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 and the rise in plasma glucose concentration
Figure 5.3 The response of erythrocyte fructose concentration to 167 changing plasma glucose concentrations following a glucose tolerance test
XI List of Tables
Page
Table 1.1 Classification of diabetes and other categories of 5 glucose intolerance
Table 1.2 Diabetic neuropathy 21
Table 2.1 Relative retention times of sugars and polyols separated 80 by gas-liquid-chromatography
Table 2.2 Monosaccharide and polyol concentrations in washed and 82 unwashed erythrocytes
Table 2.3 Purification of human placental aldose reductase 100
Table 4.1 Erythrocyte aldose reductase and sorbitol 131 dehydrogenase activities, and fructose, sorbitol, and myo-inositol concentrations in insulin-dependent diabetic patients and age-matched controls
Table 4.2 Erythrocyte aldose reductase and sorbitol 150 dehydrogenaseactivities, and fructose and sorbitol concentrations in insulin-dependent diabetic patients grouped on the basis of severity of retinopathy
Table A1 Individual cardiovascular function test results 233
Table A2 Individual vibration perception threshold results 234
Table A3 Individual retinopathy gradings 235
i Table A4 Individual urinary protein excretion results 236
XII Acknowledgements
1 would like to thank Drs Norman Palmer and Peter Wise for their invaluable guidance and supervision over the past three years, and especially for their help and advice in the preparation of this Thesis. I would also like to thank the British Diabetic Association and ICI Pharmaceuticals Ltd for their financial support.
The study described in Chapter 4 could not have been carried out without the assistance of a number of people and I would especially like to thank the following; Dr Roland Guy for organising the diabetic patients, Dr Tim Lockington and Nick Kitchin for assessing the severity of neuropathy, Drs Kinnear and Ghosh from the Department of Ophthalmology for grading the retinal photographs, and Miss Andrea Collins for measuring urinary protein excretion. Most importantly I would like to thank the diabetic patients without whose co-operation the study would not have been possible. In addition thanks must go to all those people who acted as controls in the various studies, particuarly all the members of the Department of Biochemistry who, without too much coercion, have donated both their time and blood over the last few years, not to mention Dr Janet Powell who has given up a lot of her time taking blood samples.
Thanks are also due to Nick Kitchin and Sarah Gould for their help with some of the experimental work described in this Thesis. To undertake the work presented in this Thesis has required the learning of a whole array of new techniques. In this respect I would like to thank Steve Jeremiah for explaining the black magic behind starch gel electrophoresis, and Chris Sennit and Don Mirrlees for teaching me how to measure erythrocyte monosaccharides and polyols by GLC.
I would also like to thank all the members of the Department of Biochemistry, and in particular all the members, past and present, of the TNP group, who have made my time at Charing Cross most enjoyable.
Finally I would like to say thanks to my wife Eluned for her love and support over the last three years
XIII Abbreviations
AA Amino acids AER Albumin excretion rate AGE Advanced glycation end product ANS Overall autonomic neuropathy score AR Aldose reductase ARI Aldose reductase inhibitor ATP Adenosine 5’-triphosphate ATPase Adenosine triphosphatase BB Bio-Breeding BMLM Basement-membrane-like membrane BP Blood pressure CHD Coronary heart disease cm, jim, nm Centimetre, micrometre, nanometre CSII Continuous subcutaneous insulin infusion CV Coefficient of variation CVD Cardiovascular disease CWS Cotton wool spot DNA Deoxyribonucleic acid ECG Electrocardiography EDTA Ethylenediaminetetraacteic acid ELISA Enzyme-linked immunoassay g, mg, jig, ng Gram, milligram, microgram, nanogram GC Gas chromatograph GFR Glomerular filtration rate GLC Gas-liquid-chromatography GSH Glutathione, reduced HANES Health and Nutrition Examination Study Hb Haemoglobin HbAi Glycosylated haemoglobin HE Hard exudate HLA Human leucocyte associated HMDS Hexamethyldisilazane HR Heart rate IDDM Insulin-dependent diabetes mellitus
XIV igG Immunoglobulin G IL-1 lnterleukin-1 IRMA Intraretinal microvascular abnormalities Km Michaelis constant I, dl, ml, jil Litre, decilitre, millilitre, microlitre LDL Low density lipoprotein mol, mmol Moles, millimoles MA Microaneurysm Ml Myo-inositol Min Minute mm Hg Millimetres of mercury MNCV Motor nerve conduction velocity mRNA Messenger ribonucleic acid MTT 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Thiazolyl blue Na-KATPase Sodium-potassium adenosine triphosphatase NAD+ Nicotinamide-adenine dinucleotide NADH Nicotinamide-adenine dinucleotide, reduced NADP+ Nicotinamide-adenine dinucleotide phosphate NADPH Nicotinamide-adenine dinucleotide phosphate, reduced NIDDM Non-insulin-dependent diabetes mellitus NV New vessels NVD New vessels on the disc NVE New vessels elsewhere OD Optical density PAGE Polyacrylamide gel electrophoresis PCA Perchloric acid PCR Polymerase chain reaction PMS Phenazine methosulphate RBC Red blood cell SD Standard deviation SDH Sorbitol dehydrogenase SDS Sodium dodecyl sulphate SEM Standard error of the mean Spec Act Specific activity
XV TMCS Trimethylchlorosilane TMG Tetramethylene glutaric acid TNF Tumour necrosis factor Tris Tris (hydroxymethyl) aminomethane U, ml), pU Unit, milliunit, microunit V volt VPT Vibration perception threshold
XVI Chapter One
INTRODUCTION
1 1.1 Diabetes
What is diabetes? There is no simple, concise answer to this question. Diabetes is a heterogeneous disease (Fajans, 1978), a complex syndrome characterised by hyperglycaemia, secondary to impaired secretion and/or action of insulin, and various tissue changes that have been termed the chronic complications of diabetes.
The first clear description of diabetes was made by Aulus Cornelius Celsus, a Roman physician, although polyuria had been previously reported in the Ebers papyrus (c. 1500BC). The name diabetes (8iaPaiem\), to run through; Siaprirrii; , a siphon) (Allen et al, 1919) is first known to have been used by Areteus, another Roman physician. In his writings Areteus clearly described some of the main symptoms of diabetes, its progressive course, and its fatal outcome. Despite these early descriptions the reported prevalence of diabetes in the period up to 1776 was very low (Levine, 1986). This does not necessarily indicate an absence or rarity of the disease upto this time, but most probably reflects problems of diagnosis, especially in mild cases.
The first steps to an accurate diagnosis were taken in the early 1670s when Thomas Willis made the observation that diabetic urine is "wonderfully sweet, as if imbued with honey or sugar" (Willis, 1679). This led to the discovery in 1776 by Matthew Dobson that the sweet substance in the urine was sugar. He showed that on evaporation diabetic urine yielded a whitish cake, and that this cake "smelt sweet, like brown sugar, and could not be distinguished from sugar, except that the sweetness left a slight sense of
2 coolness on the palate" (Dobson, 1776). Physicians now had a crude test for determining the amount of sugar in a patients urine and also the ability to determine the effect of various therapies on the severity of diabetes. At this time not only was the cause of the disease a subject of much speculation, but the identity of the principle organ affected was also still unclear. In 1815 the sugar present in the urine of diabetic patients was shown to be identical with glucose by Chevreul (Chevreul, 1815). Thomas Cawley provided the first description of a pancreatic lesion in diabetes. He reported that the pancreas of one of his patients was "full of calculi, which were firmly impacted in its substance" (Cawley, 1788).
Conclusive evidence for a pancreatic lesion being central to the development of diabetes came in 1889 when Minkowski and von Mering discovered that fatal diabetes developed in dogs that had been depancreatised (Mering and Minkowski, 1889). Following this Minkowski established the doctrine of the "internal secretion" of the pancreas (Minkowski, 1893), ie that diabetes was due to the loss of an "internal secretion" of the pancreas. Shortly afterwards the site of production of this "internal secretion" was shown to be the islets of Langerhans. The most important step forward in the treatment of diabetic patients came in 1921 when Banting and Best succeeded in extracting an active substance from the pancreas that was capable of lowering the raised blood glucose levels of diabetic dogs. This discovery has transformed the lives of individuals with diabetes, allowing them to lead an almost normal existence.
