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The Erythrocyte Polyol Pathway, Diabetes and Its Long-Term Complications

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The Erythrocyte Polyol Pathway, Diabetes and Its Long-Term Complications THE ERYTHROCYTE POLYOL PATHWAY, DIABETES AND ITS LONG-TERM COMPLICATIONS by Paul Anthony Lyons BSc A Thesis Submitted for the Degree of Doctor of Philosophy of The University of London Charing Cross and Westminster Medical School (February 1990) I Abstract One of the biochemical abnormalities associated with the development of the diabetic complications is elevated polyol pathway activity. It has been proposed that the erythrocyte polyol pathway might provide a good model of the polyol pathway in less accessible tissues, such as nerve, kidney and the lens. In particular it has been proposed that measuring erythrocyte polyol pathway activity might be of value in elucidating the role of the pathway in the development of complications in these less accessible tissues. This Thesis looks at the erythrocyte polyol pathway in diabetic and non-diabetic subjects, and examines the relationship between erythrocyte polyol pathway activity and the severity of neuropathy, retinopathy and nephropathy. Initial work examined the relationship between sorbitol dehydrogenase activity and age, duration of diabetes and glycaemic control in diabetic patients. In addition, red cells were examined for multiple isoenzymes of sorbitol dehydrogenase. It was established that, whereas there were marked individual differences in erythrocyte sorbitol dehydrogenase activities, these differences were the function of a single isoenzyme and did not correlate with duration of disease and other parameters. Secondly the relationship between the activities of erythrocyte aldose reductase and sorbitol dehydrogenase, and red cell fructose and sorbitol and the severity of neuropathy, retinopathy and nephropathy was examined in a cohort of long-duration (> 25 years) insulin-dependent diabetic patients. It was established that red cell aldose reductase activity was markedly increased in diabetic patients in association with a parallel increase in red cell fructose when compared to age-matched controls. However no relationship was found between erythrocyte polyol pathway activity and indices of the severity of the complications. Given that erythrocyte aldose reductase activity is increased in diabetic patients the third section of this Thesis examines whether this increase is a function of hyperglycaemia. The response of the enzyme to II glucose challenge was studied in vivo during a glucose tolerance test. There was a transient rise activity that mirrored the rise in plasma glucose. The conclusion is that aldose reductase is acutely activated in hyperglycaemia via a mechanism that may involve glycation of the enzyme. I l l To Eluned IV Contents Page Abstract II Contents V List of Figures VIII List of Tables XII Acknowledgements XIII Abbreviations XIV Chapter 1 General Introduction 1 1.1 Diabetes 2 1.2 The diabetic complications 9 1.2.1 Retinopathy 10 1.2.2 Cataract 13 1.2.3 Nephropathy 15 1.2.4 Neuropathy 19 1.2.5 Macrovascular disease 24 1.3 The pathogenesis of the diabetic complications 27 1.3.1 Glycaemic control and development of diabetic 27 complications 1.3.2 Other factors in the development of diabetic 31 complications 1.4 Nonenzymic glycation of macromolecules 36 1.5 The polyol pathway 43 1.5.1 Aldose reductase (EC 1.1.1.21) 43 1.5.2 Sorbitol dehydrogenase (EC 1.1.1.14) 47 1.5.3 Polyol pathway activity in tissues susceptible to the 48 diabetic complications 1.5.4 The polyol pathway and sugar cataract formation 51 1.5.5 The role of the polyol pathway in other diabetic 56 complications 1.5.6 Myo-inositol metabolism and the complications of 58 diabetes 1.5.7 Polyol pathway activity and altered cellular redox 65 state 1.5.8 The erythrocyte polyol pathway 67 V 1.6 Aims of project 70 Chapter 2 Materials and Methods 72 2.1 Materials 73 2.2 Methods 74 2.2.1 Krebs-Ringer bicarbonate buffer 74 2.2.2 Erythrocyte sorbitol dehydrogenase activity 74 2.2.3 Measurement of erythrocyte monosaccharide and 77 polyol concentrations 2.2.4 Measurement of erythrocyte aldose reductase 81 activity 2.2.5 Determination of plasma monosaccharides and polyols 84 by gas liquid chromatography 2.2.6 In vitro erythrocyte incubations 85 2.2.7 Starch gel electrophoresis of haemolysates and 86 staining for sorbitol dehydrogenase activity 2.2.8 Protein determination 88 2.2.9 Measurement of plasma glucose concentrations 89 2.2.10Determination of glycosylated haemoglobin 90 2.2.11 Screening for aldose reductase isoenzymes in the 90 erythrocyte 2.2.12 Statistical methods 102 Chapter 3 Ervthrocvte Sorbitol Dehvdroaenase Activity104 in Diabetic and Non-Diabetic Subjects 3.1 Introduction 105 3.2 Experimental 108 3.3 Results 109 3.4 Discussion 120 Chapter 4 Ervthrocvte oolvol pathway enzme