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Annals of Clinical & Laboratory Science 36:174-178 (2006)
© 2006 Association of Clinical Scientists

Effects of Diabetes Duration and Glycemic Control on Free Radicals in Children with Type 1 Diabetes Mellitus

Wan Ting Hsu*, Li Yu Tsai*, Shu Kai Lin, Jen Kuei Hsiao and Bai Hsiun Chen
Department of Laboratory Medicine, Faculty of Medicine, Kaohsiung Medical University, and Chung-Ho Memorial Hospital, Kaohsiung, Taiwan

Address correspondence to Bai Hsiun Chen, M.D., Department of Laboratory Medicine, Kaohsiung Medical University, Chung-Ho Memorial Hospital, No. 100, Shih Chuan 1st Road, Kaohsiung 807, Taiwan; tel 886 7 312 1101 ext 7233; fax 886 7 311 4449; e-mail: chen_bh.tw{at}yahoo.com.tw.


   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Results
 Discussion
 References
 
Parameters of lipid peroxidation, protein oxidation, and antioxidant defense systems were measured in blood samples from 47 children with type 1 diabetes mellitus and from 51 healthy controls, matched for age and sex. In the diabetic children, chemiluminescent assay of plasma superoxide anion gave photoemission (counts x 103, mean ± SD) of 674 ± 412, which were significantly higher than those in the controls (452 ± 185; p <0.05). Plasma vitamin A levels in the diabetic children (243 ± 90 µg/dl) were also higher than those in the controls (207 ± 59 µg/dl, p <0.05). In a subgroup of 24 diabetic children with blood HbA1C levels ≥8.5%, plasma lipoperoxide (LPO) and vitamin E levels were higher (p <0.05) than those in 23 diabetic children with blood HbA1C levels <8.5%. In a subgroup of 26 children with diabetes duration ≥5 yr, plasma LPO levels were higher (p <0.05) than those in 21 children with diabetes duration <5 yr. These findings confirm the presence of oxidant stress in children with type 1 diabetes mellitus and demonstrate that certain indices of oxidant stress are influenced by the duration of diabetes and by the efficacy of glycemic control. These observations suggest that supportive therapy aimed at oxidative stress may help to prevent clinical complications in children with type 1 diabetes mellitus.

Keywords: diabetes mellitus type 1, free radicals, superoxide anion, lipoperoxides, vitamins A and E


   Introduction
Top
 Abstract
 Introduction
Material and Methods
 Results
 Discussion
 References
 
Oxygen-derived free radicals are generated in aerobic organisms during physiological or pathophysiological oxidative metabolism in mitochondria. Free radicals may react with a variety of biomolecules, including lipids, carbohydrates, proteins, nucleic acids, and macromolecules of connective tissue, thereby interfering with cellular functions. Under normal physiological conditions, there is a critical balance between the generation of oxygen free radicals and the antioxidant defense systems used by organisms to deactivate and protect themselves against free radical toxicity [1].

Impairment in the oxidant/antioxidant equilibrium in favor of the former provokes oxidative stress and generally results from hyperproduction of reactive oxygen species. Oxidative stress is responsible for molecular and cellular tissue damage in a wide spectrum of human diseases [2]. Diabetic patients are exposed to increased oxidative stress due to several mechanisms, including glucose auto-oxidation and nonenzymatic protein glycation [3,4]. Nonenzymatic glycation is a spontaneous chemical reaction between glucose and the amino groups of proteins [5].

Ceriello et al [6] reported a significant increase in serum superoxide radical production in type 1 diabetes mellitus, but the study was limited to adults and involved groups of only 10 diabetic patients and controls. Elevated levels of serum thiobarbituric acid-reactive substances (TBARS), which are an indirect index of lipid peroxidation and decreased erythrocyte antioxidant activities, have been found in adult diabetic patients [7–9].

Nerup et al [10] and Rabinovitch et al [11 showed that the pathogenesis of diabetes mellitus may involve increased cytokine secretion, impaired antioxidation, and free radical damage in pancreatic beta cells. Indirect evidence and in vitro experiments support the existence of free radicals at the onset of diabetes. Yoon et al [12] reported that various chemicals may induce diabetes mellitus because of inbalance of oxidized free radicals and endogenous free radical scavengers, resulting in destruction of pancreatic beta cells.

Guzel and Seven [13] reported that plasma TBARS and lipid peroxide levels were significantly higher in long term patients (>5 yr) with type 1 diabetes than those of normal and recent onset groups (<2 months). Numerous other reports have shown relationships between type 1 diabetes mellitus and indices of oxidative stress [14–22].