Himsworth’s demonstration in 1936 that two distinct types of disease can cause the symptoms of diabetes (Himsworth, 1936) cast doubt on the
3 simplistic view that diabetes was always the result of insulin insufficiency. He showed an insulin-sensitive type, characterised by a deficiency of insulin; and an insulin-insensitive type, characterised not by an apparent lack of insulin but by the "lack of an unknown factor which sensitises the body to insulin." This classification into insulin-sensitive and insulin-insensitive types was supported by the work of Bornstein and Lawrence (1951). Using a crude assay they measured plasma insulin in 37 diabetic patients and were able to distinguish two main types of human diabetes. Patients suffering from the first type were found to lack plasma insulin. They were mainly young and their diabetes was characterised by weight loss and ketosis as well as hyperglycaemia. The second type were mainly middle- aged obese diabetics. They had available plasma insulin, showed no sign of ketosis and their diabetes could be controlled by diet alone.
Today diabetes is regarded as being a heterogeneous disease (Fajans, 1978), and the classification systems drawn up reflect this heterogeneity. The currently accepted classification system is shown in
Table 1.1. Insulin-dependent diabetes mellitus (IDDM) and non-insulin- dependent diabetes mellitus (NIDDM) are the main classes of diabetes, together accounting for 95% of all cases (Gerich, 1986). In the United
Kingdom today 749500 people (1% of the population) suffer from either
IDDM or NIDDM (IDF Directory, 1988). Diabetes may develop at almost any time of life, however the risk of developing the disease changes with age.
From Figure 1.1 it can be seen that the chance of developing diabetes does not rise linearly but peaks twice, once between the 2nd and 3rd decades and again after the age of 70 (Melton et al, 1983). This implies two processes superimposed on one another (Krowleski and Warram, 1985).
4 Table 1.1 Classification of diabetes and other categories of glucose
intolerance.
Class Characteristics
Insulin-dependent diabetes Patients who require insulin to mellitus (IDDM) prevent hyperglycaemia and ketosis.
Non-insulin-dependent diabetes NIDDM patients are not insulin mellitus (NIDDM) dependent and are not ketosis- prone.
Diabetes secondary to other In addition to the presence of the diseases. specific disease, diabetes mellitus is also present.
Impaired glucose tolerance (IGT) Nondiagnostic fasting glucose levels and glucose intolerance of a degree between normal and diabetic.
Gestational diabetes (GDM) Glucose intolerance that has its onset or recognition during pregnancy.
Previous abnormality of glucose Persons who now have normal tolerance (PreAGT) glucose tolerance but who have previously demonstrated diabetic hyperglycaemia or impaired glucose tolerance.
Potential abnormality of glucose Individuals who are at a tolerance (PotAGT) substantially increased risk for the development of diabetes.
(Adapted from National Diabetes Data Group, 1979) Age (years)
Figure 1.1 The age-specific incidence rate of diabetes mellitus (data taken from Melton et al, 1983).
6 Figure 1.2 shows the incidence rates of IDDM and NIDDM according to age at onset of disease as determined in two studies carried out in Great Britain (Paterson et al, 1983; Barker et al, 1982). The studies show that most cases of IDDM occur before the age of 25, whereas little NIDDM has occurred by that time. From Figure 1.2 it can be seen that there is a rapid rise in the
incidence rate of IDDM, that begins at around one year of age and peaks between the ages of 10 and 14. Following this peak the incidence rate falls away. Similar findings have been reported in other countries (LaPorte et al,
1981; Green and Andersen, 1983). In all of the studies men had a marginally greater chance of developing IDDM than women. Again from
Figure 1.2 it can be seen that the incidence rate of NIDDM rises steadily with
age, rising from 3 per 100000 in those less than 20 to 44 per 100000 in the age group 45-50. Studies in North America (Wilson et al, 1981) indicate that the incidence of NIDDM is higher in males until the age of 60, whereupon the incidence rates become similar.
Whereas the incidence rate for NIDDM does not appear to be changing with time (Krowleski and Warram, 1985), reports suggest an increase in the incidence of IDDM over the last 40 years (Stewart-Brown et al, 1983; Paterson et al, 1983). However a recent report suggests that the prevalence of IDDM has not changed, it is just seen at an earlier age (Kurtz et al, 1988). As well as age and sex many other factors, including geographic location, genetic factors (Krowleski and Warram, 1985), and socioeconomic status (Barker et al, 1982) also have an important effect on the chance of developing both insulin-dependent and non-insulin- dependent diabetes.
7 Age (years)
Figure 1.2 Annual incidence of diabetes in Great Britain by age and type.
IDDM denoted byn and ■ , NIDDM denoted by a (data from Barker et al, 1982; Paterson et al, 1983).
8 1.2 The diabetic complications
Over the last 60 years, since the introduction of insulin therapy, the major cause of mortality in diabetic patients has changed. Today patients
rarely die from the acute complications of diabetes, namely ketoacidosis and diabetic coma. Whereas in the era before insulin these acute complications accounted for between 40 and 70% of all deaths in diabetic patients, today they account for less than 0.5% (Vignati et al, 1985). The increased survival of diabetic patients has led to the appearance of a new and equally serious problem, the so called long-term complications of diabetes. These complications affect the eye (retinopathy and cataract), the nerves
(neuropathy), the kidney (nephropathy), and the large blood vessels (coronary artery, peripheral vascular, and cerebrovascular disease). The seriousness of the problem is highlighted by the fact that blindness as a result of proliferative retinopathy is 25 times more prevalent in the diabetic
patient than in the non-diabetic patient, cataract occurs at 5 times the frequency and at a much earlier age in diabetic patients (Caird et al, 1969), and death from renal failure is 17 times more common in diabetic patients
(Entmacher et al, 1964). In addition diabetic patients are 100 times more likely to develop neuropathy (Young, 1988), 4 times as likely to suffer a heart attack and twice as likely to suffer a stroke as their non-diabetic counterpart.
Furthermore they have 20 times the normal incidence of gangrene (Tchbroutsky, 1987).
9 1.2.1 Diabetic Retinopathy
Diabetic retinopathy is the single most common cause of blindness in the working population of England. It accounts for 16.9% of all new cases of blind registration in men, and 20.6% in women (DHSS, 1979). The principle effect of diabetes on the retina is on the retinal capillaries, and five pathologic processes may be observed: 1) Microaneurysm formation; 2)
Occlusion and closure of retinal capillaries; 3) Excessive vascular permeability; 4) Proliferation of new blood vessels; and 5) Retinal detachment. The severity of retinopathy is assessed by measuring the presence or absence of these processes, and patients are subdivided into having background, preproliferative or proliferative diabetic retinopathy (see Aiello et al, 1985). The incidence of retinopathy in insulin-dependent patients with a duration of diabetes of less than 5 years is very low, reported rates ranging from 1 to 3% (Klein et al, 1984a; Pirart, 1978). Beyond 5 years duration of diabetes there is a steady rise in the prevalence of retinopathy, reaching a value of 30-80% (Klein et al, 1984a; Pirart, 1978; Palmberg et al, 1981) after 10 years of disease; and 55-100% after 20 years (Klein et al,
1984a; Pirart, 1978; Palmberg et al, 1981). Proliferative retinopathy is uncommon in insulin-dependent diabetic patients with a duration of less than 10 years (<2%), thereafter the prevalence rate rises rapidly reaching 50% after 20 years of disease (Klein et al, 1984a). Retinopathy is less common in non-insulin-dependent patients, after 20 years of disease 50% will have some degree of retinopathy and between 5 and 15% will have proliferative retinopathy (Klein et al, 1984b).
10 The first clinical sign of background retinopathy is the appearance of
microaneurysms. Microaneurysms are dilations of retinal capillaries, which when filled with blood appear as small discrete red dots. Although a number of possible mechanisms have been proposed the exact mechanism that leads to the formation of microaneurysms remains unclear (Frank, 1984). Microaneurysms are difficult to distinguish ophthalmoscopically from a second lesion of background retinopathy, the dot haemorrhage, although they may be distinguished by fluoroscein angiography. A third feature of background retinopathy is hard exudate formation. Hard exudates are yellowish-white, discrete deposits seen particularly in the macular region of the retina. They may be discrete dots, form ring structures, or plaques. The major component of hard exudates is lipid (Gartner, 1949). This lipid is believed to leak from the plasma across the abnormally permeable walls of microaneurysms (Davis, 1988). At this stage the risk of loss of sight is slight. However as these lesions become more abundant the risk of developing proliferative retinopathy is increased. This stage of retinopathy is termed preproliferative retinopathy.
Other preproliferative lesions include "cotton wool spots", regions of retinal non-perfusion, and intraretinal microvascular abnormalities (IRMA).
