International Journal of Diabetes Mellitus
Volume 2, Issue 2 , Pages 119-121, August 2010

Erythrocyte plasma membrane redox system in first degree relatives of type 2 diabetic patients

Department of Biochemistry, University of Allahabad, Allahabad 211002, India

Received 19 December 2009; received in revised form 2 April 2010; accepted 4 May 2010. published online 14 June 2010.

Article Outline

Abstract 

Diabetes mellitus (TDM) is strongly associated with oxidative stress. Human erythrocytes contain a plasma membrane redox system (PMRS) which transfers electrons from intracellular donors (NADH, ascorbate) to extracellular acceptors outside the cell. We show that the activity of erythrocyte PMRS and AFR reductase becomes elevated in first degree relatives of type 2 diabetics and in TDM subjects. The increase in PMRS and AFR reductase signifies compensatory mechanisms to mitigate increased oxidative stress. These findings show that an impaired redox balance may be a cause the disturbance of homeostasis in type 2 diabetic families, even before the development of the disease.

Keywords: Erythrocyte, Oxidative stress, Diabetes, PMRS, AFR reductase

 

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1. Introduction 

Epidemiological studies on twins and families have provided a strong correlation for genetic factors contributing to the etiology of type 2 diabetes [1]. Diabetes mellitus is strongly associated with oxidative stress, which can be a consequence of either increased production of free radicals, reduced antioxidant defense or both [2]. Despite a large number of studies, it is not clear whether oxidative stress is a factor contributing to the development of diabetic condition or whether it is a consequence of the disease.

In diabetes mellitus, chronic hyperglycemia produces multiple biochemical sequels, and diabetes-induced oxidative stress may play a role in the onset and progression of the disease. Many complications of diabetes, including retinopathy and atherosclerotic vascular disease, the leading cause of mortality in diabetes, have been linked to oxidative stress. A rational extension of the proposed role for oxidative stress is the suggestion that the differing susceptibility of diabetic patients to microvascular and macrovascular complications may be a function of endogenous antioxidant status [2].

Studies show that human erythrocytes contain a plasma membrane redox system (PMRS) which transfers electrons from intracellular donors (NADH and/or ascorbate) to extracellular acceptors outside the cell, although the physiological acceptor is still unclear [3]. There is evidence that the intracellular ascorbate (ASC) donates electrons to extracellular ascorbate free radical (AFR) via the PMRS which incorporates an AFR reductase; such a redox system enables the cells to effectively counteract oxidative processes, thereby preventing the depletion of extracellular ASC [4].

Recent reports prove that the activity of erythrocyte PMRS and AFR reductase increase during human aging, an effect which is negatively correlated with decreased plasma antioxidant potential [5], [6]. In view of the increased oxidative stress in diabetic patients, a condition which is similar to that observed during aging, we have determined erythrocyte PMRS and AFR reductase in first degree relatives of type 2 diabetic patients (Rel-T2D) and type 2 diabetic patients in order evaluate the role of oxidative stress in the development of frank diabetic state.

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2. Materials and methods 

2.1. Selection of subjects 

The study was conducted in normotensive subjects (n=22) of age ranging between 30 and 45 with ⩾1 parent diagnosed with type 2 diabetes (average age 39years). The activity was compared with age and sex matched controls (n=25), (average age 38years) having negative family history of type 2 diabetes. Type 2 diabetic patients of average age 40years (n=21) were also included in the study. The criteria for selecting Rel-T2D and type 2 diabetic patients was the same as described earlier [7], [8]. The fasting blood glucose level of control subjects was between 76 and 90mg/dL, Rel-T2D 79–94mg/dL and T2D 142–198mg/dL. To exclude prediabetic subjects, only those Rel-T2D subjects were selected who had normal oral glucose tolerance test (OGTT) with a 2h post load of glucose <140mg/dL [9]. None of the subjects had high blood pressure or microalbuminuria. Care was also taken to exclude subjects who had a family history of hypertension. All members gave informed consent for use of their blood samples for the study. The protocol for the study was in accordance with guidelines of the institutional ethical committee.

2.2. Collection of blood, isolation of packed RBC and preparation of ghosts 

Venous blood was collected from control and type 2 diabetic patients after an overnight fast, using ACD as anticoagulant. The blood samples were centrifuged at 4°C for 10min at 1000g to remove plasma and buffy coat, and the isolated erythrocytes were washed 4–5 times with 0.154M NaCl and finally packed erythrocyte (PRBC) was obtained.

Erythrocyte trans-plasma membrane redox activity was estimated by following the reduction of ferricyanide, according to the method of Avron and Shavit [10]. A 0.2ml RBC were suspended in PBS containing 5mM glucose and 1mM freshly prepared potassium ferricyanide. The suspensions were incubated for 30min at 37°C and then centrifuged at 1800g at 4°C. The supernatant collected was assayed for ferrocyanide content using 4,7-diphenyl-1,10-phenanthrolinedisulfonic acid disodium salt and measuring absorption at 535nm (ε=20,500M−1cm−1). Results are expressed in μmol ferrocyanide/ml PRBC/30min.

