Subsequently, free radicals change lipid/protein ratio of membranes by affecting poly unsatured fatty acids and lipid peroxidation causes functional irregularities of several cellular organelles ^9,10.

Lipid peroxides are disintegrated quickly and form reactive carbon compounds. Among these, MDA is an important reactive carbon compound which is used commonly as an indicator of lipid peroxidation ¹¹. Since free radical production is increased whereas capacity of antioxidant systems is reduced in diabetes, it has been proposed that diabetic patients may require more antioxidants compared to healthy individuals ^10,12.

Since effects of free radicals in diabetes are now documented, it has been proposed to use antioxidant vitamins to block formation of free radicals and hence prevent development of diabetes ^13,14. While superoxide radicals are cleaned by enzymatic dismutation, compounds known as antioxidants clean free radicals in organism. Glutathione is a very important non-enzymatic antioxidant together with antioxidant vitamins. Vitamins A, E and C are among these important nonenzymatic antioxidants ^15,16.

It has been proposed that in diabetic patients several abnormalities related with absorption develop in the absence of antioxidant vitamins ¹⁷.

Vitamin A functions as catalyzer of removal of singlet oxygen and as a result vitamin A inhibits singlet oxygendependent reactions ^15,18. Vitamin C is also has a role in activating vitamin E when it loses its antioxidant capacity by turning into tocopherol ¹⁹.

In addition, selenium has effect on preventing decomposition, absorption and biological activity of α- tocopherol ^20,21. Selenium and vitamin E act as complementing each others function against oxidative stress ^22,23.

Selenium, functioning as part of glutathione peroxidase, has been recognized as a cellular antioxidant in addition to its protecting function against heavy metal toxicity ^24,25. Selenium has important role in vitamin E metabolism. Selenium is required for normal pancreatic functions. It is needed for absorption of lipids and vitamin E. In addition, selenium has roles to keep vitamin E within lipids ²⁰. There are intrinsic enzymatic and non-enzymatic antioxidants detoxifying mechanisms that decrease ROS concentrations in human body. Vitamins A, C, E, selenium and glutathione are some of the major non-enzymatic antioxidants in the body ¹⁶. Therefore, the idea of using antioxidant vitamins to prohibit development of diabetes as well as its complications and/or to treat diabetic patients is getting more attention than ever ^13,26.

Although there are studies reporting serum or plasma levels of antioxidant vitamins in diabetic patients, results from different groups are rather contradictory. Studies focusing on involvement of selenium in diabetic patients are rather limited. Therefore, the present study was designed to determine and evaluate changes in level of selenium, antioxidant vitamins (A, E, C) and MDA in patients with type 2 diabetes and healthy subjects. Furthermore, we examined possible relationship among four antioxidants (namely, selenium, Vitamin A, vitamin E, vitamin C) and MDA.

The quantification was made according to Miller and et al. ²⁷ utilizing absorption spectra of 326 and 296 nm for vitamin A and E, respectively. HPLC separations were accomplished at room temperature with a Cecil liquid chromatography system (Series: 1100) consisting a sample injection valve (Cotati 7125) with a 20 μl sample loop, an ultra-violet (UV) spectrophotometric detector (Cecil 68174), integrator (HP 3395) and a Techsphere ODS-2 packed (5 mm particle and 80 Å pore size) column (250 x 4.6 ID) with a methanol: acetonitril: chloroform (47: 42: 11, v/v) as mobile phase at 1 mL min- 1 flow rate.

The precipitation of proteins for vitamin C and MDA analysis were made according to the method described by Cerhata et al. ²⁸. The supernatant was filtered and the vitamin C levels were determined using the method of Tavazzi et al. ²⁹ and MDA levels were determined by the method of Karataş et al. (30) by HPLC utilizing a column (250 x 3.9 ID) packed with Tecopak C18 reversed-phase material (10 mm particle size). In the Vitamin C analysis, the mobile phase (3.7 mM phosphate buffer, pH 4.0) at 1 mL min-1 flow rate and 254 nm wavelenth were used, while MDA analysis were performed at following optimized experimental conditions: mobil phase is 30 mM KH2PO4 buffer, pH = 4 with H3PO4) and methanol (65%-35% v/v) at 1.5 mL min-1 flow rate and 254 nm wavelenth.

