Diabetes is one of the fastest growing diseases today, affecting over a million people in the UK. Numerous medical complications, such as heart disease, are regularly associated with diabetes. Despite advances in methods of diagnosis and treatment there is still a need for greater understanding of these diseases. This will include research directed towards the influence of specific treatments and reasons for the high incidence of diabetes and heart disease in 'at risk' populations. Changes in elemental status are associated as the cause or effect of various diseased states. Elemental imbalance in diabetics can result in impaired glucose tolerance and insulin resistance and in sufferers of heart disease elemental changes impair heart rate and elasticity of blood vessels. In the UK 10,000 patients with Ischaemic Heart Disease undergo coronary artery bypass grafting (CABG) surgery each year. Elemental analysis has been carried out on blood samples collected from a group of patients admitted to hospital for bypass surgery. Proton Induced X-ray Emission (PIXE) and Instrumental Neutron Activation Analysis (INAA) have been applied as complementary analytical tools for determining elemental concentrations. Differences have been examined between CABG patients with and without diabetes. Both experimental methods have been used to investigate elemental levels in whole blood, erythrocytes and plasma. Elemental concentration varied according to the blood constituent and reflected short and long-term influences on elemental homeostasis. Plasma was found to concentrate Na, Mg and Ca the highest using both experimental techniques. All blood samples were collected and prepared at St. George's Hospital, Tooting in the UK. An additional study was conducted to investigate the influence of the bypass operation on the patient's elemental status. Whole blood was obtained at pre (1h before operation), post (1-2 hours after operation) and recovery (24 hours after completion of the operation) stages of bypass surgery. Differences between the three phases were observed, individual variations have been plotted so rates of change can be seen and evaluated with the particular medical history. Concentrations of Na, Mg, Al, P, S, Cl, K, Ca and Fe in whole blood were determined. The two measurement techniques found different concentrations however results showed a general trend that post operative concentrations were elevated compared to pre operative values. Analysis of blood drawn during the recovery phase, 24 hours after the surgery, found that concentration were typically approaching pre operative levels. Both PIXE and INAA found concentrations of Na, Mg and Al peaked post operation and then decreased in the recovery phase, towards values measured pre surgery. Various factors may be responsible for the elemental changes occurring during surgery including, hormone production, routine administration of intra-operative fluids and contact of blood with non- endothelial surfaces. Hierarchical cluster analysis has been used to confirm differences between elemental levels in pre, post and recovery stages of bypass surgery. The dendograms produced indicate significant distinction between the three stages. The explosive impact of diabetes in the UK resident Asian population is discussed and the influence of diabetogenic agents introduced. Examination of research literature revealed that betel nut has been implicated as a causative agent in several medical conditions. Samples of Betel nut and six associated chewing materials widely used in Asian communities has been collected and prepared for analysis. Instrumental neutron activation analysis has been used to determine the concentration of Na, Mg, Al, Cl, Ca, V, Mn, Cu and Br in the samples by means of short-lived radionuclides.

A research report submitted to the Faculty of Health Sciences in fulfilment of the requirements for the degree of Master of Medicine, in Internal Medicine at the University of Witwatersrand, Johannesburg. September 2017 / Differentiating ischaemic left ventricular dysfunction (ILVD) from non-ischaemic left ventricular dysfunction (NILVD) is crucial since appropriately selected patients may benefit from coronary revascularisation. The aim of this study was to evaluate the diagnostic utility of myocardial perfusion imaging (MPI) in patients presenting with left ventricular dysfunction using coronary angiography (CA) as the gold standard. Methods This single centre retrospective study was conducted in 52 patients with heart failure with a reduced ejection fraction (EF< 40%) who had both MPI as well as CA at CHBAH between January 2005 and December 2012. ILVD was diagnosed when the distribution and severity of coronary disease on CA was sufficient to account for the degree of left ventricular dysfunction. Results From a total of 52 patients, 33 (63%) had ILVD and 19 (37%) had NILVD. As compared to patients with NILVD, those with ILVD were more likely to be Indian and White (p=0.0014), have more coronary risk factors (5(2) vs 3(2), p < 0.0001) and more commonly have q waves on the ECG (0% vs 55%, p < 0.0001). MPI had a sensitivity of 100% (95% CI 66-100%) and specificity of 52.63% (95% CI 30.18 - 75.08) for the diagnosis of ILVD. The presence of fixed perfusion defects on MPI was the best predictor of ILVD. Conclusion MPI has high sensitivity but low specificity for the diagnosis of ILVD. This makes it a useful screening test for the exclusion of coronary artery disease in patients presenting with heart failure. / MT2018

