Assessment of Antioxidant Vitamins Retinol and α- Tocopherol in Plasma and Ascorbic Acid in Plasma and Mononuclear Leukocytes in Type 2 Diabetics

This Article

Citations


Article Information:


Group: 2006
Subgroup: Volume 4, Issue 4, Autumn
Date: January 2006
Type: Original Article
Start Page: 202
End Page: 205

Authors:

  • M Firoozrai
  • Department of Biochemistry, College of Medicine, Iran University of Medical Sciences, Tehran, I.R.Iran
  • I Nourmohammadi
  • Department of Biochemistry, College of Medicine, Iran University of Medical Sciences, Tehran, I.R.Iran
  • K Khanaki
  • Department of Biochemistry, College of Medicine, Iran University of Medical Sciences, Tehran, I.R.Iran

      Correspondence:

      Affiliation: Department of Biochemistry, College of Medicine, Iran University of Medical Sciences
      City, Province: Tehran,
      Country: I.R.Iran
      Tel:
      Fax:
      E-mail: mfiroozrai@yahoo.com

Abstract:


The rise and along with this increase there is a growing interest in the efficacy of antioxidant vitamin therapy in the treatment of this malady.

Materials and Methods: In this case-control study, the levels of antioxidant vitamins retinol, -tocopherol and ascorbic acid were measured with HPLC and colorimetric method, in plasma and mononuclear leukocytes of patients with diagnosed Type 2 diabetes(n=62).

Results: We observed significantly lower ascorbicacid levels in mononuclear leukocytes in our study group when compared to controls.

Conclusion: While these results are not definitive, we believe they do indicate that further studies are needed to include a larger segment of the population and an evaluation of dietary intakes and habits.

Keywords: Retinol, tocopherol, Ascorbic acid, Type 2 diabetes, Antioxidants

Manuscript Body:


Introduction
The prevalence of clinically diagnosed Type 2 diabetes is expected to increase worldwide to more than 300 million by the year 2025.1 Life-style modification, obesity and changes in dietary habits and composition may predispose a person to this common form of diabetes.2,3 Much attention has been focused recently on the antioxidant micronutrients retinol (vitamin A), _-tocopherol (vitamin E) and ascorbic acid (vitamin C) for their beneficial and therapeutic effects. Even though lack of consistency remains in some results, evidence from several studies reveals that altered plasma status of these antioxidants may have a role in occurrence of oxidative insults in this chronic disease.4-7 Positive association has been documented between oxidative stress and etiology of diabetic complications.2,8 It has been postulated that an increase in the status of antioxidants by consuming fruits and vegetables rich in exogenous antioxidants can protect against oxidative stress associated with Type 2 diabetes and this would also reflect levels of intake during acute oxidative activity.9-11
This study was carried out to assess the concentrations of essential vitamins retinol and -tocopherol in plasma and water soluble vitamin C both in plasma and mononuclear leukocytes (MN) of Type 2 diabetic patients and healthy controls in small segment of our population.

Materials and Methods
All eligible participants in this study signed informed consent forms and the protocol was approved by the University Ethics Committee. Patients with Type 2 diabetes (n=62) with a mean age of 51.30±9.19 years were recruited from the diabetic center at the university hospital. Duration of disease was 9.99±7.81 years. Patients with hyperlipidemia, those who smoked or who had any unusual dietary habits or systemic disease such as nephropathy were excluded. Control subjects (n=38) consisted of non-diabetic healthy individuals with a mean age of 50.82±10.74 years and were chosen from the same sociodemographic group as the study population. None of the participants were taking vitamin or mineral supplements shortly before or during the study. Seven patients controlled their diabetes with diet alone, 37 treated with oral hypoglycemic medicine and 18 with insulin. Sufficient fasting blood samples were obtained by venous puncture for all biochemical analysis. Plasma levels of vitamins A and E were measured by reversed phase high-performance liquid chromatography (HPLC) and expressed as Ag/mL.12 Ascorbic acid concentration in plasma and MN was measured by 2,4 dinitrophenyl hadrazine as described by Roe and Keuther and expressed as mg/dl and mg/g TP, respectively.13 Laboratory assay for MN preparation was according to Cunningham.14
Plasma glucose concentration was measured by glucose-oxidase technique; glycated Hemoglobin (HbA1c) was measured by calorimetric and total protein (TP) content of cell protein precipitate was determined by the biuret method in our laboratory. Statistical calculations were performed using SPSS-10 for Windows software package and all results are presented as mean±SD. Comparison between diabetic and control subjects was made with unpaired Student's T-test. For other variables, analysis of variance ANOVA was used when necessary. P value of < 0.05 was considered as significant.

