Indmedica Home | About Indmedica | Medical Jobs | Advertise On Indmedica
Search Indmedica Web
Indmedica - India's premier medical portal

Biomedical Research

Effect of antioxidant vitamin supplementation on erythrocyte membrane composition in Type I diabetes mellitus in context of oxidative stress

Author(s): Sarita A. Shinde, Adinath N. Suryakar, Alka N. Sontakke, Umesh K. More

Vol. 21, No. 2 (2010-04 - 2010-06)

(1)Sarita A. Shinde٭, (2)Adinath N. Suryakar, (1)Alka N. Sontakke, (1)Umesh K. More

(1) Department of Biochemistry, Pad.Dr.D.Y.Patil Medical College Pimpri, Pune- 411018: Maharashtra: India
(2) Department of Biochemistry, Dr. V. M. Medical College, Solapur, Maharashtra: India


In diabetes mellitus, the high incidence of microvascular and atherosclerotic disorder has been seen which are associated with abnormalities of erythrocyte composition, rheological function and with increased oxidative stress among many other factors. Human erythro-cytes are perhaps the cells most exposed to peroxidation damage by free radicals. In view of this erythrocyte membrane composition of type I diabetic patients was analyzed before and after supplementation of vitamin (E +C) along with insulin and compared with those patients who were on only insulin treatment. All Subjects were selected from Out Patient department of Pad. Dr. D.Y. Patil Medical College: Pimpri and Dr. V.M. Medical College: Solapur during the period of April 2006 to April 2008. The result showed significantly decreased (p< 0.001) concentration of Glycated hemoglobin, serum MDA and membrane cholesterol and significantly increased erythrocyte reduced glutathione, membrane phospholipids ( p<0.05,p<0.001 respectively) after three months follow up of vitamin ( E+C) supplementation along with insulin in type I diabetic patients. The present study may conclude that supplementation of vitamin ( E+C) along with insulin protect the erythrocyte membrane composition against such oxidative deterioration and may minimize variety of hema-tological abnormalities which further causes the complications in type I diabetic patient.

Key Words: Diabetes mellitus, oxidative stress, membrane cholesterol, phospholipids, vitamin E and C.
Accepted August 26 2009


Diabetes mellitus (DM) refers to a group of common metabolic disorders that share the phenotype of hyperglycemia. Type 1 DM is the result of interactions of genetic, environmental, and immunologic factors that ultimately lead to the destruction of the pancreatic beta cells and insulin deficiency [1]. Reactive Oxygen Species (ROS) play a central role in β cell death and disease progression [2]. A variety of hematological abnormalities are seen in diabetes. These include increased erythrocyte aggregation, decreased deformability of erythrocytes, increased platelet aggregation, and adhesion predisposed to sluggish circulation, endothelial damage and focal capillary occlusion [3]. De novo oxidative damage, a result of increased protein glycosylation could participate in the mechanism, whereby diabetic erythrocytes may acquire membrane abnormalities [4]. Enhanced glycosylation by elevated glucose concentration may induce the formation of oxygen derived free radicals through protein glycosylation, which releases early and late glycosylation end products, contributing to enhancement of oxidative stress seen in diabetes [5]. Human erythrocytes are perhaps the cells most exposed to peroxidation damage by free radicals. The mechanisms by which the erythrocyte defends itself against oxidative damage are very efficient and are located in both cytosol and membrane domains. The membrane itself contains only vitamin E, as the major lipid soluble chain breaking antioxidant [6].

Many investigators reported lower concentrations of glu-tathione in the erythrocytes, aorta, and lenses of diabetic patients compared with healthy subjects [7]. Lower glu-tathione and elevated lipid peroxidation concentrations are risk factors for the development of pathological states such as neuropathy, cataracts, and atherosclerosis in dia-betes [8].

Hence the present study was undertaken to observe the effect of supplementation of antioxidant vitamins i.e.

(E+C) conjointly on erythrocyte membrane composition in type 1 diabetes mellitus in context of oxidative stress.

Material and Methods

Subjects and study design

In all 100 subjects were enrolled in the present study. Control group comprising of 40 healthy age and sex (16-30 years) matched subjects. Test group comprising of 60, type I diabetic patients of age (14-32) years selected from Dr. V. M. Medical College and Chatrapati Shivaji Maharaj Sarvopchar Rugnalaya; Solapur and Pad. Dr. D.Y. Patil Medical College, Hospital and Research Centre, Pune. The patients were diagnosed based on the clinical and laboratory data. Patients selected for the study were controlled diabetics. Patients were stabilized with insulin injections (long acting and short acting dosage) and did not show any insulin resistance and antihypertensive drugs as required. None were current users of vitamin.