Ischaemia reperfusion injury (IR-injury) occurs when blood supply to a certain area of the body is blocked, and is subsequently followed by reperfusion. During the period of ischaemia, tissue is damaged as a result of lack of oxygen. Rapid reperfusion is mandatory, but unfortunately causes damage in addition to the damage induced by ischaemia alone. While a prolonged period of ischaemia is harmful to the bodily tissue, short periods of ischaemia interspersed with short bouts of reperfusion have protective effects. This mechanism is called ischaemic preconditioning (IPC). In this thesis, the impact of co-morbidity and age on IR-injury and IPC are explored. Moreover, the possible role of IPC to enhance exercise performance is investigated. Finally an attempt is made to understand the interchangeable effects of IPC and exercise performance in the prevention of IR-injury. Using the brachial artery endothelial function as a surrogate marker, first the consequences of IR-injury in both young and older individuals on endothelial function were studied. It was also assessed whether IPC could prevent endothelial IR-injury. It was found that endothelial function in both groups declined, when IR-injury was not preceded with IPC. However, when IPC was applied prior to IR-injury, a protective effect was detected in young subjects, but not in older participants. In chapter 5, this study was repeated in patients with heart failure, as they are at an increased risk for IR-injury. While in both groups a significant decline in endothelial function was observed, a much larger decline was established in the heart failure group. Moreover, IPC failed to protect against endothelial dysfunction in heart failure patients after IR-injury. The third study presented in this thesis, focused on the question whether exercise performance enhancement during a 5-km time trial was comparable when IPC on the upper legs was applied immediately before the time trial versus 24 hours (24-IPC) prior to exercise. Interestingly, a significant and strong correlation was found in finish time between acute IPC and 24-IPC, suggesting comparable effects of IPC and 24-IPC on exercise performance. In a follow-up study, it was determined whether local IPC applied on the upper arm, or remote IPC applied on the legs, would lead to an improved maximum incremental arm crank exercise test in individuals with a complete spinal cord lesion. The main finding was that upper arm IPC led to an increased performance enhancement, whilst remote IPC (stimulus below the lesion) did not lead to any significant differences. These studies help to inform the best or most practical application of IPC in daily life situations. Some previous work has suggested that exercise may resemble some of the effects of IPC. More specifically, acute exercise might possess the same protective effects against ischaemia-reperfusion injury as IPC. Therefore, in young healthy individuals it was studied, whether an acute bout of endurance or interval exercise is able to protect against brachial endothelial IR-injury. It was established that interval exercise prevented endothelial dysfunction after an IR stimulus, while no protective effect of endurance exercise was found. It was concluded that interval exercise, but not endurance exercise, prevented endothelial dysfunction after an ischaemic period. In conclusion, this thesis provides further evidence for the protective effects of (remote) IPC, both on the prevention of endothelial IR-injury as well as improvement in exercise performance. However, effects may depend on the protocol and population studied.

