A free radical is any species with one or more unpaired electrons in its outer orbital, and is thus electronically unstable and tends to react with other neighbouring species to achieve a more thermodynamically stable configuration with no net spin. These radicals and other non-radicals that are themselves easily converted to free radicals are collectively termed the reactive species (Evans et al., 2002).
The molecular oxygen (O2) itself is a less reactive bi-radical with two unpaired electrons located in a parallel spin at a different π orbital. Such configuration confers a spin restriction on the reacting molecules (Yu, 1994) and eventually creates a one-electron reduction transfer that leads to the generation of ROS, notably the superoxide (O2•-), hydrogen peroxide (H2O2) and the hydroxyl radicals (•OH) (Figure 1).
2. Generation of Free Radicals in Diabetes Mellitus
In a hyperglycaemic state, excess electron donors (NADH and FADH2) will be generated from the TCA cycle during the catabolism of glucose and lead to a high mitochondrial membrane potential, ∆μH+ by pumping H+ across the inner mitochondrial membrane. However, beyond a certain threshold value of ∆μH+, electron transport to complex III will be inhibited and electron is instead transferred to O2 for reduction to O2•- (Korshunov et al., 1997). The overexpression of uncoupling protein-1 (UCP-1) that dissipates ∆μH+ has been shown to prevent hyperglycaemia-induced overproduction of ROS (Nishikawa et al., 2000). Similarly, elevated FFAs has also been noted to induce uncoupling of oxidative phosphorylation that leads to the generation of more mitochondrial O2•- (Bakker et al., 2000).
The overproduction of mitochondrial O2•- is proposed to be the unifying source that diverts the propagation of oxidative stress in diabetes mellitus into various pathways of glucose overutilisation as described after forth, and hence more free radical production (Figure 2).
Figure 2: Alternative pathways of glucose overutilisation in diabetes mellitus that lead to the generation of ROS.
2.3 Formation of AGEs
Surplus production of glyoxal, methylglyoxal and 3-deoxyglucosone from the auto-oxidation of glucose and the fragmentation of glyceraldehyde-3-phosphate (G3P) has been shown to react non-enzymatically with the amino groups of proteins to form cross-links and adducts called AGEs (Brownlee, 1992). The production of AGEs damages target cells by inducing receptor-mediated generation of ROS upon binding with RAGEs and leads to the activation of the nuclear transcription factor, NF-ĸB that mediates pathological changes in diabetic complications (Yan et al., 2003).
2.4 Activation of PKC
Accumulation of G3P due to the inhibition of glyceraldehye-3-phosphate dehydrogenase (GADPH) by mitochondrial O2•- is shown to stimulate the de novo synthesis of diacylglycerol from dihydroxyacetone phosphate, through reduction of the latter to glycerol-3-phosphate and stepwise acylation (Wolf et al., 1991). The diacylglycerol thus formed will activate PKC isoforms that stimulate NAD(P)H-dependent oxidases and lead eventually to the generation of more ROS (Inoguchi et al., 2003).
3. Pathophysiology of Diabetes and Diabetic Complications
There is considerable evidence that the ROS generated during diabetes may activate stress-sensitive signalling pathways that lead to the pathogenesis and pathophysiology of diabetes and diabetic complications. In fact, ROS-mediated insulin resistance, endothelial dysfunctions and β-cell destruction have been reported with diabetic subjects and experimental animals (Evans et al., 2002).
3.1 Free Radicals and Insulin Resistance
Oxidative stress has been shown to activate multiple serine kinase cascades that target both the insulin receptor (IR) and the insulin receptor substrate (IRS) family of proteins. Increased phosphorylation of IR and IRS on discrete serine or threonine residues decreases the extent of insulin-stimulated tyrosine phosphorylation and leads eventually to the impairment of insulin action (Paz et al., 1997). Furthermore, the serine/threonine phosphorylated forms of IRS are less able to associate with IR and downstream target molecules, and result overall, in insulin resistance (Birnbaum, 2001).
3.2 Free Radicals and Endothelial Dysfunctions
Superoxide overproduction has been shown to stimulate the expression of inducible nitric oxide synthase that leads to an overall surplus production of nitric oxide. This in turn, will be quenched by O2•- to form the stronger oxidant, peroxynitrite that is capable of initiating DNA single-strand breakage. DNA damage on the other hand, is an obligatory stimulus for the activation of poly (ADP-ribose) polymerase that results in a cascade mechanism leading to acute endothelial dysfunctions (Ceriello, 2003).