"Cotton wool spots" are areas of intracellular oedema, within the retinal nerve fibre layer, associated with closed retinal arterioles. IRMA are patches of acellular capillaries that lie within the retina. More often than not they are associated with more normal looking vessels of a larger calibre. It is unclear whether these vessels are pre-existing capillaries or intraretinal new vessels
(Davis et al, 1969). Although the probability of progression from preproliferative to proliferative retinopathy is not known, once preproliferative lesions become prominent neovascularisation (the appearance of new blood vessels) on the surface of the retina or optic disc is very likely.
The major cause of impaired vision and blindness in non-proliferative retinopathy is macular oedema. Macular oedema is a thickening of the macular region of the retina, and it is often associated with the presence of hard exudates, which may be spread diffusely across the macular or organised into a circular pattern. Another form of macular oedema, cystoid macular oedema, involves the formation of fluid-filled cysts within the retinal nerve fibre layer. Macular oedema may occur at any age in patients with retinopathy and its prevalence does not appear to vary according to the type of diabetes (Klein et al, 1984c).
The beginning of the most severe form of retinopathy, proliferative retinopathy, is characterised by the appearance of new blood vessels.
Vessels that appear on or within one disc diameter of the optic disc, or in the vitreous cavity anterior to the disc are termed new vessels on the disc (NVD); whereas vessels arising from other areas of the retinal circulation are designated as new vessels elsewhere (NVE). The optic disc is the most common site of new vessels and the development of NVD carries the greatest risk for loss of vision. Data indicates that an eye with NVD covering 25% or more of the optic disc has a 30% chance of deteriorating from good vision to severe blindness in less than three years if not treated (Diabetic Retinopathy Study Group, 1981).The cause of neovascularisation is unclear.
It is believed that non-perfused areas of the retina release an angiogenic stimulus as a result of hypoxia (Henkind, 1978). Whatever the basis, the rate of growth of new vessels is variable and appears to follow a cycle of proliferation and regression (Dobree, 1964). Proliferation of new vessels is accompanied by proliferation of fibrous tissue. These fibrovascular proliferations often attach themselves to the posterior surface of the vitreous. Even when regression of the blood vessels occurs these attachments remain in place. There are a number of important consequences of neovascularisation. In normal eyes posterior vitreous detachment is a process that occurs late in life. However, in diabetic patients with proliferative retinopathy it occurs much earlier. Posterior vitreous detachment occurs as a result of shrinkage of the posterior (retinal) surface of the vitreous. In the normal eye this results in the vitreous being pulled cleanly away from the retina. However, where fibrovascular proliferations are present, they too are pulled forward and with them the retina from which they originate. This often produces regions of retinal detachment and loss of vision (Davis, 1988). A second consequence is haemorrhage. As shrinkage occurs force is exerted on the new blood vessels and this can lead to vitreous haemorrhage. The extent of impairment of vision that results depends on the severity of haemorrhage (Davis, 1988). A third consequence is neovascular glaucoma. This results from the formation of new vessels on the surface of the iris and in the angle of the eye (Aiello et al,
1985).
1.2.2 Cataract
The crystalline lens is a transparent, elastic structure located behind the iris. Its transparency results from the optical homogeneity of its component parts. Disruption of this optical homogeneity leads to a loss of transparency or cataract formation. Cataracts may be classified using the system developed by Chylack (1978). Cataracts are classified as one of three types: 1) Immature cataract in which internal morphologic structures are preserved in at least some parts of the lens; 2) Mature cataract in which no normal morphologic details are apparent but where there is no swelling of the lens; 3) Hypermature cataract which resembles mature cataract but is associated with a large increase in lens volume. Hypermature cataracts may be further subdivided into subcapsular, cortical, or nuclear cataract
(Straatsma et al, 1985).
A number of studies have reported an increased prevalence of cataract in diabetic patients. Hiller and Kahn (1976) and Caird et al (1964) reported that cataract extraction was 4 to 6 times more common in diabetic patients. These studies have been criticised on the grounds of a possible referral bias, ie diabetic patients are more likely to be seen by an ophthalmologist and therefore more likely to have their cataracts identified and removed (Sommer, 1977), thereby overestimating the number of diabetic patients with cataract. To avoid such a referral bias it is necessary to examine the relationship between cataract and diabetes in the general population. Data from two large epidemiological studies, the Framingham study and the Health and Nutrition Examination Survey (HANES) has demonstrated an increased prevalence of senile cataracts in diabetes (Ederer et al, 1981). In diabetic patients of less than 65 years old there was a marked excess prevalence of cataract, with a relative risk of developing cataract of 4.02 (Framingham study) and 2.97 (HANES) compared to non diabetics. For older diabetics the prevalence was less marked, with a relative risk of 1.63 (HANES) and 1.02 (Framingham study). Morphologically cataracts seen in adult diabetic patients are similar to senile cataracts seen in non-diabetic subjects. The most common kinds of cataract seen in both the diabetic and non-diabetic populations are the nuclear sclerotic and posterior subcapsular types. In nuclear sclerotic cataracts the nucleus of the lens becomes more dense, turning a brownish- yellow colour and preventing light from passing through. The posterior subcapsular cataract results from an opacity on the back surface of the lens, just under the capsule (Aiello et al, 1985). A distinctive, but today uncommon, form of cataract is the juvenile diabetic cataract. Seen in young onset insulin-dependent diabetic patients (Nielsen and Vinding, 1984), it is characterised by dense white subcapsular opacities ('snow-flake' cataract).
While the cause of juvenile cataract has been shown to be osmotic swelling (Kinoshita et al, 1962), the mechanism of adult diabetic cataract formation remains obscure, although research to date suggests that a number of mechanisms are probably involved (Bron and Cheng, 1986).
1.2.3 Diabetic Nephropathy
Renal disease is a major cause of sickness and death in diabetic patients. Studies have shown that renal disease accounts for 5.3-12% of all deaths in diabetic patients (Bell, 1953; Warren et al, 1966). Moreover death as a result of renal disease is more common in patients diagnosed at a young age (Lundbaek, 1965). Glomerular lesions are common in long- duration diabetes (Decked, 1988), although the reported prevalence depends very much on the diagnostic criteria employed (Knowles, 1974).
Glomerulosclerosis is however of little clinical significance, the earliest clinical sign of nephropathy being the appearance of proteinuria (Mann et al,
1949).
Despite glomerular lesions being common only 40-50% of all insulin- dependent patients will develop nephropathy (Andersen et al, 1983; Knowles, 1974). The incidence of diabetic nephropathy rises rapidly in the first 10 years following diagnosis of diabetes, reaching a peak at 16 years after which it declines (Andersen et al, 1983). Diabetic nephropathy is more common in men than women, particularly in those that develop proteinuria
16-20 years after diagnosis of diabetes (Andersen et al, 1983). Survival after the onset of persistent proteinuria is variable, but on the whole poor, the average time lag between development of proteinuria and death being 7 years irrespective of sex (Decked et al, 1981). The chief cause of death in diabetics with nephropathy is renal failure. A study in the UK by Moloney et al (1983) reported that 60% of deaths in patients with nephropathy was due to renal failure. This finding is in keeping with other reported studies (Kussman et al, 1976; Andersen et al, 1983). Thus 20-30% of all insulin- dependent patients will develop renal failure. Diabetic nephropathy is also seen in non-insulin-dependent diabetic patients. However death as a result is much lower in this group (WHO Expert Committee on Diabetes Mellitus,
1980).
Kidney disease in insulin-dependent diabetic patients develops in three stages (Keen and Viberti, 1981). The initial or silent phase is marked by definite structural and functional changes that include increased glomerular filtration rate (GFR), an increase in kidney size, and microproteinuria. The second stage is marked by the appearance of proteinuria, which though intermittent initially becomes persistent with time. The third stage is characterised by persistent proteinuria, followed by a progressive decline in renal function and finally renal failure.
Characteristic of the silent phase of diabetic kidney disease is the development of glomerular basement membrane thickening. At the onset of diabetes capillary basement membrane thickness, mesangial volume, and mesangial basement membrane-like membrane (BMLM) thickness are all normal (Osterby, 1972). Basement membrane thickening becomes demonstrable after two to three years of diabetes and by five years a thickening of 25-30%, compared to normal, is apparent (0sterby, 1972).