Objective: The aim was to determine if in vitro ischaemic preincubation can precondition cardiomyocytes and if the responses to adenosine receptor antagonists are similar to those previously determined during 'metabolic' preconditioning with glucose deprivation or adenosine agonists. Methods: Isolated rabbit cardiomyocytes were preconditioned with 10 min of ischaemic preincubation, followed by a 30 min postincubation before the final sustained ischaemic period. The protein kinase C inhibitor calphostin C or the adenosine receptor antagonists 8-sulphophenyltheophylline (SPT), BW 1433U, and 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) were added either during the preincubation or into the final ischaemic pellet. Adenosine deaminase (10 U·ml-1) was added during ischaemic preincubation. Rates of contracture and extent of injury were determined by sequential sampling and assessment of trypan blue permeability following 85 mOsM swelling. Results: Myocytes were preconditioned by a 10 min in vitro ischaemic preincubation. Preincubation with 100 μM SPT or with adenosine deaminase, or addition of 200 nM calphostin C into the final ischaemic pellet did not alter rates of rigor contracture but nearly abolished protection. A significant degree of protection was maintained following ischaemic preincubation with the highly selective adenosine A1 receptor blocker DPCPX (10 μM), while the A1/A3 antagonist BW 1433U (1 μM) severely limited protection. SPT and BW 1433U added only into the final ischaemic pellet of preconditioned cells significantly blocked protection, while protection was maintained in the presence of DPCPX. Conclusions: Ischaemic preconditioning of cardiomyocytes is blocked by adenosine receptor antagonists known to bind to A3 receptors but not by DPCPX which has high affinity for A1 receptors, but little affinity for A3 receptors. Maintenance of protection during the final ischaemic phase has a similar receptor specificity. Blockade of protein kinase C activity abolishes protection. Ischaemic and metabolic preconditioning in vitro appear to occur through similar pathways.

Objective: The aim was to determine if in vitro ischaemic preincubation can precondition cardiomyocytes and if the responses to adenosine receptor antagonists are similar to those previously determined during "metabolic" preconditioning with glucose deprivation or adenosine agonists. Methods: Isolated rabbit cardiomyocytes were preconditioned with 10 min of ischaemic preincubation, followed by a 30 min postincubation before the final sustained ischaemic period. The protein kinase C inhibitor calphostin C or the adenosine receptor antagonists 8-sulphophenyltheophylline (SPT), BW 1433U, and 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) were added either during the preincubation or into the final ischaemic pellet. Adenosine deaminase (10 U · ml-1) was added during ischaemic preincubation. Rates of contracture and extent of injury were determined by sequential sampling and assessment of trypan blue permeability following 85 mOsM swelling. Results: Myocytes were preconditioned by a 10 min in vitro ischaemic preincubation. Preincubation with 100 μM SPT or with adenosine deaminase, or addition of 200 nM calphostin C into the final ischaemic pellet did not alter rates of rigor contracture but nearly abolished protection. A significant degree of protection was maintained following ischaemic preincubation with the highly selective adenosine A1 receptor blocker DPCPX (10 μM), while the antagonist BW 1433U (1 μM) severely limited protection. SPT and BW 1433U added only into the final ischaemic pellet of preconditioned cells significantly blocked protection, while protection was maintained in the presence of DPCPX. Conclusions: Ischaemic preconditioning of cardiomyocytes is blocked by adenosine receptor antagonists known to bind to A3 receptors but not by DPCPX which has high affinity for A1 receptors, but little affinity for A3 receptors. Maintenance of protection during the final ischaemic phase has a similar receptor specificity. Blockade of protein kinase C activity abolishes protection. Ischaemic and metabolic preconditioning in vitro appear to occur through similar pathways.

Objective: The aim was to further characterise an experimental model of preconditioning of isolated rabbit cardiomyocytes and to determine the role of adenosine receptor subtypes in initiation of the protective response. Methods: Isolated myocytes were subjected to 5 min preincubation in the presence or absence of glucose and various agonists and antagonists of adenosine receptors. Ischaemic pelleting was preceded by a 30 min postincubation period. Rate and extent of injury during ischaemia was determined by sequential sampling of the pelleted cells and assessment of trypan blue permeability following 85 mOsm swelling. Results: Myocytes were preconditioned with a 30-50% reduction of injury by a 5 min glucose-free preincubation. Substitution of 5 mM pyruvate for glucose during preincubation did not prevent the protective response. Protection was maintained over a 60-180 min postincubation period. Protection was blocked by 100 μM of the non-specific adenosine A1A2, antagonist SPT, both when added only during preincubation or only into the ischaemic pellet. Calphostin C, a specific protein kinase C inhibitor at 200 nM, added to the ischaemic pellet blocked protection. Preincubation with R-PIA, the adenosine A1 agonist, did not precondition at an A1 selective dose of 1 μM, but did at 100 μM. The selective A2 agonist CGS 12680 (1 μM) did not precondition. The selective A1/A3 adenosine agonist, APNEA, preconditioned at 1 μM and 200 nM dose levels. Preconditioning induced either by 200 nM APNEA or by glucose-free preincubation was not blocked by 200 nM or 10 μM of the A1 antagonist DPCPX, which has extremely low affinity for A3 receptors, but was blocked by 1 μM of the A1/A3 adenosine antagonist BW 1433U83. Conclusions: Preconditioning can be induced in isolated myocytes by a 5 min preincubation/30 min postincubation protocol, and a similar protection induced by adenosine agonists with A3, but not A1 selectivity. Preconditioning is blocked by non-selective or selective A1/A3 adenosine antagonists and a specific protein kinase C inhibitor, but not by A1 antagonists with little affinity for A3 receptors. The results suggest that preconditioning in isolated rabbit myocytes requires participation of adenosine receptors with agonist/antagonist binding characteristics of the A3 subtype, and is likely to be mediated by activation of protein kinase C.Cardiovascular Research 1994;28:1049-1056.

