Emerging Biomarkers of Oxidative Stress and Inflammation as Indicators of Progression to Type 2 Diabetes Mellitus and the Association with Cardiovascular Disease

The recognition by global health authorities that Type 2 diabetes (T2DM) and cardiovascular disease (CVD) is a pandemic health issue has brought about an urgent search for better risk predictors of T2DM and CVD. What has also emerged is that the prediabetic state with elevated glycaemia may go undiagnosed, or is detected incidentally during clinical investigation. A current challenge is to predict the development of T2DM and progression to CVD in these patients. Emerging biomarkers reflecting oxidative stress, inflammatory cytokines, and coagulation and fibrinolytic markers are continuing to provide insight into the pathogenicity of the hyperglycaemic state and its comorbidities. This thesis proposes an investigation into biomarkers (traditional and emerging) related to hyperglycaemia/prediabetes, T2DM and CVD to observe any correlation, concordance or predictive capacity in the hyperglycaemic state. An initial proposal, presented as a hypothesis, was recognising the availability of traditional screening programs for prediction (e.g. Framingham study) of CVD and diabetes (Paper 1). The hypothesis was to present an alternate model of assessment of diabetic macrovascular complication that will discriminate prediabetes and undiagnosed diabetes. Considering the validity of conventional markers the additional emerging biomarkers: glutathione (GSH), malondialdehyde (MDA), D-dimer and homocysteine (Hcy) were originally proposed. Nwose (2007, PhD dissertation) observed that GSH levels with established CVD compared with those that have either established DM or prediabetes was significantly higher (p <0.0001). D-dimer levels were highest in DM patients (p <0.003), but increased in pre-DM with CVD (p <0.007). MDA and Hcy were also significant in the prediabetic state (p =0.05 and p <0.01 respectively). As differing parameters may reflect alternative biochemical processes we recommended that analysis of the emerging biomarkers be conducted on a larger cohort, and a longitudinal study subjected to a binomial logistic regression in order to refit modelling for CVD risk also be carried out. To follow through on emerging biomarkers an investigation into the balance between GSH, glutathione disulphide (GSSG) and 8-hydroxy-2ˊdeoxyguanosine (8-OHdG) and how this was disturbed in patients with impaired fasting glucose was explored (Paper 2). All emerging biomarkers in this and subsequent papers were analysed by enzyme linked immunosorbent assay (ELISA), general biochemistry was conducted at a nationally accredited pathology laboratory and anthropometric data was conducted at the Diabetes Health (DiabHealth) Initiative at Charles Sturt University (CSU). In this study we observed the GSH:GSSG ratio was significantly lower in the impaired fasting glucose group when compared to controls (p =0.04). A pro-oxidant response to mild-moderate hyperglycaemia with a significant rise in oxidative stress was concluded. A further study involved a cross sectional cohort of patients, enrolled at DiabHealth. The cohort was divided with respect to Framingham CVD risk categories. Inflammatory and oxidative stress markers, anthropometric and general biochemical markers were reviewed (Paper 3). Significant correlations were again observed with GSH:GSSG between control and type 2 diabetes (T2DM) (p<0.05), and IL-6 was significantly increased in T2DM compared to the control and pre-diabetic groups (p <0.05). This research supported a postulate proposed that a rebound de novo synthesis of erythrocyte GSH is a response to prolonged hyperglycaemia and that raised Interleukin -6 (IL-6), due to increased reactive oxygen species (ROS), is a result of the up regulation of pro- inflammatory mediators. In paper 4, oxidative stress and inflammatory markers were further explored in increasing blood glucose concentrations with GSH (p <0.001), GSH:GSSG (p <0.05), 8-OHdG (p <0.05) and Interleukin 1β (IL-1β) (p <0.05) providing significant results when 1 st (glucose <4.5mmol/L) and the combined 4th and 5th quintile (glucose >6.1mmol/L) groups were compared. Importantly clients in this study were not discriminated on the basis of medication and results, none-theless, replicated previous findings for screened patients with no comorbidities. This finding was considered important when conducting community based screening as biomarkers need to be informative for all persons being tested and in identifying T2DM and CVD disease progression. Ratios of inflammatory markers were also explored in this study, as a review of the literature revealed no previous