Serum samples (1.0 mL) obtained for determination of selenium levels were destroyed in Teflon bomb according to Breyer & Gilbert ³¹ and were determined according to modified methods of Watkinson ³² and Whetter  Ullrey ³³. Se levels were determined fluorometrically by a Perkin Elmer 100 fluorescence spectrophotometer at 570 nm according to Standard Addition Method.

All the chemicals used in the study were of analytical grade and purchased from Merck (Darmstad, Germany).

Statistical analysis was carried out using SPSS for Windows, Ver.10 (SPSS Inc. Chicago, IL, USA). The data obtained are expressed as mean values ± S.D. Student’s t-test and pearson test was used to correlations determine whether differences between the means were significant, with p<0.05 taken as the significance level.

Click Here to Zoom

Table 1: Demographic features of diabetic patients and controls.

Click Here to Zoom

Table 2: Comparison of blood glucose, HbA1c, MDA, Se, Vit. A, E, C levels

When fasting blood glucose and percentage HbA1C of controls and diabetic patients were compared, there is a two fold increase in diabetic patients (p<0.005). There was a five-fold increase in serum MDA levels in patients with diabetes compared to controls (p<0.005). On the other hand, levels of antioxidant vitamins (A, E, C) and selenium were observed to be significantly reduced in diabetic patients ( p<0.005).

While there was a positive correlation between percentage HbA1C and MDA (r=0.87 p<0.005), there were a negative correlation between percentage HbA1C and vitamin A, vitamin E, vitamin C and Se (r=-0.72 p<0.005, r=-0.82 p<0.005, 0.74 p<0.005, r=-0.85 p<0.005) respectively.

Furthermore there were a negative correlation between MDA and vitamin A, vitamin E, vitamin C and Se (r=-0.68 p<0.005, r=-0.79 p<0.005, 0.70 p<0.005, r=0.81 p<0.005) respectively.

Jorge et al. ³⁶ reported that a minor oxidative stress was observed in female type 2 diabetic patients after α- tocopherol treatment interfered from the reduced levels of erythrocyte MDA and the increase total antioxidant status. On the other hand no beneficial change was levels serum MDA.

Results of vitamin E levels in blood, plasma and serum levels of patients with type 2 diabetes mellitus are contradictory. Several groups reported that there are increases in vitamin E levels compared to controls ^13,37-39. On the other hand, some reports indicate that no significant changes occur ^40,41. In addition, several studies documented that vitamin E levels in blood, plasma and serum were decreased in type 2 diabetic patients ^{14,34,35,42-44}. In the present study we also observed the latest trend. Table 2.

Controversy regarding blood, plasma and serum vitamin A levels in type 2 diabetic patients also exists. Havivi et al. ⁴⁵ claimed that levels of vitamin A in type 2 diabetic patients were increased whereas several reports documented that there is reduction in vitamin A levels rather than increase compared to controls ^35,37,38,46. In the present study, serum vitamin A levels of diabetic patients were determined to be lower than controls.

Vitamin C levels in diabetic patients were also reported to be increased ^14, decreased ^35,44 or unchanged ⁴⁷. In our study we found that vitamin C levels in patients with diabetes were found to be lower compared to controls.

Although glucose itself can initiate oxidative stress, deficiency of essential trace elements such as selenium may exacerbate this oxidative stress in diabetic rats ⁴⁸. Serum selenium levels of diabetic patients were also reported to be increased ⁴⁹ decreased (50-52 or unchanged 53) compared to controls. In the present study we determined a significant decrease in selenium levels of diabetic patients compared to controls.

There are several reports indicating increased MDA levels in patients with type 2 diabetes ^34,54-57 or unchanged ³⁶. Results of present study support these findings.

Our findings about increase in serum MDA levels in patients with type 2 diabetes are probably due to oxidative events caused by metabolic stress. Significant reductions in the levels of antioxidant vitamins (A, E, C) and selenium are indicators of metabolic response to oxidative stress in patients with type 2 diabetes.