Objective: The aim was to discriminate among several hypotheses of preconditioning of isolated rabbit cardiomyocytes and to determine if ischaemic preincubation would evoke a protective response. Methods: Isolated myocytes were subjected to 5 min of preincubation, in the presence or absence of glucose, and incubated in the presence of 1 mM iodoacetic acid during the final sustained ischaemic period. In a second series, the protein kinase C (PKC) activators phorbol 12-myristate 13-acetate (PMA), ingenol 3, 20-dibenzoate, and thymeleatoxin were added during preincubation. In a third series, preincubation periods were substituted by brief ischaemic pelleting of cells. Final prolonged ischaemic pelleting was preceded by a 30 min postincubation period. Rate and extent of injury was determined by sequential sampling and assessment of trypan blue permeability following 85 mOsM swelling. Results: Myocytes were preconditioned by a 5 min glucose-free preincubation. Addition of iodoacetic acid into the final ischaemic pellet increased the rates of rigor contracture and injury, but did not abolish the protective response. Direct protein kinase C activation with PMA, a non-selective phorbol ester, and ingenol, an ε, δ-PKC isozyme selective activator, protected cells, but thymeleatoxin, an α,β,γ-PKC isozyme selective activator, did not. A 10 min ischaemic preincubation preconditioned, but the protection was not enhanced when ischaemia was extended to 30 min, or when PMA was included during the initial ischaemic preincubation. Adenosine partially inhibited the response. Conclusions: (1) Preconditioning of isolated myocytes is not dependent on glycolysis or glucose transport. (2) Preconditioning appears dependent on activation of the ε-PKC isoformn. (3) Ischaemia is capable of preconditioning isolated myocytes in vitro, and initiation of this effect is modified by simultaneous additional of adenosine but not by direct protein kinase C activation with PMA. Induction of protection by PMA and ingenol shows that protection requires protein kinase C activation, but direct potassium channel activation by regulatory G proteins is not critical.Cardiovascular Research 1994;28:1700-1706.

Objective: The aim was to determine if isolated rat cardiomycytes could be protected from ischaemic cell death by preincubation with adenosine or adenosine agonists. Methods: Cardiomyocytes isolated from rat hearts were preincubated in the presence of adenosine, CCPA (2-chloro-N6-cyclopentyladenosine), or carbachol prior to concentration into an ischaemic slurry. Effects of glycolysis and of isoprenaline were determined by addition of iodoacetic acid or isoprenaline to the ischaemic incubates and by exclusion of glucose from all media. Rates of ischaemic contracture were determined and survival of the myocytes versus paired control preparations was determined after various times of ischaemia, following resuspension of the cells in isotonic or hypotonic media. Results: Adenosine and CCPA produced only a small reduction of the rates of contracture and death of isolated myocytes. Carbachol gave no significant protection. Neither the degree of injury of control cells nor the amount of protection by CCPA was altered in the presence of added isoprenaline. Protection was abolished by the A1 receptor blocker sulphophenyl theophylline, iodoacetic acid, and exclusion of glucose. Conclusions: Adenosine and adenosine agonists afford a minimal degree of protection to ischaemic isolated myocytes by a glucose dependent mechanism. This protection does not appear to account for the larger degree of protection seen in intact hearts, following similar preconditioning protocols. The failure of adenosine to protect may be related to the quiescent state of isolated cardiomyocytes, or be species specific in that adenosine may not be the trigger for preconditioning in rats.Cardiovascular Research 1993;27:1670-1676.