Eventually this thickening of the glomerular basement membrane leads to glomerular closure (Gundersen and 0sterby, 1977). By a variety of techniques insulin-dependent diabetic patients have been shown to have enlarged kidneys when compared to non-diabetic subjects (Mogensen and Andersen, 1973; Christiansen et al, 1981; Wiseman and Viberti, 1983). It has now been established that 40% of insulin-dependent diabetic patients have larger than normal kidneys (Wiseman and Viberti, 1983). Examination of kidney biopsies has shown that this increase in kidney size is associated with an increase in glomerular size, glomerular volume being 70% greater than in non-diabetic kidneys (0sterby and Gundersen, 1975). This increase in glomerular size, furthermore, is mirrored by an increase in filtration surface area. Kroustrup et al (1977) showed that glomerular filtration area was increased by 80% in patients with early diabetes.
A second feature of this silent phase of diabetic kidney disease is elevated GFR. GFR has been shown to be elevated on average by 20-40% (Ditzel and Schwartz, 1967; Christiansen et al, 1981; Wiseman et al, 1984). This increase in GFR is of the same magnitude as the increase in kidney size (Christiansen et al, 1981; Mogensen et al, 1979). The increase in GFR is believed to have a number of determinants including elevated renal plasma flow (Tucker and Blantz, 1977), an increased transglomerular pressure gradient (Hostetter et al, 1981), and increased filtration surface area (Flirose
et al, 1980).
An ominous sign of the silent phase is the development of microalbuminuria. Microalbuminuria is defined as an albumin excretion rate
of between 30 and 300 mg/24 hours (Viberti et al, 1984). The presence of microalbuminuria has been shown to indicate an elevated risk of developing clinical nephropathy (Mathiensen et al, 1984; Mogensen and Christensen, 1984; Viberti et al, 1982b). The mechanism behind the increased excretion of albumin is unclear. However, it has been proposed that it is due to a loss
of anionic charge on the glomerular basement membrane (Decked et al, 1988). The development of intermittent proteinuria (albumin excretion > 300
mg/24 hours) marks the second stage of diabetic kidney disease. This
period of intermittent proteinuria may last for several years before the
proteinuria becomes persistent (Ireland et al, 1982). As with
microalbuminuria the structural basis of proteinuria is unclear. Its onset marks the beginning of a decline in renal function leading to renal failure (Viberti et al, 1984). GFR, which may be normal or even raised at the onset of proteinuria, steadily falls, the rate of decline varying between 1.0 ml/min/month to 2.4 ml/min/month (Viberti et al, 1984). This decline in GFR
may be slowed by antihypertensive treatment (Parving and Hommel, 1989), and is most probably due to closure as a consequence of basement membrane thickening of the glomeruli (Gundersen and 0sterby, 1977). As GFR declines, total protein excretion in the urine increases as does the fractional clearance of albumin (Viberti et al, 1983a). Renal failure usually develops around 7 years after the start of persistent proteinuria (Deckert et al, 1981), at this point serum creatinine is greater than 8 mg/dl, and creatinine clearance below 20% of normal (Molinoff, 1987).
Patients with advanced diabetic nephropathy very often suffer from other diabetic complications. One third of insulin-dependent diabetic patients die of cardiovascular disease before they reach end-stage renal failure (Moloney et al, 1983). Peripheral vascular disease is also a major cause of morbidity in diabetic patients with nephropathy (Grenfell and Watkins, 1986). A large proportion of such patients also have neuropathy and this tends to get worse as nephropathy progresses (Gonzalez-Carrillo et al, 1982). Retinopathy is always present in advanced nephropathy. The majority of patients (>70%) with renal failure from nephropathy have proliferative retinopathy (Moloney et al, 1983; Gonzalez-Carrillo et al, 1982).
In addition 25-35% are blind and about 35% have severely impaired vision
(Moloney et al, 1983; Gonzalez-Carrillo et al, 1982).
1.2.4 Neuropathy
Compared to the other long-term complications of diabetes the symptoms of diabetic neuropathy are diverse. This diversity implies that the underlying causes of neuropathy will be equally as diverse. Since the aetiology of neuropathy has not been elucidated it is only possible to classify neuropathy in terms of clinical features. A simple and commonly used classification system (Table 1.2) divides neuropathy into two broad groups, both of which may be further subdivided (Thomas and Elaisson, 1984).
Irrespective of diagnostic criteria, the prevalence of neuropathy increases with age (Thomas and Ward, 1975), and with duration of disease (Pirart, 1978). Sensory neuropathy is usually insidious in onset. Long nerve fibres are most vulnerable and the symptoms often appear in a ’stocking and glove’ distribution. That is to say the symptoms first appear in the feet and spread proximally, later affecting the hands and arms. In the most severe cases changes may also affect the anterior abdominal wall (Gillon et al, 1986). The symptoms felt by the patient include tingling, muscle cramps, numbness, and pain. Although in most cases the symptoms are relatively mild, impaired pain perception is an important contributing factor in the development of neuropathic ulcers (most commonly found on the feet) and neuropathic joint degeneration (Ellenberg, 1976). Acute painful neuropathy is a distinct and relatively uncommon syndrome. It has a very sudden onset and is characterised by severe pain in the legs associated with muscle loss and severe weakness (Ward, 1988). Newly diagnosed diabetic patients, irrespective of whether they show clinical neuropathic symptoms, frequently show abnormalities in functional tests. These include reduced nerve motor conduction velocities (Ward et al, 1971), and impaired sensory perception
(Terkidsen and Christensen, 1971).
A prominent histopathological feature of sensory polyneuropathy is loss of both large and small myelinated nerve fibres. This loss in seen predominantly in the distal portions of nerves of the lower limbs (Johnson et al, 1986). It has been suggested that the neurological symptoms seen may
2 0 Table 1.2 Diabetic Neuropathy
Asymmetric neuropathy Mononeuropathy
Diabetic amyotrophy
Polyneuropathy Distal symmetric sensory polyneuropathy
Autonomic neuropathy
(Adapted from Thomas and Eliasson, 1984) reflect the class of nerve fibres that are predominantly involved (Brown and Greene, 1984). Loss of large fibres leads to a diminution in proprioception and vibration perception, whereas loss of small fibres leads to decreased pain and temperature perception (Brown and Greene, 1984). The precise nature of the fibre loss has not been fully determined. Axonal degeneration occurs in peripheral nerves (Dyck et al, 1986) and it is believed that this degeneration occurs in a 'dying-back' manner (Said et al, 1983). That is degeneration begins distally and progresses centripetally towards the cell body. Another feature of sensory polyneuropathy is segmental demyelination. However it is unclear whether this is a primary event in neuropathy or secondary to axonal degeneration (Dyck et al, 1986). Other axonal changes seen include axonal atrophy, and paranodal abnormalities such as swelling and axo-glial dysjunction (Sima et al, 1988). In contrast to axonal loss, axonal regeneration and remyelination is also more prominent in the nerves of diabetic patients than controls (Sima et al, 1988).
The other major class of polyneuropathy is autonomic neuropathy.
Autonomic neuropathy affects between 17 and 40% of diabetic patients
(O'Brien and Corrall, 1988; Ewing 1984), with its prevalence peaking in the fifth decade of life (O'Brien and Corrall, 1988). Abnormalities in autonomic nerve function are often found in patients who are asymptomatic (Pfeifer et al, 1982). Prior to the 1970s the diagnosis and classification of autonomic neuropathy was based on the presence of clinical symptoms. The advent of simple, non-invasive cardiovascular reflex tests has permitted a more precise approach. The most commonly used tests are the heart rate response to the Valsalva manoeuvre, standing, and deep breathing, and the blood pressure response to standing and sustained handgrip (Ewing and
2 2 Clarke, 1986). Using the combined results of these tests it is possible to classify autonomic neuropathy by severity of damage (Ewing and Clarke, 1986).
The symptoms of autonomic neuropathy are very often vague and as a result may go unrecognised for some time. Common symptoms of autonomic neuropathy include cardiovascular abnormalities such as resting tachycardia (resting heart rate in excess of 95 beats per minute), postural hypotension (systolic blood pressure fall of 30 mm Hg or more on standing); gastrointestinal disorders, including delayed gastric emptying (Kassander, 1958), diarrhoea (Feldman and Schiller, 1983), and constipation (Feldman and Schiller, 1983); bladder dysfunction (Bradley, 1980); impotence (Ewing et al, 1980); gustatory sweating (Watkins, 1973); and respiratory changes (Williams et al, 1984).