The patients suffering from hepatic disease, cardiovascu-lar disease (CVD) and any chronic or acute inflammatory illness, cancer of all types, pulmonary tuberculosis, alcoholics and smokers were excluded from the study. All the subjects included in the study volunteered after proper consent and reported for follow-up at right time. The study was approved by ethical committee.

The test group was further categorized randomly into Group I (n=30) were treated by only insulin and group II (n=30) were received both vitamin [E (evinal 400 mg/day +C (celin 500mg/day) ] conjointly and insulin for a period of three months. All had avoided aspirin and other platelet active agents during the study period.

Collection of Specimen

Fasting venous blood sample were collected at morning 8.00am in different bulbs under aseptic conditions. Blood glucose was estimation by glucose oxidase peroxidase [9] and glycated hemoglobin (HbA1c) by resin bind-ing method [10]. Measurement of erythrocyte reduced glutathione (GSH) by method of Beutler et al [11]. Esti-mation of serum malon di aldehyde( MDA) by Wilber et al method [12]. Erythrocyte ghost preparation was done by Dodge et al method [13]. The membrane cholesterol was estimated by zak et al [14], membrane phospholipids by fiske subbarow [15] and membrane proteins by Lowry’s method [16].

Baseline levels of all of the above biochemical parameters of all subjects were measured at the time of enrollment. But group I and II again reassessed for the above parameters after follow up of three months period. The glycated hemoglobin (HbA1c) levels were used as an index of metabolic control.

Statistical Analysis

Statistical comparison of data was done using student’s ‘t’ test. Values were expressed as mean ± standard deviation (SD). Sigma version 3.0 was used for statistical analysis

Observation and Results

The study revealed (Table 1), significantly increased con-centration of glycated hemoglobin (p<0.001), serum MDA (p<0.001), erythrocyte membrane cholesterol (p<0.001) and significantly decreased concentration of erythrocyte membrane phospholipids (p<0.001), mem-brane proteins ( p<0.001) and erythrocyte reduced glu-tathione (p<0.05) in type I diabetics as compared with controls.

Table 1. Comparison of biochemical parameter in between controls and Type 1 diabetics.

Parameters Control
(n = 40)
Type 1 Diabetics
SL (F) mg/dl 76.00 ± 8.33 150.43±10.74 ٭٭
Glycated Hb (%) 5.37 ± 0.45 7.29 ± 0.43 ٭٭
Membrane Proteins (mg/ml of packed cell) 4.98 ± 0.31 3.26 ± 0.54 ٭٭
Membrane Cholesterol (μg/mg of proteins) 100.55 ± 7.46 174.13 ± 15.44 ٭٭
Membrane Phospholipids (μg/mg of proteins) 200.25 ± 7.85 192.03 ± 8.8 ٭٭
Serum MDA (nmole/ml) 3.59 ± 0.98 7.94 ±1.09 ٭٭
Erythrocyte GSH (μmole/gm of Hb) 5.04 ± 0.89 4.72 ± 0.35 ٭

Values are expressed as Mean ± SD ٭٭ indicates p<0.00; ٭ p<0.05

Table 2. Effect of antioxidant vitamin supplementation on erythrocyte membrane

Table 2

Values are expressed as Mean ± SD : ٭٭indicates p<0.001 and ٭ p<0.05

Table 2 shows significantly decreased concentration of glycated hemoglobin after 3 months follow up in both group as compared to their baseline levels (p<0.001). Membrane proteins were nonsignificantly increased in both group after 3 months as compared to their baseline levels. No change in concentration of membrane choles-terol in group I but significant reduction was observed in group II (p<0.001) after 3 months follow up as compared to its baseline levels. Membrane phospholipids were non-significantly increased in group I and significantly increased in group II (p<0.001) after 3 months as compared to their baseline levels. Significant rise was observed in serum MDA concentration after 3 months follow up as compared to its baseline levels in group I (p<0.05). But serum MDA is significantly decreased in group II (p<0.001) after 3 months follow up as compared to its baseline levels. Concentration of reduced glutathione significantly increased in group I (p<0.001) and group II (p<0.001) after 3 months follow up as compared to its baseline levels.


In diabetes mellitus, the high incidence of microvascular and atherosclerotic disorder has been associated with abnormalities of erythrocyte composition and rheological function and with increased oxidative stress among many other factors [17,18].

The red cell membrane (RBC) contains approximately equal amount of lipids and protein. Cholesterol is intercalated between the phospholipids molecules. The relative amounts of cholesterol and phospholipids are responsible for the fluid properties of the erythrocyte membrane [19].