3.3 Free Radicals and β-cell Destruction
Autoimmune destruction of pancreatic β-cells typical of type 1 diabetes mellitus is greatly contributed by ROS and other proinflammatory cytokines such as
, 
, whose synergistic interaction results in the ultimate apoptosis and necroticism of pancreatic cells (Hoorens et al., 2001). This is due to the fact that β-cells contain very low free radical-scavenging enzymes that render them more vulnerable to ROS-mediated oxidative damage (Tiedge et al., 1997). On the other hand, transgenic mice with β-cell-targeted overexpression of Cu/Zn-superoxide dismutase, are resistant to alloxan-induced diabetes, thus providing direct in vivo evidence that ROS metabolism can affect susceptibility to oxidative stress-mediated diabetogenesis (Kubisch et al., 1997).4. Biomarkers of Oxidative Stress
Direct measurement of ROS as biomarkers of oxidative stress is made complicated due to their high reactivity and short half-lives (Jakus, 2000). Nevertheless, the low chemical specificity of ROS that renders them highly damaging to cellular macromolecules, is worth an indirect assessment of oxidative stress. This method evaluates not only levels of damaged biological products, but also reflects an overall antioxidant status of a system.
4.1 Lipid peroxidation
4.2 Oxidation of proteins
Primary products of protein oxidation from the direct reaction of proteins with ROS often leads to accompanying changes in the structures of amino acids such as o-tyrosine, nitrotyrosine, methionine sulfoxide and oxohistidine. Secondary oxidative damage of proteins in contrast, results from the reaction of proteins with reactive carbonyl compounds that arise from the oxidation products of lipids, carbohydrates and proteins. MDA adducts of lysine, carboxymethyllysine and pentosidine are typical examples of such damage (Baynes and Thorpe, 1999).
4.3 Oxidation of nucleic acids
Attack of different reactive species on DNA may be distinguished from the pattern of damage inflicted to the bases. For instance, O2•- and H2O2 are quite latent towards DNA bases while •OH produces a multiplicity of products from all the four bases. In fact, guanine is the most oxidisable base in the DNA and an increase in the level of its oxidation product, 8-hydroxy-2’-deoxyguanosine in the urine of diabetic patients is an indication of oxidative damage (Leinonen et al., 1997).
5. Antioxidant networks
Antioxidants entail their protective mechanisms against oxidative damage by scavenging free radicals or interrupting radical chain reaction. Their protective roles involve a series of network of antioxidants that are strategically compartmentalised in subcellular organelles within the cell to provide maximum protection. For instance, both the Mn- and Cu/Zn-superoxide dismutase remove O2•- by catalysing a dismutation reaction to H2O2 in the mitochondria and cytoplasm, respectively. The reactive H2O2 will be further removed by catalase in the peroxisome or coupled to the oxidization of GSH into GSSG by the mitochondrial and cytosolic glutathione peroxidase (Yu, 1994).
However, the antioxidant defence system is often breached in a diseased state and rapid decline from the baseline level has been noted in animal models with diabetic history compared to normal rats (West, 2000). Nevertheless vitamin E supplementation has been shown to protect LDL against oxidation, which is a critical step in the development of atherosclerosis in late diabetes mellitus. In addition, this vitamin has also been experimentally indicated to improve significantly diabetes-induced abnormal contractility and endothelial dysfunction in diabetes mellitus (Karasu et al., 1997). Vitamin C on the other hand, scavenges ROS in the interstitial fluids and bloodstream and acts to regenerate vitamin E and to increase cellular GSH (Stall and Sies, 1997).
Alpha-lipoic acid in contrast, exists in both the oxidised and reduced forms and acts as an antioxidant in both the membrane and aqueous phase. It establishes cellular antioxidant network by raising intracellular GSH levels and regenerating vitamins E and C. Apart from that, it has also been shown to prevent diabetic retinopathy, alleviates cataract and inhibits aldose reductase activity (Nawroth et al., 2000). In fact, the supplementation of antioxidants and phytochemicals from dietary sources may help to alleviate and delay diabetes induced-oxidative stress.
6. References
Brownlee, M. 2001. Biochemistry and molecular cell biology of diabetic complications. Nature 414: 813-820.
Sitasawad, S.L., Shewade, Y. and Bhonde, R. 2000. Role of bitter gourd fruit juice in STZ-induced diabetic state in vivo and in vitro. Journal of Ethnopharmacology 73: 71-79.
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