The following sequence for the development of autonomic abnormalities has been proposed by Ewing et al (1980). Initial symptoms include impaired sweating and thermoregulation function, followed by impotence and bladder problems. These are followed by abnormalities of the cardiovascular reflex tests. The final phase is the development of the so- called late severe symptoms, which include postural hypotension and gastric problems. It is still unclear whether autonomic neuropathy progresses inevitably through these stages or whether some diabetic patients show a halted development demonstrating only a few of the early features.
Development of the late symptoms has a poor prognosis. In a study by
Ewing et al (1980) 50% of the diabetic patients with abnormal autonomic function died within two and a half years. In addition diabetic patients with
23 the greatest number of abnormal test results had the highest mortality rate (Clarke and Ewing, 1982).
Little is known about the histopathological changes associated with autonomic neuropathy. Axonal degeneration leading to a loss of unmyelinated and small myelinated axons has been reported in a number of studies (Martin, 1953; Low et al, 1975). However in none of these studies was a quantitative assessment made. In a more extensive study Olsson and Sourander (1968) demonstrated diffuse degenerative changes in the sympathetic preganglionic communicating rami. These changes included axonal degeneration and segmental demyelination.
1.2.5 Macrovascufar Disease
Atherosclerotic large vessel disease is another complication associated with diabetes that is seen in both insulin-dependent and non- insulin-dependent diabetic patients. A number of prospective studies have shown convincingly that the prevalence of cardiovascular disease (CVD) is greater in diabetic patients than in non-diabetic patients. The Framingham study (Kannel and McGee, 1979) found that male diabetic patients were twice as likely to develop CVD as aged-matched non-diabetic patients, and diabetic women were almost three times as likely to develop CVD. When adjusted for other major risk factors, such as hypertension, cholesterol, and smoking, the relative risk of developing coronary heart disease (CHD), cerebrovascular disease and peripheral vascular disease are the same for both sexes, with the relative risk of CHD and cerebrovascular disease being doubled, and the relative risk of peripheral vascular disease being quadrupled in the diabetic population. Furthermore the relative risk of death as a result of CVD is almost doubled in diabetic men and nearly tripled in women.
The Bedford study (Jarrett et al, 1982) similarly reported an increased mortality from CHD in diabetic subjects compared to control subjects, with borderline diabetic subjects being intermediate between the two. A study of non-insulin-dependent patients (Uusitupa et al, 1985) found symptoms of CHD to be twice as high in male and female diabetic patients compared to controls. Furthermore, the prevalence of definite myocardial infarction was increased 1.7-fold in males and 4.4-fold in female diabetic patients.
CVD in diabetic patients is similar to that seen in non-diabetics, and its incidence is influenced by the same risk factors (WHO Expert Committee on Diabetes Mellitus, 1980). However diabetes itself is a risk factor for CVD (Epstein, 1967), and where present it accelerates the natural progression of CVD ( Robertson and Strong, 1968). That diabetes is not a primary cause of
CVD is emphasised by the fact that in countries, such as Japan, where the incidence of CVD is low in the general population, the incidence is also low in the diabetic population (WHO Expert Committee on Diabetes Mellitus,
1980). Diabetes also removes the relative protection that females have against developing CVD when compared to their male counterparts. In the
Bedford study (Jarrett et al, 1982) the control group showed the normal higher mortality rate from CVD in men compared to women, but in both the borderline and definite diabetic groups the mortality rate was the same if not higher in the women. This finding is in agreement with the Framingham
25 study (Garcia et al, 1973) which reported a higher mortality rate in women than in men, particularly in those receiving insulin treatment.
26 1.3 The pathogenesis of the diabetic complications
Those tissues that develop the diabetic complications are freely permeable to glucose (Gabbay, 1973), and glucose uptake is insulin- independent. Therefore since intracellular glucose concentrations mirror blood glucose concentrations hyperglycaemia has its maximum effect on cellular metabolism in these tissues. However the precise role of hyperglycaemia in the development of the diabetic complications is not fully understood.
1.3.1 Glycaemic control and development of diabetic
complications
1. Retinopathy
A number of studies have examined the relationship between glycaemic control and the development of retinopathy. Pirart (1978) in a retrospective study of 4398 diabetic patients found the prevalence of retinopathy to be higher in the group of patients with chronic poor control.
Moreover, the development of proliferative retinopathy was rare in patients with satisfactory chronic control. The Wisconsin study (Klein et al, 1984b) found that patients with a glycosylated haemoglobin value in the highest quartile had a significantly greater prevalence of retinopathy than those with values in the lowest quartile. Dornan et al (1982a) found mean blood glucose levels to be significantly lower in patients who remained free of retinopathy for 30 years than in patients who developed either background or proliferative retinopathy. These authors also found an inverse relationship between blood glucose levels and the duration of diabetes prior
27 to the development of both background and proliferative retinopathy, suggesting that development of retinopathy is a function of the degree and duration of glycaemic exposure.
The advent of more effective methods of treatment that allow better long-term control of glycaemia has permitted controlled prospective trials to be carried out to ascertain the relationship between quality of glycaemic control and the development of complications (Raskin and Rosenstock, 1986). The Steno study (Steno study group, 1982) found that, although treatment by continuous subcutaneous insulin infusion (CSII) led to a near normalisation of blood glucose and glycosylated haemoglobin levels, it was associated with a deterioration in retinal morphology, the frequency of deterioration being greatest in the CSII-treated group. Despite this initial deterioration, after six months the CSII treated group showed a significant improvement in retinal function compared to the conventionally treated group and after two years of CSII treatment improvement of retinopathy was more frequent than in the conventionally treated patients (Lauritzen et al, 1983). Similarly the Kroc Collaborative study group (1984) found that treatment for one year by CSII did not retard the progression of established retinopathy, but after 2 years retinopathy in the CSII-treated group had stabilised, whereas it had continued to progress in the conventionally treated group (The Kroc Collaborative Study Group, 1985). These findings suggest that the development of retinopathy is related to poor glycaemic control, although intensive treatment may cause a transient acceleration of established retinopathy.
28 2. Nephropathy
Pirart (1978) found that the prevalence of nephropathy was greater in patients with the worst glycaemic control. Whereas the Kroc (1984) and Steno (1982) study groups both showed transient acceleration of retinopathy with CSII treatment, in both studies kidney function was improved in CSII treated patients. Both groups reported a fall in urinary albumin excretion rate and the Steno group also reported a fall in GFR. Other studies have also shown that early structural and functional changes characteristic of diabetic nephropathy respond to improved glycaemic control. Mogensen and Andersen (1975) demonstrated a reduction in kidney volume following improved glycaemic control in newly diagnosed insulin-dependent diabetic patients. By contrast Wiseman et al (1985) however showed that treatment with CSII although leading to a fall in GFR, caused no change in kidney volume.
Once overt clinical nephropathy has developed, strict glycaemic control appears to have little effect on the progressive decline in renal function. Viberti et al (1983a) were unable to show any significant beneficial effect of CSII treatment in six patients with nephropathy, although in one patient treatment slowed considerably the rate of fall in GFR.
3. Neuropathy
Studies have shown that there is a positive correlation between glycaemic control and both the clinical and electrophysiological symptoms of neuropathy. Pirart (1978) reported that the prevalence of neuropathy increased with poor control and that after 25 years duration of diabetes the prevalence of neuropathy in patients with satisfactory control was between 10 and 20%, whereas in patients with chronic poor control the prevalence was just below 70%. At the time of diagnosis of insulin-dependent diabetes nerve conduction velocity is slightly lowered (Ward et al, 1971), a defect that improves rapidly with the onset of insulin therapy, but deteriorates without it. Graf et al (1979) found that glycosylated haemoglobin levels were inversely correlated with nerve conduction velocity in untreated NIDDM patients. The reduction in hyperglycaemia with the initiation of treatment led to a proportional improvement in motor nerve conduction velocity, but no improvement in sensory nerve conduction velocity (Graf et al, 1981).
CSII therapy has also been reported to improve nerve conduction velocity in a number of studies. Troni et al (1984) reported an increase in H- reflex conduction velocity following 2 days CSII treatment in 10 insulin- dependent patients. Normal H-reflex conduction velocity was only achieved in one patient however. In another 12 insulin-dependent subjects, CSII treatment for 6 months caused a progressive rise in H-reflex conduction velocity, the increase paralleling improved glycosylated haemoglobin levels. Similar findings have been reported by Boulton et al (1982). In their study near normalisation of blood glucose levels by CSII treatment for 4 months resulted in significant improvements in motor nerve conduction velocity and vibration perception threshold.
