control hyperglycemia, suggesting an in-

C (12)

D

D+I

(12)(ii)

Fig. 3. Vitamin E-quinone in the heart of control (C), diabetic (D), and insulin-treated diabetic (D + i) rats. There was a significant increase in the vitamin E-quinone in heart of diabetic rats. This increase was prevented in the insulin-treated diabetic (D + 1) rats.

creased membrane oxidative stress in the heart ventricles exposed to chronic hyperglycemia.

DISCUSSION Previous studies have reported increased levels of lipid peroxidation products in the plasma, RBC, kidney, retina, lens, and sciatic nerve of diabetic animals and patients. 16-28'32-37 However, lipid peroxidation damage in whole heart homogenate has been reported to be similar at 1 week of diabetes in rats treated with streptozotocin 38 and decreased at 6 weeks in rats treated with alloxan. 2° Diabetic cardiomyopathy is characterized by the contractile dysfunction of the ventricles. However, there is no study on the membrane oxidative damage in the ventricles associated with diabetes. This study with heart ventricles free of the atria and other vascular tissues found the accumulation of vitamin E-quinone and lipid peroxidation products at 2 months of diabetes in rats treated with streptozotocin. Increased levels of lipid peroxidation products and tocopherol-quinone suggests the occurrence of oxidative damage and MDA accumulation in the heart ventricles of diabetic rats. The increased level of vitamin E-quinone in heart ventricles of diabetic rats could be due to increased utilization of vitamin E in scavenging oxygen radicals generated by the hyperglycemia. Quinones can undergo reduction by enzymatic or nonenzymatic mechanisms to semiquinone and readily converted to

340

S.K. JAIN and S. N. LEVINE

free radical metabolites, which can interact with dioxygen to produce highly toxic active oxygen species. 39 A c c u m u l a t i o n of small quantities of q u i n o n e s can, therefore, generate large quantities of active oxygen species, which deplete intracellular glutathione and inhibit key enzymes, such as those i n v o l v e d in maintaining intracellular calcium and vitamin E - q u i n o n e conversion to vitamin E. 39 Vitamin E levels in the heart ventricles of diabetic rats are higher than control rats; this does not seem to be caused by the hyperglycemia because control of h y p e r g l y c e m i a with i n s u l i n does not affect the v i t a m i n E levels in diabetic heart ventricles. Increased v i t a m i n E level has been previously reported in the liver of streptozotocin-treated diabetic rats. 4° Suklaski et al. 4° suggest that increased v i t a m i n E levels associated with diabetes may be due to an alteration in metabolism or storage of vitamin E by the diabetic rats compared to normal rats. The uncontrolled lipolysis associated with the insulin-deficient state in diabetes could result in mobilization of the alpha-tocopherol from adipose tissue to the liver, both of which are important storage sites for alpha-tocopherol. 4t In contrast, we found that insulin treatment of diabetic rats did not lower the v i t a m i n E level in the heart ventricles; further, whether the heart is a significant site for the alpha-tocopherol storage is not known. It seems possible that alpha-tocopherol a c c u m u l a t i o n is a defense m e c h a n i s m by the heart. Various studies have reported that exogenous administration of lipid peroxides produces concentration-dependent vasoconstriction in isolated and intact arterial, gut, lung, and cardiac smooth muscle samples. 5-~2'4~ Effect of lipid peroxides and peroxidation products on vasoconstriction have been thought to be caused by the inhibition o f prostacyclin synthetase and the resulting lower levels of prostacyclin, a potent vasorelaxant. 5 If this is the case, then increased oxidative damage and accumulation of lipid peroxides may have a role in the altered contractile property of heart ventricles associated with diabetes.

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14. 15. 16. 17. 18. 19. 20. 21.

Acknowledgements--This study was supported by the Research

Award from the National American Diabetes Association Inc. The authors are grateful to Karen Michelle Kircus for editing of the manuscript.

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