In conclusion; diets of diabetic patients should contain recommended daily allowance of antioxidant vitamins and selenium to allow non-enzymatic as well as enzymatic antioxidant systems to respond oxidative stress observed in diabetic patients.

Acknowledgement
This study was supported partially by a grant (No: 1847 (199T079) from The Scientific and Technical Research Council of Turkey (TUBITAK).

1) Templar J, Kon SP, Milligan TP, Newman DJ, and Raftery MJ: Increased plasma malondialdehyde levels in glomerular disease as determined by a fully validated HPLC method. Nephrol Dial Transplant 14(4):946-951, 1999.

2) Ozben T, Nacitarhan S and Tuncer N: Plasma and urine malondialdehyde levels in non- insulin dependent diabetic patients with and without microalbuminuria. Int J Clin Lab Res 25:162-164, 1995.

3) Halifeoğlu İ, Karataş F, Çolak R, Canatan H, Telo S. Tip 2 diyabetik hastalarda tedavi öncesi ve tedavi sonrası oksidan ve antioksidan durum. Fırat Tıp Dergisi 10(3): 117- 122, 2005;

4) Alan WS: The role of advanced glycation in the pathogenesis of diabetic retinopathy. Exp Mol Pathol 75:95-108, 2003.

5) Singal PK, Bello-Klein A, Farahmand F, Sandhawalia, V: Oxidative stress and functional deficit in diabetic cardiomyopathy. Adv Exp Med Biol 498:213-220, 2001.

6) Mercuri F, Quagliaro L, Ceriello A: Oxidative stress evaluation in diabetes. Diabetes Technol Ther 2(4):589- 600¸2000.

7) Kar M, Chakraborti AS: Release of iron from hemoglobin-a possible source of free radicals in diabetes mellitus. Indian J Exp Biol 37(2):190-192, 1999.

8) Jain SK, Palmer M: The effect of oxygen radicals metabolites and vitamin E on glycosylation of proteins. Free Radic Biol Med 22(4):593-596, 1997.

9) Yagi K: Assay for blood plasma or serum. Methods Enzymol, 109: 328-331, 1984.

10) Cheesman KH and Slater TF: Introduction to free radical biochemistry. Br Med Bull 49(3): 481-493, 1993.

11) Jacob RA and Burri BJ: Oxidative damage and defense. Am. J. Clin. Nutr. 63, 985-990, 1996.

12) Langenstroer P, and Pıeper GM: Regulation of Spontaneous EDRF rebase in diabetic rat aorta by oxygen free radical. Am J Physiol 263:257-265, 1992.

13) Ceriello A, Bortolotti N, Falleti E, Taboga C, Tonutti L, Crescentini A, Motz E, Lizzio S, Russo A, Bartoli E: Total radical-trapping antioxidant parameter in NIDDM patients. Diabetes Care 20(2):194-197, 1997.

14) Maxwell SR, Thomason H, Sandler D, Leguen C, Baxter MA, Thorpe GH, Jones AF, Barnett AH: Antioxidant status in patients with uncomplicated insülin-dependent and noninsülin- dependent diabetes mellitus. Eur J Clin Invest 27(6):484-490, 1997.

15) Dıplock AT: Antioxdant nutrients and disease prevention: An Overview. Am J Chim Nutr 53:1895-1935, 1991.

16) Halliwell B: Free radical antioxidants in human disease. Curiosity, cause or consequence. Lancet, 344:721-724, 1994.

17) Sinha M, Grosh AK: Influence of dietary vitamin A deficiency on rat digestive & absorptive functions during diabetes mellitus. Indian J Med Res 100:196-200, 1994.

18) Jound AF: Data on Oxidants and Antioxidants. Bull Eur Pysiopathol Respir 22:2535-2555, 1986.

19) Levine M: New concepts in biology and biochemistry of ascorbic acid. New Engl J Med 314:892-901, 1997.

20) Oldfield JE: The two faces of selenium. J Nutr 117:2002- 2008, 1987.

21) Van Metre DC, Callan RJ: Selenium and vitamin E. Vet Clin North Am Food Anim Pract. 17(2):373-402, 2001.