Further evidence linking poor glycaemic control to the development of neuropathy has come from 2 short controlled intervention studies. Holman et al (1983) in a study on 74 insulin-dependent patients randomly assigned to conventional or intensive therapy, found at the end of 2 years that vibratory perception threshold had increased in those patients conventionally treated, and decreased in those that received intensive
30 treatment. This suggests that intensive treatment prevents a deterioration in sensory perception that occurs in conventionally treated patients. Service et al (1985) demonstrated a statistically significant difference in nerve conduction velocity and vibratory perception threshold between a group of CSII treated patients and a group of conventionally treated patients, the differences being in favour of the CSII treated patients.
4. Macrovascuiar disease
Even minor elevations in blood glucose levels appear to lead to an elevated risk of macrovascuiar disease. The Bedford study (Jarrett et al, 1982) found that even borderline diabetic patients showed an increased mortality rate when compared to controls. Furthermore Pirart (1978) found that the prevalence of atherosclerosis was as high in patients with good control as it was in those with poor control.
1.3.2 Other factors in the development of diabetic complications
The evidence reviewed above strongly suggests a relationship between the quality of glycaemic control and the development of the diabetic complications. However further long-term prospective studies, such as the
Diabetes Control and Complications Trial (Diabetes Control and
Complications Trial, 1983), are required to determine whether strict glycaemic control will prevent the development of clinical complications.
A point that warrants special emphasis is that the diabetic complications do not develop in all diabetic patients. Epidemiological studies show that the prevalence of complications does not reach 100% (Palmberg et al, 1981; Pirart, 1978; Anderson et al, 1983). This is particularly striking in the case of diabetic nephropathy. Whereas more than 90% of diabetic patients show signs of glomerulosclerosis, less than 50% go onto develop clinical nephropathy (Deckert and Poulsen, 1981). Thus some diabetic patients, regardless of duration or severity of disease, do not develop clinically apparent complications. This group makes up approximately 20-25% of all diabetic patients (Raskin and Rosenstock, 1986).
In addition a small number of patients (<5%) develop complications after a short duration of disease and despite apparently good glycaemic control (Raskin and Rosenstock, 1986; Boulton et al, 1984). This implies that other factors, both genetic and non-genetic, may influence individual susceptibility to the diabetic complications, a view that is supported by a number of clinical studies. One such clinical study has looked at retinopathy in identical twins (Leslie and Pyke, 1982). In a study of 95 pairs of identical twins these authors found that in 37 pairs of non-insulin-dependent diabetic twins 35 pairs had the same degree of retinopathy. The results were not as clear cut for 58 pairs of twins with insulin-dependent diabetes. In this group 31 pairs were concordant for diabetes, 21 out of the 31 pairs having similar degrees of retinopathy. It was concluded that genetic factors were involved in the determination of frequency and severity of retinopathy in NIDDM, but that in the case of IDDM this relationship was obscured by the involvement of non-genetic factors. Recent work by Seaquist et al (1989) supports the idea that genetic factors are important in the development of nephropathy. They found in a study of 37 families that there was a familial clustering of nephropathy. That is to say, the presence of kidney disease in a sibling is significantly related to the renal status of the proband. 17% of the siblings of probands free of nephropathy showed signs of kidney disease themselves, compared to 83% of the siblings of probands with nephropathy.
Other groups have looked for associations between retinopathy and nephropathy , and genes located in the HLA region. A number of groups have reported an association between HLA-DR4 and severe retinopathy. However this has not been corroborated by all groups (reviewed by Barbosa and Saner, 1984). One study examined the relationship between glycaemic control and HLA-DR4 (Dornan et al, 1982b). They found the prevalence of
HLA-DR4 to be highest in patients with good control and retinopathy, and lowest in those with no retinopathy, despite poor control. In addition two groups (McCann et al, 1983; Mijovic et al, 1985) have reported an association between C4B3, one of the components of the complement system, and retinopathy. Evidence for any HLA association with nephropathy is poor.
An association has also been reported between microangiopathy and a phenotype of the immunoglobulin G heavy chain in insulin-dependent diabetic patients (Mijovic et al, 1986). In a study of 122 insulin-dependent diabetic patients there was an increased frequency of the Gm(zafnbg) phenotype in the group with microangiopathy compared to the group without. Furthermore Baldwin et al (1988) have reported an increased prevalence of the of the phenotype Gm(zafxnbg) in non-insulin-dependent diabetics with retinopathy compared to those without. Other markers that have been looked at include acetylator phenotype, and red cell sodium-lithium countertransport. Initial findings by McClaren et al (1977) suggested that diabetic patients with a fast acetylator phenotype may be protected against developing neuropathy. However this has not been confirmed by subsequent studies (Boulton et al, 1984). A number of recent studies (Mangili et al, 1988; Krowleski et al, 1988) have shown elevated red cell sodium-lithium countertransport activity to associated with diabetic nephropathy. Red cell sodium-lithium countertransport activity is believed to be a marker of familial hypertension (Hilton, 1986), and it has been proposed that the association between it and diabetic nephropathy is in part genetically determined (Mangili et al, 1988).
There is a growing body of evidence suggesting that poorly defined genetic and non-genetic factors may well play a role in the development of the diabetic complications. It has been proposed therefore that the interaction of hyperglycaemia with genetic and environmental factors initiates a sequence of events in susceptible tissues that culminate in the development of the clinical complications of diabetes mellitus (Figure 1.3). Thus hyperglycaemia is presumed to produce the biochemical, functional and structural alterations in target tissues which lead to the onset of complications in susceptible patients, individual susceptibility being influenced by genetic and environmental factors.
34 Independent Genetic or Environmental Variables
GLUCOSE — ■►BIOCHEMICAL------—► ALTERATIONS IN---- ► STRUCTURAL LESIONS-----► C LINICAL ABNORMALITIES IN TARGET TISSUE IN TARGET TISSUES COMPLICATIONS TARGET TISSUES FUNCTION LA T Polyol pathway i Nerve conduction Axonal degeneration Neuropathy Mesangial proliferation Nephropathy 1 Myo-inositol i ANS function Retinopathy Glycosylation i Sensory function T g fr Blood-vitreal leak
Figure 1.3 The glucose hypothesis of diabetic complications (taken from Greene, 1986a). 1.4 Nonenzvmic alvcation of macromolecules
The formation of advanced glycation end products on tissue macromolecules is one potential mechanism linking recurrent hyperglycaemia with the development of diabetic complications.
The first stage of advanced glycation end product (AGE) formation is the generation of early glycation products (Figure 1.4). Glycation begins with the direct nucleophillic addition of a glucose molecule to an amino group (Beswick and Harding, 1985). The reaction of glucose with the amino groups of lysine, or N-terminal amino acid residues, or the amines of nucleic acid bases gives rise to the formation of unstable Schiff base adducts. Schiff base formation reaches equilibrium within a matter of hours (Baynes et al, 1984), with the steady-state level being determined by the glucose concentration over this time. Once formed Schiff bases undergo a slow chemical rearrangement over a number of weeks to form the more stable Amadori product (Higgins and Bunn, 1981). Like Schiff bases, Amadori products are chemically reversible equilibrium products, with equilibrium being reached over a period of around 28 days. The amount of Amadori product formed depends on the integrated glucose concentration over this time. Thus the two chemical determinants of nonenzymic glycationin vivo are glucose concentration and length of macromolecule exposure to glucose. Therefore as glucose concentration rises so does the rate of Amadori product accumulation. Amadori products may be broken down by oxidative degeneration (Figure 1.4). The oxidative cleavage of Amadori products results in the formation of protein-bound carboxymethyllysine
36 • I'» > c h 2o h * I • • NH I ®NH I (CHOH)3 II c h 2 I V CH
0 I O 0 I II c = A (CH0H)4 NH. I —; I (CH0H)4 + (CHOH)3 c h 2 c h 2o h 1 I c h 2o h NH c h 2o h Glucose Protein • SchKf Base ■■ Amadori Product Amadori Product Imidazoles i I Glucose-Derived Protein Crosslinks H - C = O
^ ^ — I COOH c = o NH2 NH I U> 1 c h CHOH - 2 CHO -► Pvrroles 1 I + Protein ! c h 2 H - C -OH CHOH + 1 I COOH H - C -OH R1 R2 1 I c h 2o h CH2OH Electrophillic Pyrrole ----- Intermediate Ervthronic Acid Carboxvmethvllvsine 3:.Deoxy.fllucasgne
Figure 1.4 The formation of early and late non-enzymatic glycosylation products (Adapted from Brownlee et al, 1988). molecules and free erythronic acid (Ahmed et al, 1986). Alternatively they may act as precursors for AGE formation.