The purposes of this study were as follows: (i) to document the nature and extent of oxidative damage in 47 Chinese children with type 1 diabetes mellitus, compared to 51 control subjects, (ii) to assess the oxidant/antioxidant balance in the patient group in relation to other metabolic control parameters, and (iii) to evaluate the influence of diabetes duration and glycemic control (HbA1C) on the free-radical indices in diabetic patients. This study included 10 indicative parameters, including superoxide anion (SOA), lipid peroxide (LPO), enzymatic antioxidants (ie, superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione reductase (GRx)), oxidized glutathione (GSSG), and free radical scavengers (ie, glutathione (GSH), vitamin C, vitamin A and vitamin E).


   Material and Methods
Top
 Abstract
 Introduction
 Material and Methods
Results
 Discussion
 References
 
This study was approved by the Ethics Committee of Kaohsiung Medical University, and informed consent was obtained from all participants. The patient population comprised 47 unrelated Chinese children with type 1 diabetes mellitus who were receiving insulin therapy at Kaohsiung Medical University’s Chung-Ho Memorial Hospital. Fifty-one healthy age- and sex-matched children served as controls. Blood samples from each patient and control subject were drawn into an EDTA tube and a heparin tube. The blood samples were processed within 1 hr of collection.

Vitamin C.  Plasma vitamin C was determined by the method of Lee et al [23]. Plasma (200 µl) was added to a microcentrifuge tube that contained 50 µl of 25 mmol/L dithiothreitol (DTT). Proteins were precipitated by adding 100 µl of 12% trichloroacetic acid. A P/ACE-MDQ capillary electrophoresis system (Beckman Coulter, Brea, CA, USA) was used to measure vitamin C concentration in the protein-free extract.

Vitamin A.  Plasma vitamin A was measured by the fluorometric method of Hansen and Warwick [24]. Vitamin A was extracted from plasma by addition of 5 ml of hexane and vortexing for 1 min. After centrifugation at 2500 rpm for 10 min at 4°C, the supernatant was placed in a quartz cuvette to measure vitamin A using a Hitachi spectrofluorimeter.

Vitamin E.  Plasma vitamin E was determined by a modified flurometric method [24,25]. The excitation wavelength was 295 nm, and the emission wavelength was 340 nm.

Glutathione peroxidase (GPx) and glutathione reductase (GRx).  Erythrocyte GPx and GRx activities were measured by commercial Ransel kits (Randox Laboratories, UK) based on the report of Paglia and Valentine [26]. In the presence of glutathione reductase (GRx) and NADPH, GSSG is converted to the reduced form (GSH) with concomitant oxidation of NADPH to NADP+.

Glutathione (GSH).  Erythrocyte GSH and GSSH were determined by capillary electrophoresis, as described by Serru et al [27] and Ciriaco et al [28].

Superoxide dismutase (SOD).  Erythrocyte SOD activity was estimated by a commercial Ransod kit (Randox Laboratories). This spectrophotometric method [29] is based on the generation of O2 by xanthine and xanthine oxidase.

Superoxide anion (SOA).  Plasma SOA was measured by lucigenin-based chemiluminescence (LBCL) as described by Tsai et al [30]. Photo-emission was counted at 37°C for 10 min under atmospheric conditions.

Lipid peroxide (LPO).  Plasma LPO was determined by the spectrophotometric method of Berge and Aust [31].

Statistics.  Data were expressed as mean ± SD and analyzed by unpaired t test (p <0.05 = statistical significance).


   Results
Top
 Abstract
 Introduction
 Material and Methods
 Results
Discussion
 References
 
The clinical characteristics of the patients with type 1 diabetes mellitus and control subjects are shown in Table 1Go. There were no significant differences in age or gender of the diabetic patients vs controls.


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  		Table 1. Clinical characteristics of type 1 diabetes mellitus patients and control subjects (mean ± SD).
 
Indices of free radical status in the patients with type 1 diabetes mellitus and controls are shown in Table 2Go. The plasma superoxide anion level (counts x 103, mean ± SD) was 674 ± 412 in the diabetic children, which was significantly higher than the controls (452 ± 185, p <0.05). The plasma vitamin A level of 243 ± 90 µg/ml in the diabetic children was significantly higher than those of the controls of (207 ± 58.9 µg/ml, p <0.05). The other indices of free radical status revealed no significant differences between the diabetic children and controls.


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  		Table 2. Comparison of free radical indices between patients with type 1 diabetes mellitus and controls.
 