In present study as per Table 1, a significant alteration was observed in concentration of membrane cholesterol and phospholipids. The increased membrane cholesterol reflects the increased serum cholesterol concentration. The decreased membrane phospholipids in diabetics could have occurred from the damage induced by nonenzymatic glycation of the cell membrane and subsequent loss of phospholipids from the RBC membrane [20]. Alteration in the membrane cholesterol- phospholipids ratio results in morphologically abnormal erythrocytes with decreased life span. Membrane proteins were decreased as compared with controls and this could be due to damage of membrane proteins by nonenzymatic glycation [21]. Increased serum MDA reflects increased oxidative stress. Mechanisms involved in the increased oxidative stress in diabetes include not only oxygen free radical generation due to nonenzymatic glycation, autooxidation of glycation products, but also changes in the tissue content and activ-ity of antioxidant defense systems [22]. The present study also shows fall in concentration of erythrocyte GSH in type I diabetics. Vitamin E has been improved to be the major lipid soluble chain breaking antioxidant and protect biological membrane from lipid peroxidation [7]. Vitamin C, has the potential to protect both cytosolic and mem-brane components of cells from oxidant damage [23].

The aim of present study was to investigate whether supplementing type I diabetic patients with dose of vitamin (E+C) along with insulin would improve erythrocyte membrane composition and antioxidant status in context of oxidative stress.

Table 2 showed significant rise in concentration of serum MDA after 3 months follow up in only insulin treatment group along with no significant change in concentration of membrane cholesterol, phospholipids and proteins. Alteration in membrane lipid composition may be associated with oxidative stress which is not improved by only insulin treatment but may be deteriorated further.

In group II after vitamin (E+C) supplementation along with insulin, membrane cholesterol concentration is sig-nificantly decreased and phospholipids were significantly increased. Hence ratio is maintained for proper blood rheological pattern. Serum MDA was significantly reduced after 3 months of vitamins supplementation and antioxidant status i.e. GSH is significantly increased as compared to its baseline levels. Vitamin E and C act synergistically when the initiating radicals are generated within the lipid core. Vitamin C improves the antioxidant status by i) increasing erythrocyte GSH levels by being a cofactor for NADP reductase. This enzyme plays a key role in the regeneration of GSH from oxidized glu-tathione. ii) Regenerating alpha tocopherol [24].

A major protective mechanism against oxidative damage is the membrane integrity. Oxidation induces change in the membrane permeability resulting in hemolysis would relate to the degree of intravascular RBC destruction. Ex-travascular mechanism(s) of RBC destruction may involve changes in cell deformability and antigenicity [25]. Lipid peroxidation causes polymerization of membrane components and decreases cell deformability [26]. Products of lipid oxidation, such as oxidized cholesterol and oxidized unsaturated fatty acyl groups of phospholipids, may affect structure and function of the membrane. Each phospholipids class consists of a variety of phospholipids molecules characterized by their different fatty acyl side chains. Alterations in the phospholipids molecular species composition as a consequence of oxidant damage can be deleterious to the RBC membrane [27]. Since the RBC lacks the ability for de novo synthesis of proteins and lipids, the uptake of phospholipids from plasma takes place as a part of repair mechanism of oxidized membrane lipids [28]. In general, the overall effect of lipid peroxidation is to decrease membrane fluidity, deformability, viscoe-lasticity and life span of erythrocyte which may causes complications in type I diabetes mellitus.


In IDDM, increased serum lipid peroxidation, decreased antioxidant status and altered erythrocyte membrane composition was observed. This change in the membrane composition could be due to oxidative stress, which may affect membrane fluidity and deformability. So supplementation of vitamin (E+C) along with insulin protect the erythrocyte membrane composition against such oxidative deterioration and may minimize variety of hematological abnormalities and hence complications in type I diabetic patient.


I would like to acknowledge the kind cooperation ex-tended by the staff of biochemistry, medicine and PSM department of Dr. V. M. Medical College: Solapur and Pad. Dr. D.Y. Patil Medical College Hospital and Re-search Centre: Pune. Also I would like to acknowledge the Shivaji University: Kolhapur for their kind permission to research project.