Calcium tolerant rabbit cardiomyocytes, isolated by collagenase perfusion, were preincubated for varying periods of time followed by resuspension in fresh media and centrifugation into an ischaemic pellet with restricted extracellular fluid. Pellets were incubated for 240 min under oil at 37°C to mimic severe ischaemia. Time to onset of ischaemic contracture (rod to square transformation) and trypan blue permeability following resuspension in 85 mOsm media were monitored at sequential times. The protocol of Series 1 was a 5-10 min pre-incubation, immediately followed by ischaemic pelleting. Preincubation with pinacidil (50 μm) protected cells from ischaemic insult, but pinacidil added only into the ischaemic pellet did not protect. Protection was abolished by the protein kinase (PKC) inhibitors chelerythrine (10 μm) added with pinacidil and calphostin C (200nm) added only into the ischaemic pellet. Neither PKC inhibitor had an effect on injury of untreated ischaemic myocytes (data not shown). Series 2-5 were preconditioning protocols with a 10 min intervention period, followed by a 30 min oxygenated drug-free period, prior to ischaemic pelleting. In series 2 pinacidil protected cells from ischaemic insult and this protection was abolished when glyburide (10 μm) was present during preincubation, or during post-incubation and ischaemia. Glyburide only partially inhibited the protection when glyburide was added only into the ischaemic pellet. In Series 3, 8-sulfophenyltheophyline (SPT)(100 μm) or adenosine deaminase during preincubation, or SPT only added into the ischaemic pellet abolished pinacidil’s protection. In Series 4, cardiomyocytes were ischaemically preconditioned by pelleting for 10 min followed by 30 min reoxygenation. Glyburide during initial ischaemic blocked protection, but when added during post incubation and into the final pellet protection was not reduced. In Series 5 8-cyclopentyl-1,3, dipropylxanthine (DPCPX) (10 μm) added into the final pellet abolished protection by pinacidil, but not protection following ischaemic preconditioning. In contrast to pinacidil, ischaemically preconditioned cells maintain protection in the presence of glyburide, indicating that: (1) pinacidil does not exactly mimic preconditioning and (2) ischaemically preconditioned cells do not require opened K+ATP channels for protection, although they appear to be important during initiation of the preconditioned state. It is hypothesized that pinacidil opening of K+ channels may facilitate induction of preconditioning.

Strategies to reduce infarct size in ischaemia-reperfusion (IR) syndromes such as acute myocardial infarction are of high clinical and scientific interest. Remote ischaemic preconditioning (rIPC) is one such strategy but its mechanisms remain incompletely understood. Multiple lines of evidence from animal studies suggest that the endogenous purine nucleoside adenosine is a key mediator of preconditioning pathways but no evidence exists as to adenosine’s role in the more complex physiology of humans. The work in this thesis aims to elucidate the role of endogenous adenosine in the physiological phenomenon of rIPC and to examine the role of exogenous adenosine in triggering preconditioning-like states. In a randomised, placebo controlled study using healthy volunteers and the human forearm model of ischaemia-reperfusion injury, I demonstrate that delivery of the adenosine receptor antagonist caffeine, prior to the initiation of a rIPC stimulus abrogates the protective effect of rIPC on IR. By then selectively infusing caffeine to achieve high local but low systemic concentrations, I also demonstrate that adenosine receptor activation is important in the ‘trigger’ phase of rIPC rather than in the ‘effector’ phase and that blockade of the trigger phase effectively inhibits the release of a circulating humoral protective factor. These studies provide evidence of the crucial role of adenosine receptor activation in human rIPC, demonstrating their sites of action and illuminating their potential mechanism of action. To study whether exogenously delivered adenosine can recapitulate preconditioning-like states, in initial studies in a large mammal model of acute myocardial infarction, I demonstrate that adenosine, given after the onset of ischaemia, but prior to reperfusion, significantly reduces myocardial infarct size. In a subsequent study, translating these findings to humans with coronary disease, I demonstrate that the delivery of adenosine in a range of concentrations is able to illicit the release of a circulating preconditioning factor which is transferrable across species and can reduce infarct size in a murine model of myocardial IR.