AGE formation occurs on macromolecules with low turnover rates, such as matrix components like collagen, and DNA molecules in terminally differentiated cells. On such molecules Amadori products undergo an extensive series of dehydrations, reactions and rearrangements to form complex AGEs (Monnier and Cerami, 1983). In contrast to Amadori products AGEs are irreversibly bound to proteins. Therefore the amount of AGE in tissues does not return to normal levels when hyperglycaemia is corrected, but continues to increase over the lifetime of the tissue (Monnier et al, 1984).
At the present time two general types of AGE have been characterised (Figure 1.4). One is a heterocyclic imidazole derivative, 2-furoyl-4(5) 1-H- imidazole (FFI), which appears to form from the condensation of two Amadori products (Pongor et al, 1984). The other type of AGE appears to be formed from the reaction of an Amadori product with 3-deoxyglucasone, an Amadori-derived compound (Njoroge et al, 1987). The reaction product cyclises to form electrophillic pyrrole intermediates, which may then react with further amino groups to form pyrrole-based cross-links.
The consequences of AGE formation that may result in the development of diabetic complications can be subdivided into those involving cross-links on extracellular matrix proteins, those involving interactions with AGE receptors, and finally those involving macromolecule cross-links within cells.
38 Accelerated AGE cross-link formation as a result of hyperglycaemia leads to the enhanced extracellular accumulation of plasma proteins such as low density lipoprotein (LDL), immumoglobulin G (IgG) and albumin. Brownlee et al (1985) have shown that LDL binding to collagen is increased by nonenzymic glycation of the collagen. This suggests that excessive AGE formation by hyperglycaemia may contribute to the accelerated development of atherosclerosis in diabetic patients. Furthermore the deposition of IgG and albumin in the basement membrane of retinal and glomerular arterioles in diabetes can also be explained by AGE formation, since glycated collagen has been shown to covalently bind both these proteins (Sensi et al, 1986). Moreover, the continued accumulation of plasma proteins by AGE cross- linking could contribute directly to the progressive narrowing and eventual occlusion of blood vessels seen in both diabetic retinopathy and nephropathy. The occlusion of blood vessels is also enhanced by the fact that AGE formation reduces the susceptibility of collagen and basement membrane to enzymatic degradation (Lubec and Poliak, 1980; Schnider and Kohn, 1981). AGE formation also leads to impaired basement membrane assembly (Yurchenco et al, 1986), and a decrease in anionic proteoglycan content (Rohrbach et al, 1982). Such changes have been implicated in the pathogenesis of diabetic nephropathy (Decked et al, 1988).
Another characteristic of diabetic vascular disease is the increased synthesis of matrix components (Brownlee and Spiro, 1979). This matrix proliferation is often accompanied by cellular proliferation. The link between
AGE accumulation and increased proliferation of various cell types appears to be the macrophage. Recently both mouse and human macrophages have been shown to have a unique high-affinity receptor for AGE-proteins (Vlassara et al, 1988). AGE-protein binding to this receptor is believed to initiate a cascade of cytokine-mediated events in the blood vessel wall. AGE-protein binding leads to the production of tumour necrosis factor (TNF), interleukin-1 (IL-1), and possibly other cytokines (Vlassara et al, 1988). These cytokines then bind to a number of cell types where they initiate both a degradative and proliferative response. TNF and/or IL-1 binding to mesenchymal cells initiates the secretion of a number of extracellular
hydrolases, which include collagenase and mesangial neutral protease (Le and Vilcek, 1987; Lovett et al, 1983). The proliferative response is mediated through both the direct growth-promoting effects of these cytokines (Le and Vilcek, 1987), and indirectly through the growth-promoting effects mediated by endothelial cells (Nawroth and Stern, 1986).
The final mechanism through which AGE formation may lead to tissue damage is AGE cross-linking of DNA molecules, and cross-linking between DNA and proteins. Formation of such cross-links has been demonstrated to cause mutation (Bucala et al, 1985), and decreased gene expression (Bucala et al, 1984) which may ultimately lead to cell death.
The identification of a role for nonenzymic glycation in the pathogenesis of the complications of diabetes has prompted the search for inhibitors of AGE formation. One compound that has been selected for study as a possible AGE inhibitor is aminoguanidine (Brownlee et al, 1986). Aminoguanidine reacts with Amadori products to form unreactive substituted early glycation products rather than AGEs (Figure 1.5). Preliminary in vitro and in vivo data (Brownlee et al, 1988) has demonstrated that
40 H O l ."j # > - - — —| • » - ■■ - • ■ | ... \ /^ 1 c r nh 2 ®NH NH 1 II 1 (CHOH)4 CH ch 2 | c h 2oh (CHOH)4 c = 0
c h 2oh (CHOH)3
c h 2oh
Glucose Plots in Schiff-Basa Amadori Product
4^ »—■*
Glucose-Derived Protein Crosslinks Figure 1.5 Prevention ot advanced glycosylation end-product-protein crosslinking by aminoguanidine. (Adapted from Brownlee et al, 1988) aminoguanidine prevents AGE formation, and may well prevent some of the structural changes seen in diabetic complications.
4 2 1.5 The polvol pathway
A second biochemical abnormality associated with hyperglycaemia is elevated flux through the polyol pathway (Gonzalez et al, 1984b). The polyol pathway comprises two enzymes (Figure 1.6): Aldose reductase (alditol : NADP+ 1-oxidoreductase, EC 1.1.1.21), which catalyses the conversion of glucose to its sugar alcohol sorbitol, utilising the coenzyme NADPH; and sorbitol dehydrogenase (L-iditol :NAD+ 5-oxidoreductase, EC 1.1.1.14), which catalyses the conversion of sorbitol to fructose, using the coenzyme
NAD+.
1.5.1 Aldose reductase (EC 1.1.1.21)
The presence of aldose reductase in mammalian tissues was first demonstrated in 1956 by Hers (Hers, 1956). He showed that the seminal vesicles of sheep contained an enzyme capable of converting glucose to sorbitol, which he termed aldose reductase. Since then the presence of aldose reductase has been demonstrated in a wide variety of other mammalian tissues. Its presence has been demonstrated in most human tissues, including brain (Wermuth et al, 1982; Srivastava et al, 1984;
Grimshaw and Mathur, 1989), kidney (Wirth and Wermuth, 1985a; Grimshaw and Mathur, 1989), liver (Wirth and Wermuth, 1985a; Grimshaw and Mathur,
1989), muscle (Grimshaw and Mathur, 1989; Morjana and Flynn, 1989a), erythrocyte (Basak Haider et al, 1980; Srivastava et al, 1984), lens
(Jedziniak et al, 1981, Conrad and Doughty, 1982; Grimshaw and Mathur,
1989), retina (Akagi et al, 1984; Vinores et al, 1988), optic nerve (Akagi et al, 1984), aorta (Srivastava et al, 1984), and placenta (Clements and Winegrad, GLUCOSE A
ALDOSE REDUCTASE (EC 1.1.1.21)
SORBITOL i
SORBITOL DEHYDROGENASE (EC 1.1.1.14)
FRUCTOSE
Figure1.6 The polyol pathway.
44 1972; Kador et al, 1981). Moreover, using immunohistochemical techniques the localisation of aldose reductase within these tissues has been unraveled. Akagi et al (1984) have shown aldose reductase to be present within the epithelium and endothelium cells of the cornea, the epithlial cells of the lens, and the axon of the optic nerve. Wirth and Wermuth (1985a) have demonstrated the presence of aldose reductase in the medulla of the kidney, the cortex of the adrenal gland, and in the Kupffer cells, bile ducts and hepatocytes of the liver. The cellular location of aldose reductase is the cytoplasm (Wermuth and von Wartburg, 1982).
Aldose reductase belongs to a larger family of NADPH-dependent oxidoreductases, known as the aldehyde reductases. Four distinct aldehyde reductases have now been identified in man, these are: 1) Aldose reductase; 2) L-hexonate dehydrogenase; 3) Carbonyl reductase; and 4) Succinic semialdehyde reductase. The two major aldehyde reductases are aldose reductase and L-hexonate dehydrogenase, and both have similar substrate specificities. They may however be distinguished immunologically (Wirth and Wermuth, 1985b), by their relative inhibition by aldose reductase inhibitors (Poulsom, 1986), and by their relative substrate specificities
(Wermuth and von Wartburg, 1982). Aldose reductase has a greater affinity for D-glucose than D-glucuronate, whereas L-hexonate dehydrogenase has a higher affinity for D-glucuronate.