activities122 and the severity of the diabetic complications 4.1 Introduction 123 4.2 Experimental 125 4.2.1 Patient selection 125 4.2.2 Clinical evaluation 125 VI 4.3 Results 130 4.3.1 Erythrocyte polyol pathway enzyme activity and 130 metabolite concentrations: Diabetic patients versus controls 4.3.2 Clinical status of the diabetic cohort 135 4.3.3 Relationship between polyol pathway enzymeactivities 144 and metabolite concentrations and the severity of diabetic complications 4.4 Discussion 154 Chapter 5 Activation of Aldose Reductase 160 5.1 Introduction 161 5.2 Experimental 163 5.3 Results 164 5.4 Discussion 168 Chapter 6 General Discussion 171 Chapter 7 References 183 Appendix 232 VII List of Figures Page Figure 1.1 The age specific incidence rate of diabetes mellitus 6 Figure 1.2 Annual incidence of diabetes in Great Britain by age 8 and type Figure 1.3 The glucose hypothesis of diabetic complications 35 Figure 1.4 The formation of early and late non-enzymatic 37 glycosylation products Figure 1.5 Prevention of advanced glycosylation end-product- 41 protein crosslinking by aminoguanidine Figure 1.6 The polyol pathway 44 Figure 1.7 Glucose metabolism in tissues susceptible to the 50 diabetic complications Figure 1.8 The intracellular biochemical changes that accompany 53 diabetic cataract formation Figure 1.9 Proposed self-perpetuating defect involving polyol 62 pathway activity, phosphoinositide metabolism, protein kinase C and sodium potassium ATPase Figure 2.1 The elution profile on carboxybenzaldehyde coupled AH-93 sepharose 4B. Figure 2.2 The elution profile on Matrex Gel Orange A 95 Figure 2.3 Separation of lactate dehydrogenase isoenzymes by 99 native-Page Figure 2.4 SDS-PAGE and Western blotting of purified placental 101 aldose reductase VIII Figure 3.1 SDFi activity in insulin-dependent (IDDM) and non- 11 0 insulin-dependent (NIDDM) diabetic patients and non-diabetic controls Figure 3.2 The variation of erythrocyte sorbitol dehydrogenase 111 activity with time Figure 3.3 The effect of age on erythrocyte sorbitol dehydrogenase 11 3 activity in A) insulin-dependent (o)and non-insulin- dependent (•) diabetic patients, and B) non-diabetic controls Figure 3.4 The effect of sex on erythrocyte sorbitol dehydrogenase 114 activity in insulin-dependent (IDDM) and non-insulin- dependent (NIDDM) diabetic patients, and non­ diabetic controls Figure 3.5 The effect of duration of diabetes on erythrocyte 115 sorbitol dehydrogenase activity in insulin-dependent (o) and non-insulin-dependent (•) diabetic patients Figure 3.6 The relationship between HbA1 and erythrocyte 11 6 sorbitol dehydrogenase activity in insulin-dependent (o) and non-insulin-dependent (•) diabetic patients Figure 3.7 Correlation of erythrocyte sorbitol dehydrogenase 11 7 activity and plasma glucose levels in insulin-dependent (o) and non-insulin-dependent (•) diabetic patients Figure 3.8 Starch gel electrophoretic pattern of sorbitol 119 dehydrogenase in the erythrocytes of insulin-dependent diabetic patients Figure 4.1 Erythrocyte aldose reductase activity in insulin- 132 dependent (IDDM) and non-diabetic controls Figure 4.2 The correlation between erythrocyte aldose reductase 133 activity and fructose concentration IX Figure 4.3 The correlation between erythrocyte sorbitol 134 dehydrogenase activity and fructose concentration Figure 4.4 The correlation between erythrocyte aldose reductase 136 activity and sorbitol concentration Figure 4.5 The correlation between erythrocyte sorbitol 137 dehydrogenase activity and sorbitol concentration Figure 4.6 The relationship between erythrocyte aldose reductase 138 activity and age Figure 4.7 The relationship between erythrocyte aldose reductase 139 activity and duration of diabetes Figure 4.8 The relationship between erythrocyte aldose reductase 140 activity and glycosylated haemoglobin levels Figure 4.9 The correlation between Valsalva ratio and the overall 142 autonomic neuropathy score Figure 4.10 The relationship between erythrocyte aldose reductase 145 activity and the overall autonomic neuropathy score Figure 4.11 The relationship between erythrocyte sorbitol 146 dehydrogenase activity and the overall autonomic neuropathy score Figure 4.12 The correlation between erythrocyte aldose reductase 148 activity and vibration perception threshold Figure 4.13 The correlation between erythrocyte sorbitol 149 dehydrogenase activity and vibration perception threshold Figure 4.14 The relationship between erythrocte aldose reductase 151 activity and albumin excretion rate Figure 4.15 The relationship between erythrocyte sorbitol 152 dehydrogenase activity and albumin excretion rate X Figure 5.1 The response of erythrocyte aldose reductase activity 165 to changing plasma glucose concentration following a glucose tolerance test Figure 5.2 The relationship between the increase in erythrocyte 166 aldose reductase activity
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