investigations had been conducted for their association with hyperglycaemia. IL-1β/IL-10, IL-6/IL-1β and CRP/IL-6 provided significant results (p <0.05), affirming ratios could be included in routine investigations. As paper 4 did not exclude patients on the basis of medication use in the outpatient community, a study on medication use with respect to metabolic syndrome (MetS) (as defined by ATPIII) was investigated (Paper 5). Antidiabetic, antihypertensive and Statin use differed significantly between a MetS and No MetS group defined by ATPIII (p <0.0001). When medication use was separated into anti-diabetic, anti-hypertensive and statins, similar significant differences were found between the Metabolic Syndrome (MetS) and No MetS groups. The focused outpatient community showed medication is relatively well controlled for MetS, however statin use in the MetS group indicated usage may be below that recommended. The investigation in paper 6 further tested the role of homocysteine, which remains a controversial biomarker, for disease progression and associated oxidative stress processes in the categories of no MetS (<3 factors) vs MetS (≥3 factors). A caveat of this study was to include antihypertensive, antihyperlipidaemic and antihyperglycaemic medication, which are not part of the ATPIII definition for MetS. Hcy (p =0.03) and 8-OHdG (p =0.0001) were significantly elevated in the MetS group compared to the no MetS. The conclusion was the significant Hcy result and clustering of Mets factors are likely the result of complex metabolic pathophysiology interactions associated with 8- OHdG and Hcy. Traditional markers have an important place in aiding diagnosis. Increased or decreased creatinine, a traditional renal function marker, has been postulated as a risk factor for T2DM. In paper 7, 102 patients derived from a pathology data base archive was categorised into three groups premised upon glucose tolerance test results i.e. Control (No diabetes), Prediabetes and Diabetes. Glomerular filtration rate (GFR) and serum creatinine results were analysed using ANOVA and post-hoc analysis with no significant difference observed at any level. We concluded that low creatinine should not be used as a T2DM predictive biomarker pending further investigation and clarification on a larger cohort. Peripheral vascular disease (PVD), possibly a first indicator of atherosclerosis was investigated in paper 8. Arterial stenosis and calcified arteries can be generally differentiated according to low or increased ankle brachial pressure index (ABPI) respectively. Oxidative stress and inflammatory markers were measured to observe any differences between low (<1.07) and high (>1.23) ABPI determined as the cut-off for the 1st and 3rd tertile. D-dimer (p <0.001) and GSH (p <0.05) were significant between groups. C-reactive protein (CRP), D-dimer and IL-6 combined with traditional biomarkers BGL, HbA1c and systolic blood pressure were the best model for predicting ABPI class (p<0.05). The results suggest that emerging oxidative stress and inflammatory biomarkers are important contributors in identifying different pathophysiological processes involved in peripheral vascular pathophysiology. Cognitive decline as a consequence of T2DM is recognised, however mild cognitive and inflammation is not conclusive. In paper 9 participants undertook the Stroop testing battery i.e. reading and naming interference tests (RIT and NIT) and inflammatory markers were measured on each patient. CRP was significant for RIT (p = 0.022); IL-1β (p = 0.039), MCP-1 (p <0.01) was significant for NIT. IL-10 (p <0.01); IL-6/IL-10 (p <0.03) were significant in RIT and NIT. This is the first study to demonstrate a connection between inflammatory markers and behavioural data. Exercise is known to reduce comorbidities in T2DM. In paper 10 the effect of endurance testing (ET) and high-intensity interval training (HIIT) on inflammatory cytokines and IGF-1 were tested on young adults. Blood was drawn before the interventions, 30 min and 2 days after the training sessions. Significant results were obtained with IL-6/IL-10 ratio (p =0.047), and a decrease of MCP-1 (p =0.03). HIIT may present a valid alternative to ET which has implications on obesity derived from inactivity on populations where ET is not an option. The research conducted in this thesis has contributed to the expansion of our understanding of the role oxidative stress and inflammatory biomarkers have in the hyperglycaemic state, T2DM and CVD. Further evaluation of emerging biomarkers by increasing participant numbers and providing a longitudinal study is recommended in our efforts to obtain more informative and predictive information in the hyperglycaemic state.