Results
The observation (mean or %), characteristics and biochemical measurements of all participant are summarized in Table 1. These results indicate, comparing the mean plasma concentration of retinol from diabetic patients (0.59±0.17 Ag/mL) and control subjects(0.51±0.13 Ag/mL), no statistically significant difference. Plasma concentration for -tocopherol among patients (19.57±6.8 Ag/mL) and control subjects (20.08±7.39 Ag/mL) was also not statistically significant. MN ascorbic acid level in diabetic patients was statistically lower than that of controlsubjects (1.35±0.55 vs. 1.77±0.61 mg/g TP, P < 0.05). However, the plasma ascorbic acid level between two groups we observed showed no significant differences (0.99±0.28 vs. 1.00 ± 0.23 mg/dL).

Table 1. Baseline characteristics and biochemical measurement of Type 2 diabetic and control subjects

----

Healthy controls

N=38

Type 2 patients

N=62

Age (y)

50.8± 10.7

51.3 ± 9.1

Gender (M/F)

24/14

33/29

Duration of Diabetics (year)

-------

9.99 ± 7.81

Fasting Plasma glucose(mg/dL)

89±74

200 ± 52*

HbA1c (%)

----------

6.67 ± 1.04

Blood Pressure(mmHg):

 

130 ± 21

Systolic

110±12

79.4 ± 11.6

Diastolic

71.0±4.3

 

Retinol (Ag/mL)

0.51±0.13

0.59 ± 0.17

- Tocopherol (Ag/mL)

20.08±7.39

19.57 ± 6.8

Ascorbic Acid:

 

 

Plasma (mg/dL)

1.00±0.23

0.99 ± 0.28

MN (mg/g TP)

1.77±0.61

1.35 ± 0.55*

Data is presented as mean ± SD or %; Comparison between groups has been made using student t-test;
* P<0.05

 

Discussion
Our data did not reveal any statistically significance when considering mean plasma concentrations of retinol, -tocopherol and ascorbic acid among type 2 diabetic patients and our control volunteers. Similar results on plasma concentration of retinol and - tocopherol was reported by Ahmad and Basualdo from Type 2 diabetic subjects.4,15
Merzouk showed ascorbic acid levels in plasma of Type 2 diabetics and non- diabetics to be normal.7 Kim demonstrated that there were no significant differences between plasma ascorbic acid levels in diabetic patients and control group.16 However, several authors have reported both a decrease or higher levels of retinol, - tocopherol and ascorbic acid concentrations when comparing Type 2 diabetics to control groups.4,6,7,16,17 Epidemiological studies have confirmed that increased serum _-tocopherol levels are associated with decreased risk of Type 2 diabetes. 18 Pharmacological doses have been shown to improve glycemic control and insulin- mediated glucose disposal.19 However, there have been suggestions that dietary in take of _-tocopherol is beneficial and has a protective effect on glucose metabolism.9
There is convincing evidence that ascorbic acid metabolism is altered in conditions such as hyperglycemia and insulin insufficiency and patients with diabetes have about 30% lower plasma ascorbic acid levels than persons without.8,20 Decrease in active transport of reduced ascorbic acid and uptake inhibition of the oxidized form been observed in hyperglycemia.21,22
Unlike plasma concentration of ascorbic acid in our patients, concentration of this water soluble vitamin in MN, comparing to our controls, was lower (P < 0.05). Other investigators have also shown MN ascorbic acid to be reduced. Chen reported a significant reduction in Type 2 diabetics relative to nondiabetics (1.44 mg/g TP vs. 2.44 mg/g TP)23
Cunningham observed a 30% reduction in adults with Type 1 diabetes.14 Mononuclear leukocytes can accumulate a much higher concentration of ascorbic acid than plasma and is considered to be the best index and more reliable index of tissue ascorbic acid stores.24
Plasma concentration of antioxidant vitamins of our study population was normal when comparing to that of controls however, interpretation of these result must be taken with caution and further research on a larger scale is encouraged which should include dietary intake of the population under investigation.

Acknowledgements
We are indebted to the head and staff of the university research division and also to The Cellular Molecular Research Center for financial support and technical assistance. We also gratefully acknowledge Ehsan Noormohammadi, BSc (Department of Biochemistry, University of Oklahoma) for his skillful editing of the manuscript and S. Teemsar for typing. All authors contributed to the interpretation of results.