The erythrocyte AFR reductase activity was assayed following the method as described by May et al. [11]. The washed erythrocytes were hemolysed and diluted 100% (v/v) by addition of water followed by centrifuging for 10min in the cold. AFR was generated in diluted hemolysates by incubating them at 37°C in PBS (pH 7.0), containing 1mM ascorbate, 5units/ml ascorbate oxidase, and 0.1mM of NADH. The rate of NADH oxidation was measured spectrophotometrically at 340nm for 3min at 37°C. The change in NADH concentration was calculated from the slope of the resulting line, using an extinction coefficient ε=6.22mM−1cm−1. The values were corrected in each experiment for the rate observed with lysate and reduced nucleotide alone. AFR reductase activity is reported in terms of μmol NADH oxidised/min/ml PRBC.

2.3. Statistical analysis 

Statistical analyses were performed using GraphPad Prism version 4.00 for Windows, GraphPad Software, San Diego, California, USA. The results were reported as means±SD from individual magnitudes. Statistical differences were analyzed with Student’s t-test, and the differences were considered to be significant when P<0.05.

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3. Results and discussion 

There is much evidence that oxidative stress is involved in the etiology of several diabetic complications [12], [13]. Several mechanisms have been proposed to explain the development of diabetic complications: increased polyol pathway, increased advanced glycation end product (AGE) formation, activation of protein kinase C, and increased hexoamine pathway flux. All seem to reflect a single hyperglycemia-induced process of oxidative stress [14].

We present evidence that erythrocyte PMRS activity is increased in Rel-T2D (14.76%) and in type 2 diabetic patients (37.53%) (Fig. 1). The activity of PMRS has been shown to be elevated in erythrocytes from patients with diabetic nephropathy [15] and in lymphocytes from insulin dependent diabetes mellitus patients, a disease in which mitochondrial activity is depressed [16]. A few recent studies have shown that the activity of PMRS is modulated during aging in animals, providing an efficient additional cell protection from oxidative stress [5], [6], [17].

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  • Fig. 1. 

    Erythrocyte PMRS activity in normal (control), first degree relative of type 2 diabetic patients (Rel-T2D), and type 2 diabetic (T2D) patients. aPMRS activity expressed in terms of μmol ferrocyanide/ml PRBC/30min. *P<0.05 compared to normal.

Erythrocytes, being the most abundant cells in the blood, have a crucial role in recycling ASC in blood plasma. Erythrocytes can take up DHA from the plasma through the GLUT1 glucose transporter. Inside the cell, DHA can be recycled to ASC via a direct reduction by glutathione, by glutathione dependent enzymes such as glutaredoxin and protein-disulfide isomerase, and by NADPH-dependent thioredoxin reductase. Since the rate of release of ASC from erythrocytes to plasma is slow, the role of the recycling of ASC in the maintenance of plasma ASC levels assumes importance.

Fig. 2 shows an increase in the activity of erythrocyte AFR reductase in Rel-T2D (23.16%) and T2DM (38.34%). The AFR reductase driven reduction of extracellular ascorbate free radical has been shown to be an electrogenic process, indicating that the vectorial electron transport is involved in the reduction of extracellular ascorbate free radical [18]. There is evidence for a decrease in the free radical scavenging capacity of plasma in diabetics, concomitant with a significant decrease of plasma vitamin C and an increase of AFR/vitamin C [19]. Our observation of an increase in the activity of AFR reductase during T2DM may thus provide increased recycling of ascorbate between erythrocyte and plasma and thus help to maintain the antioxidant potential of the plasma during increased oxidative stress which is encountered during diabetes. The increased activity of erythrocyte AFR reductase in Rel-T2D is an interesting aspect of the current study, and suggests that an underlying oxidative stress condition is present in Rel2D subjects even before the onset of frank diabetic state.

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  • Fig. 2. 

    Erythrocyte AFR reductase activity in normal (control), first degree relatives of type 2 diabetic patients (Rel-T2D), and type 2 diabetic (T2D) patients. aAFR reductase activity expressed in terms of μmol NADH oxidised/min/ml PRBC. *P<0.05 compared to normal.

In conclusion, we present evidence that erythrocyte PMRS and AFR reductase are elevated in Rel-T2D and T2DM. The increase in PMRS and AFR reductase signify compensatory mechanisms to mitigate increased oxidative stress. These findings show that an impaired redox balance may be a cause for disturbance of homeostasis in type 2 diabetic families even before the development of the disease.

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Conflict of interest 

None declared.

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References 

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PII: S1877-5934(10)00027-5

doi:10.1016/j.ijdm.2010.05.005

International Journal of Diabetes Mellitus
Volume 2, Issue 2 , Pages 119-121, August 2010

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