22) Underwood EJ: Trace Elements in Human and Animal Nutrition, Acedemic Press. New York, 1977.

23) Zintzen H: A summary of the vitamin E/selenium problem in ruminants, News and Reviews, Roche, 1-18, 1978.

24) Al-Saleh IA & Al-Doush I: Selenium levels in wheat grains grown in Saudi Arabia. Bull Environ Contam Toxicol 59:590-594, 1997.

25) Oster O& Prellwitz W: Are Germans Selenium - Deficient? In Selenium in Biology and Medicine. Springer - Verlag, Berlin Heidelberg, New York, 1989.

26) Ceriello A, Giugliano D, Quatraro A, Dello Russo P, Torello R: A preliminary note on inhibiting effect of *-tocopherol on protein glycation. Diabete Metab 14:40-52, 1988.

27) Miller KW, Lorr NA, Yang CS: Simultaneous determination of plasma retinol *-tocopherol, Iycopene, *-carotene, and *- carotene by high performance liquid chromatography. Anal Biochem 138:340-345, 1984.

28) Cerhata D, Bauerova A, Ginter E: Determination of ascorbic acid in blood serum using high performance liquid chromatography and its correlation with spectrophotometric (colorometric) determination. Caska Slov Farm 43(4):166- 168, 1994.

29) Tavazzi B, Lazzarino G, Di-Pierro D, Giardina B: Malondialdehyde Production and Ascorbate Decrease are Associated to the Eperfusion of the İsolated Postischemic Rat Heart. Free Radic Biol Med 13(1):75-78, 1992.

30) Karatas F, Karatepe M and Baysar A. Determination of free malondialdehyde in human serum by high performance liquid chromatography. Analytical Biochemistry, 2002; 311: 76-79.

31) Breyer PH, and Gılbert BP: Determination of Selenium (IV) Differential Puls Voltametry of the 3,3’-Diaminobenzidine piazselenol. Analytica Chimica Acta 201:23-32, 1987.

32) Watkinson JH: Fluorometric Determination of Traces of Selenium. Anal Chem 32(8):981-983, 1960.

33) Whetter PA, and Ullrey DE: Improved Fluorometric Metod for Determining Selenium. J Assoc Anal Chem 61(4):927- 930, 1987.

34) Noberasco G, Odetti P, Boeri D, Maiello M, Adezati L: Malondialdehyde (MDA) level in diabetic subjects. Relationship with blood glucose and glycosylated hemoglobin. Biomed Pharmacother 45, (4-5):193-196, 1991.

35) Sundaram RK, Bhaskar A, Vijayalingam S, Viswanathan M, Mohan R: Antioxidant status an lipid peroxidation in type II diabetes mellitus with and without complications. Clin Sci (Colch) 90(4):255-260, 1996.

36) Jorge L. Ble-Castillo, Elizabeth Carmona-Díaz, José D. Méndez, Francisco J. Larios-Medina, Roberto Medina- Santillán, Guadalupe Cleva-Villanueva, Juan C. Díaz- Zagoya. Effect of α-tocopherol on the metabolic control and oxidative stress in female type 2 diabetics. Biomedicine & Pharmacotherapy 59 (2005) 290–295

37) Krempf M, Ranganathan S, Ritz P, Morin M, Charbonnel B: Plasma vitamin A and E in type 1 (insulin-dependent) and type 2 (non-insulin-dependent) adult diabetic patients. Int J Vitam Nutr Res 61(1):38-42, 1991.

38) Vatassery GT, Morley JE, Kuskowski MA: Vitamin E in plasma and platelets of human diabetic patients and control subjects. Am J Clin Nutr 37:641-644, 1983.

39) Caye-Vaugien C, Krempf M, Lamarche P, Charbonnel B, Pieri J: Determination of alpha-tocopherol in plasma, platelets and erythrocytes of type I an type II diabetic patients by high-performance liquid chromatography. Int J Vitam Nutr Res 60(4):324-30, 1990.

40) Rautalahti M, Albanes D, Haukka J, Roos E, Gref CG, Virtamo J: Seasonal variation of serum concentrations of *- carotene and *-tocopherol. Am J Clin Nutr 57:551-556, 1993.