  1. Braunwald B, Longo FH, Jameson. Diabetes mellitus in Harrison’s principles of internal medicine, 17th Ed, Mc Graw-Hill publication , I : 2005; 1463-1464
  2. Emily H, Tammy MB. Antioxidants, NFKB Activation and Diabetogenesis. Expt Biol Med 1999; 222: 205-213.
  3. Biswas BR. Occular complication of diabetes mellitus. The journal of general medicine. 2002; 14: 21-27.
  4. Scwartz RS, Madsen JW, Anne C, Raybicky, Nagel RL. Oxidation of spectrin and deformability defects in diabetic erythrocytes. Diabetes 1991; 40: 701-708.
  5. Resm H, Peketin C, Guner G. Erythrocyte membrane cytoskeletol protein glycation and oxidation in short term diabetic rabbits. Clin. Exp. Med 2001; 1: 187-193.
  6. Constantinescu A, Derick H, Packer L. Vitamin E recycling in human erythrocyte membranes. The journal of biological chemistry 1993; 268 (15): 10906-10913.
  7. Selvam R. Anuradha CV. Lipid peroxidation and anti-peroxidative enzymes changes in erythrocytes in diabe-tes mellitus. Indian J Biochem Biophysics 1988; 25: 268-272.
  8. Baynes JW. The role of oxidative stress in development of complications in diabetes. Diabetes 1991; 40: 405-411.
  9. Trinder P. Estimation of blood sugar level by GOD-POD method. Ann. Clin Biochem 1969; 6: 24-24.
  10. Trivelli LA, Ranney PH, Lai HT. Estimation of glycated hemoglobin by resin binding method. New Eng J Med 197; 284: 353-353.
  11. Fairbanks V, George K. Biochemical aspects of hematology in Teitz Text Book of Clinical Chemistry editors Carl Burtis and Edward Ashwood 3rd edn. WB Saunders Co Philadelphia: PA 1999; 1652-1653.
  12. Wilbur KM, Bernheim F, Shapiro OW. The thiobarbituric acid method for mlaondialdehyde estimation. Arch Biochem Biophy 1943; 250; 305-313.
  13. Dodge JT, Mitcheli C, Hanahan DJ. The preparation and chemical characteristics of hemoglobin free ghosts of human erythrocytes. Archives of biochemistry and biophysics 196; 100: 119-130.
  14. ZaK B. The colorimetric estimation of serum cholesterol. Am. J. Clin Pathol 1957: 27: 583-585
  15. Varley H Gowenlock AH and Bell M. Practical clinical biochemistry. 4th Edn .William Heinemann Medical Book Ltd. London 1976; 473-474
  16. Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin-phenol reagent. J Biol Chem1951; 193: 265-275.
  17. McMillan DE. Plasma protein changes, blood viscosity and diabetic microangiopathy. Diabetes 1976; 25 suppl 2): 858-864.
  18. Schut NH, Van Arkel EC, Hardeman MR, Bilo HJG, Michels RPJ, Vreekan J. Blood and plasma viscosity in diabetes: possible contribution to late organ complications? Diabetes Res 1992; 19: 31-35.
  19. Nagle JF and Tristram- Nagle S. Structure of lipid bi-layers. Biochim Biophys Acta 2000; 1469: 159-195.
  20. Ellenberg M, Rifkin H. Diabetes mellitus theory and practice. 3rd Edn. New York: Medical examination Publishing Co. 1983.
  21. Watala C, Pluta J, Golanski J. Increased protein glycation in diabetes mellitus is associated with decreased aspirin- mediated protein acetylation and reduced sensi-tivity of blood platelets to aspirin. J Mol Med 2005; 83: 148-158.
  22. Ceriello. A hyperglycemia: the bridge between non enzymatic glycation and oxidative stress in the patho-genesis of diabetic complications. Diabetes Nut Metab 1999; 12(1): 42-46.
  23. May J. Is ascorbic acid an antioxidant for the plasma membrane? FASEB 1999; 13: 995-1006.
  24. Murray RK, Keeley FW. Micronutrients and vitamins In: Murray RK, Granner DK, Mayes PA, Rodwell VW, Harper’s illustrated biochemistry, 27th Edn. Lange medical Books, Mc Graw hill (2006); 494-495
  25. Chiu D, Lubin B, Shonet S. Erythrocyte membrane lipid reorganization during sickling process. Br J Hae-matol 1979; 41: 223-234.
  26. Haest CWM, Plasa G, Kamp D, Deuticke B. Spectrin as astabilizer of the phospholipid asymmetry in the human erythrocyte membrane. Biochem Biophys Acta 1978: 509: 21-32.
  27. Ricther C. Biophysical consequences of lipid-peroxidation in membranes. Chem Phys Lipids 1987: 44: 175 -189.
  28. Lubin B, Chiu D, Bastaky J, Roelafsen B, Van Deenen LLM. Oxidative hemoglobin denaturation and RBC destruction. Semin Haematol 1989: 26: 128-135.

Sarita Shinde

Department of Biochemistry
Pad. Dr. D.Y.Patil Medical College Pimpri
Pune 411018, India
E-mail: snc_unc(at)

Biomedical Research 2010: 21 (2): 156-160

Access free medical resources from Wiley-Blackwell now!

About Indmedica - Conditions of Usage - Advertise On Indmedica - Contact Us

Copyright © 2005 Indmedica