Table 3Go compares the indices of free radical status between 24 diabetic children with poor glycemic control (blood HbA1C levels ≥8.5%) and 23 diabetic children with better glycemic control (HbA1C levels <8.5%). This table reveals that plasma LPO levels in the HbA1C ≥8.5% group were significantly higher (p < 0.05) than those of the HbA1C <8.5% group. The HbA1C ≥8.5% group also had higher values of plasma vitamin E than those of the HbA1C <8.5% group (p < 0.05).


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  		Table 3. Comparison of free radical values between diabetic patients with HbA1C ≥8.5% and HbA1C <8.5%.
 
Table 4Go compares the indices of free radical status in the 26 diabetic children with duration of diabetes ≥5 yr vs the 21 diabetic children with duration of diabetes <5 yr. This table reveals that plasma LPO levels (3.67 ± 1.75 µM) in the patients with diabetes duration ≥5 yr were significantly higher than those of patients with diabetes duration <5 yr (2.40 ± 1.16 uM, p <0.05).


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  		Table 4. Comparison of free radical values between patients with diabetes duration > 5 years and < 5 years.
 

   Discussion
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
References
 
In this study, there were no significant differences of erythrocyte SOD, GRx, or GPx activities between the diabetic patients and the control subjects. The studies of Jos et al [32] in young patients with type 1 diabetes mellitus agree with our observations. Jain and McVie [22] and Murakami et al [33] also found no difference of GPx activity between type 1 diabetic patients and control subjects. Dominguez et al [19] found that type 1 diabetes mellitus patients had significantly higher erythrocyte SOD and lower erythrocyte glutathione peroxidase activities than did the normal group. They attributed this discrepancy to disruption of the functional relationship between the two enzymes, which might be harmful to cells. Dominguez et al [19] also noted a significant diminution of blood glutathione content at the recent onset of diabetes.

Our observations revealed no significant difference in vitamin E levels in type 1 diabetic patients vs controls. Likewise, Dominguez et al [19] reported no significant difference of vitamin E levels in young patients with type 1 diabetes mellitus vs controls. Other investigations in diabetic patients have led to discordant results. Unchanged, elevated, or decreased plasma vitamin E concentrations have been reported, independent of the type of diabetes [15,33–35]. The discrepancies may possibly be explained by the fact that circulating lipid levels were not always considered when interpreting plasma vitamin E concentrations.

In this study, no significant difference in vitamin C levels was found between the diabetic patients and the control group. Asayama et al [14] reported elevated vitamin C levels in type 1 diabetic subjects, while others [36–37] found lower values in diabetic patients than controls.

In this study, plasma vitamin A levels of diabetic patients were significantly higher than those of the controls. Dominquez et al [19] reported no significant difference of vitamin A levels between type 1 diabetes mellitus patients and controls. Vitamin A, a lipid-soluble and chain-breaking anti-oxidant, is an effective quencher of singlet oxygen and can inhibit lipid peroxidation, exhibiting effective radical trapping antioxidant behavior only at low physiological oxygen pressure [38].

Our study revealed that type 1 diabetic children had significantly higher plasma superoxide anion levels (p < 0.05) compared to controls. Ceriello et al [6] found similar results in serum specimens from type 1 diabetic patients.

The measurement of free radicals is difficult because of their high reactivities, short half-lives, and low concentrations in body fluids. Therefore, indirect markers are commonly used to evaluate secondary products of lipid peroxidation, such as plasma TBARS and hydroperoxide. In our study, there was no significant difference of LPO values between diabetic patients and control subjects. Greimacher et al [7] and Ruiz et al [39] found increased plasma TBARS levels in type 1 diabetic patients. Our data showed significantly higher LPO values in diabetic patients with disease duration ≥5 yr vs those with duration <5 yr. Ruiz et al [39] reported that plasma TBARS concentrations were higher in diabetic patients with vascular complications. In our studies, there were significantly higher plasma LPO values in patients with poor glycemic control (HbA1C ≥8.5%) vs those with HbA1C <8.5%. Grismacher et al [7] observed that diabetic patients with good glycemic control (HbA1C <6.5%) had lower plasma TBARS concentrations than those with poor glycemic control (HbA1C >8.5%).

The results of the present study and previous reports provide ample evidence that oxidant stress is present in patients with type 1 diabetes mellitus. Moreover, certain indices of oxidant stress are influenced by the duration of diabetes and the efficacy of glycemic control. These observations suggest that supportive therapy aimed at oxidative stress may help to prevent clinical complications in children with type 1 diabetes mellitus.


   Footnotes
 
* These authors contributed equally to this study. Back


   References
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 References
 

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