41) Abahusain MA, Wright J, Dickerson JW, de Vol EB: Retinol, alpha-tocopherol and carotenoids in diabetes. Eur J Clin Nutr 53(8):630-636, 1999.

42) Nourooz-Zadeh J, Rahimi A, Tajaddini-Sarmadi J, Tritschler H, Rosen P, Halliwell B, Betteridge DJ: Relationships between plasma measures of oxidative stress and metabolic control in NIDDM. Diabetologia 40(6):647-653, 1997.

43) Knekt P, Reunanen A, Marniemi J, Leino A, Aromaa A: Low vitamin E status is a potential risk factor for insulindependent diabetes mellitus. J Intern Med 245(1):99-102, 1999.

44) Ceriello, PA: Oxidative stress and diabetes associated complications. Endo Prac 12, 60−62, 2006.

45) Havivi E, Bar On H, Resehef A, Stein P, Raz I: Vitamins and trace metals status in non insülin dependent diabetes mellitus. Int J Vitam Nutr Res 77(4):26-28, 1991.

46) Granado F, Olmedilla B, Gil-Martinez E, Blanco I, Millan I, Rojas-Hidalgo E: Carotenoids, retinol and tocopherols in patients with insulin-dependent diabetes mellitus and their immediate relatives. Clin Sci(Colch) 94(2):189-195, 1998.

47) Ceriello A, Bortolotti N, Pirisi M, Crescentini A, Tonutti L, Motz E, Russo A, Giacomello R, Stel G, Taboga C: Total plasma antioxidant capacity predicts trombosis-prone status in NIDDM patients. Diabetes Care 20(10):1589- 1593, 1997.

48) Reddi AS, Bollineni JS: Selenium-deficient diet induces renal oxidative stress and injury via TGF-beta1 in normal and diabetic rats. Kidney Int 59(4):1342-1353, 2001.

49) Ashour M, Salem S, Hassaneen H, El-Gadban H, Elwan N, Awad A, Basu K: Antioxidants status and insulindependent diabetes mellitus (IDDM). J Clin Biochem Nutr 26:99-107, 1999.

50) Navarro-Alarcon M, Lopez-G de la Serrana H, Perez- Valero V, Lopez-Martinez C: Serum and urine selenium concentrations as indicators of body status in patients with diabetes mellitus. Sci Total Environ 228(1):79-85, 1999.

51) Ruiz C, Alegria A, Barbera R, Farre R, Lagarda J: Selenium, zinc and copper in plasma of patients with type 1 diabetes mellitus in different metabolic control states. J Trace Elem Med Biol 12(2):91-95, 1998.

52) Kruse-Jarres JD, Rukgauer M: Trace elements in diabetes mellitus. Peculiarities and clinical validity of determinations in blood cells. J Trace Elem Med Biol 14(1):21-27, 2000.

53) Kljai K, Runje R: Selenium and glycogen levels in diabetic patients. Biol Trace Elem Res 83(3): 223-229, 2001.

54) Jiang ZS, Zhang SL, Cai XJ: Effect of salvia miltiorrhiza composita on superoxide dismutase and malondialdehyde in treating patients with non-insulin dependent diabetes mellitus. Chung Kuo Chung Hsi I Chieh Ho Tsa Chih.17(1):32-34, 1997.

55) Dierckx N, Horvath G, van Gils C, Vertommen J, van de Vliet J, De Leeuw I, Manuel-y-Keenoy B: Oxidative stress status in patients with diabetes mellitus: relationship to diet. Eur J Clin Nutr. 57 (8): 999-1008, 2003.

56) Pasaoglu H, Sancak B, Bukan N. Lipid peroxidation and resistance to oxidation in patients with type 2 diabetes mellitus. Tohoku J Exp Med. 203 (3): 211-218, 2004

57) Ceriello A, Bortolotti N, Motz E, Crescentini A, Lizzio S, Russo A, Tonutti L, Taboga C: Meal-generated oxidative stress in type 2 diabetic patients. Diabetes Care 21(9):1529-1533¸ 1998.