References: (24)

  1. Zimmet P, Alberti KG, Shaw J. Global and societal implications of the diabetes epidemic. Nature 2001; 414: 782-7.
  2. Ruh C R, McDonald B R. Use of antioxidant nutrients in the prevention and treatment of type 2 diabetes: Review. JAm Coll Nutr 2001; 20 (5 ): 363S -9S.
  3. Cheng D. Prevalence, predisposition and prevention of type II diabetes: Review. Nutrition & Metabolism 2005; 2: 29.
  4. Ahmad M, Khan MA, Khan AS. Naturally occurring antioxidant vitamin levels in patients with type-II diabetes mellitus. J Ayub Med Coll Abbottabad 2003; 15(1): 54-7.
  5. Yamada H, Yamada K, Waki M, Umegaki K. Lymphocyte and plasma vitamin C levels in type 2 diabetic patients with and without diabetes complications. Diabetes Care 2004;27 (10): 2491- 2.
  6. 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 1991;61(1): 38-42.
  7. Merzouk S, Hichami A, Madani S, Merzouk H, Berrouiguet AY, Prost J, et al. Antioxidant status and levels of different vitamins determined by high performance liquid chromatography in diabetic subjects with multiple complications. Gen Physiol Biophys 2003;22(1 ): 15-27.
  8. Will C J, Byers T. Dose Diabetes Mellitus Increase the Requirement for Vitamin C?. Nutr Rev 1996;54(7): 193-202.
  9. Mayer-Davis J E, Costacou T, King I, Zaccaro J D, Bell A R. Plasma and dietary vitamin E in relation to incidence of type 2 Diabetes. Diabetes Care 2002;25 (12) :2172-4.
  10. Cao G, Booth SL, Sadowski JA, Prior RL. Increased in human plasma antioxidant capacity after consumption of controlled diets high in fruits and vegetables.Am J Clin Nutr 1998; 69: 1081-7.
  11. Ylonen K, Alfthan G, Groop L, Saloranta C, Aro A, Suvi M. Dietary intakes and plasma concentrations of carotenoids and tocopherols in relation to glucose metabolism in subjects at high risk of type 2 diabetes: the Botnia Dietary Study. Am J Clin Nutr 2003;77: 1434-41.
  12. Catignani GL, Bieri JG. Simultaneous determination of retinol and -tocopherol in serum or plasma by liquid chromatography. Clin Chem 19983;29(4): 708-12.
  13. 13- Roe JH, Keuthe CA. The determination of ascorbic acid in whole blood and urine through the 2, 4- nitrophenylhydrazine derivative dehydro- ascorbic Acid J Biol Chem 1943;147:399-403.
  14. Cunningham J J, Ellis L S, McVeigh L M, Levine E R, Calles–Escandon J. Reduced mononuclear leukocyte ascorbic acid content in adults with insulin- dependent diabetes mellitus consuming adequate dietary vitamin C. Metabolism 1991;40 (2): 146-9.
  15. Basualdo CG, Wein EE, Basu TK. Vitamin A (retinol) status of first nation adults with non- insulin- dependent diabetes mellitus. J Am Coll Nutr1997; 16(1): 39-45.
  16. Kim JH, Kim MJ. Dietary intakes and plasma antioxidant vitamins levels in Korean elderly with diabetes. Asia Pac J clin Nutr 2004;13(Suppl): S152-4.
  17. Maxwell J RS, Thomason H, Sandler D, Leguen C, Baxter A M, Thorpe G H G, et al. Antioxidant status in patients with uncomplicated insulin- dependent and non-insulin- dependent diabetes mellitus. Eur J Clin Invest 1997; 27: 484-90.
  18. Salonen JT, Nyyssonen K, Tuomainen T_P, Maenpaa PH, Korpela H,Kaplan GA, Lynch LJ, Helmrich SP,Salonen R. Increase risk of noninsulin dependent diabetes mellitus at low plasma vitamin E concentration; a four year follow up study in men. Br J Med 1995; 311:1124-7.
  19. Ceriello A, Varricchio M D, Onefrio F. Pharmacologic dose of vitamin E improve insulin action in healthy subjects and nonsulin-dependent diabetic patients Am J Clin Nutr 1993;57:650-6.
  20. 20- Mann GV. Hypothesis; the role of vitamin C in diabetic angiopathy. Perspect Biol Med 19974;17: 210-17.
  21. Stankova L, Riddle M, Larned J, et al. plasma ascorbate concentrations and blood cell dehydroascorbate transport in patients with diabetes mellitus. Metabolism 1983; 33: 347-53.
  22. Verlangieri AJ, Sestito J. Effect of insulin on ascorbic acid uptake by heart endothelial cells; possible relationship to retinal atherogensis. Life Sci 1981; 29: 5-9.
  23. Chen S M. Hutchinson L M,Pecoraro E R,Lee L Y W.labbe F R. Hyperglycemia -induced intracellular depletion of ascorbic acid in human Mononuclear Leukocytes. Daibetes 1983; 32:1078-81.
  24. Jacob RA, Skala JH, Omaye ST. Biochemical indices of vitamin C statues. Am J Clin Nutr 1987; 46:818-26.