The general reaction catalysed by aldose reductase is shown below:
R - CHO + NADPH + H + ^ R - CH^OH + NADP+ Aldose reductase appears to require its substrate to be present in free aldehyde form, since its Km for sugars varies according to the amount of sugar present as free aldehyde in solution (Hayman and Kinoshita, 1965).
The coenzyme NADPH is essential for the aldose reductase reaction. The hydrogen transfer has been shown to be A-side stereospecific, ie during catalysis the pro 4R "A" hydrogen is transferred from the nicotinamide ring to the substrate (Feldman et al, 1977).
Purification of aldose reductase from a number of sources has revealed it to be a monomeric protein, with a molecular weight between
33000 and 39000 daltons (Srivastava et al, 1984; Kador et al, 1981; Clements and Winegrad, 1972; Wermuth et al, 1982). Two isoenzymes of aldose reductase have been reported in rabbit and rat muscle (Cromlish and Flynn, 1983). Flowever, only one form of aldose reductase was found in
human psoas muscle (Morjana and Flynn, 1989a).
Despite being immunologically distinct , analysis of the amino acid compositions of aldose reductase and L-hexonate dehydrogenase suggests structural homology between the two enzymes (Wermuth et al, 1982; Yoo and McGuinness, 1987). Furthermore analysis of the primary amino acid sequences of human placental aldose reductase and human liver L- hexonate dehydrogenase shows there to be 51% homology between the two (Bohren et al, 1989). Interestingly the amino acid sequences of both enzymes show good homology with p-crystallin, a major lens protein found in the frog. This suggests that the three proteins belong to the same superfamily and have similar evolutionary origins (Carper et al, 1987).
46 Similar relationships have been observed between the amino acid sequences of other crystallins and other enzymes. For example, 8-crystallin
from birds and the enzyme argininosuccinate are 55% identical, and e-
crystallin from birds and reptiles has a similar sequence to the glycolytic enzyme lactate dehydrogenase (Doolittle, 1988). The reason for this
recruitment of enzymes to act as structural proteins in lens is unclear, but presumably involves gene duplication and divergent evolution.
1.5.2 Sorbitol dehydrogenase (EC 1.1.1.14)
Sorbitol dehydrogenase has been identified in a number of mammalian tissues, including human, rat,sheep and horse liver (Leissing and McGuinness, 1978; Jeffery et al, 1984; Porter and McGuinness, 1987;
Maret and Auld, 1988), human and rat brain (O'Brien et al, 1983; Cao Danh et al, 1985), human and rat lens (Varma and Kinoshita, 1974a; Jedziniak et al, 1981), human erythrocyte (Charlesworth, 1972; Barretto and Beutler, 1975), and pig and rat kidney (Op't Hoff, 1969; Cao Danh et al, 1985).
Sorbitol dehydrogenase belongs to the larger alcohol dehydrogenase family (Jeffery et al, 1981), individual members being characterised by the
presence of a zinc atom involved in the reaction mechanism, oligomeric
structures, and distantly related primary structures (Jeffery and Jornvall,
1988). Reported values for the relative molecular mass of sorbitol dehydrogenase vary from 95,000 to 155,000 (Leissing and McGuinness, 1978; Maret and Auld, 1988), with values for the subunits varying from 27,500 to 49,700 (Jeffery and Jornvall, 1988). Sorbitol dehydrogenase catalyses the transfer of a hydrogen atom from the C-2 position of sorbitol to the C-4 position on there side of NAD+.
The sorbitol dehydrogenase gene has been assigned to a single locus on chromosome 15 in humans (Donald et al, 1980). In a study of 665 blood donors one electrophoretic variant of erythrocyte sorbitol dehydrogenase was described (Charlesworth, 1972). However three forms
of sorbitol dehydrogenase have been shown to exist in human liver (Maret and Auld, 1988), although it is unclear whether these represent true isoenzymes. There is however evidence for multiple forms of the enzyme in pig (Op't Hoff, 1969).
1.5.3 Polyol pathway activity in tissues susceptible to the diabetic complications
Apart from in seminal vesicles and placentas, where the polyol pathway provides fructose for the nutrition of sperm and fetuses (Hers, 1960), no definite physiological role has been identified for the polyol pathway. One proposed physiological function for the polyol pathway is as a transhydrogenase system (Kuck, 1961), transferring hydrogen from NADPH to NAD giving rise to the production of NADH. A second proposed function of the pathway is as a route of glucose metabolism that by-passes the control points of glycolysis, namely hexokinase and phosphofructokinase (Jeffery and Jornvall, 1983). This pathway is however only likely to occur in the liver due to the absence of the enzyme fructokinase in other tissues.
48 Another proposed role for the polyol pathway is osmoregulation in the renal medulla (Burg, 1988). Renal cells grown in culture in a hyperosmotic
medium contain high levels of both aldose reductase and sorbitol when compared to similar cells grown in medium with normal osmolality (Bagnasco et al, 1987). Moreover the intracellular sorbitol concentration is high enough to balance the osmotic force exerted on the cells by the medium. Further investigation has revealed that the increase in aldose reductase activity is due to an increase in the amount of enzyme protein (Bedford et al, 1987). In isosmotic conditions low levels of aldose reductase
are present in the cell, whereas under conditions of increased osmolality aldose reductase accounts for more than 10% of the soluble cell protein. The increase in aldose reductase and sorbitol levels following an increase in medium osmolality is slow, becoming maximal after three or four days (Uchida et al, 1987). Reverting to isosmotic conditions results in a slow fall in aldose reductase levels and activity, but a rapid fall in sorbitol levels.
Thus alterations in polyol pathway activity may be important in osmoregulation in the renal medulla.
Whatever its physiological role, at physiological glucose concentrations flux through the polyol pathway is negligible (Gonzalez et al,
1984b), since aldose reductase has a low affinity (high Km) for glucose.
Under such conditions glucose is metabolised primarily by hexokinase, the
first enzyme of the glycolytic pathway (Figure 1.7). In tissues insulin-
independent for glucose uptake, rises in extracellular glucose level are
mirrored by rises in intracellular glucose. Since hexokinase has a low Km
for glucose and is saturated even at physiological concentrations, such rises do not lead to an increase in glycolytic activity (Fujii and Beutler, 1985), and Unregulated transport in
NAD+
Sorbitol dehydrogenase T T NADH Glycolysis Fructose Pentose phosphate pathway
Figure 1.7 Glucose metabolism in tissues susceptible to the diabetic complications (taken from Clarke et al, 1984).
50 glucose accumulates in the cell. The fate of this excess glucose was unclear until Van Heynigen (1959) observed the presence of sorbitol in the lenses of alloxan-diabetic rats. This suggests that excess glucose is metabolised via the polyol pathway. An increase in polyol pathway activity during hyperglycaemia has been confirmed by other studies. Morrison et al (1970) demonstrated that glucose utilisation by the polyol pathway increases from 3% to upto 11% in erythrocytes incubated in 50mM glucose, and Gonzalez et al (1984b) demonstrated that flux via the polyol pathway accounts for 1/3 of total glucose turnover in rabbit lenses incubated in 35.5mM glucose. Elevated polyol pathway activity has now been established as a factor in the development of the diabetic complications, and a number of mechanisms have been proposed by which glucose metabolism, via the pathway, causes the onset of complications.
1.5.4 The polyol pathway and sugar cataract formation
An understanding of how polyol pathway activity leads to the onset of diabetic complications stems from studies on the development of sugar cataract in the lens. In 1955 Friedenwald and Rytel observed that the earliest morphological change in the lens during experimental cataract development is the appearance of hydropic lens fibres, and that in later stages of development these hydropic cells rupture leading to interfibrillar clefts filled with protein aggregates (Friedenwald and Rytel, 1955). Later
Dische et al (1957) demonstrated that the increase in lens weight in galactose-fed rats is due almost entirely to water uptake. Together with Van
Heynigen's finding (1959) that polyols accumulate in the lenses of rats with sugar cataract, these observations led Kinoshita et al (1962) to propose the osmotic stress hypothesis of sugar cataract formation. This proposed that the accumulation of polyols, such as sorbitol and galactitol, in lens fibres as a result of polyol pathway activity might be sufficient to create an osmotic disturbance. An increase in such osmotically active substances would cause an influx of water into the lens fibres. Since cell membranes are relatively impermeable to polyols, their concentrations would reach a level that would increase the volume of water sufficiently to cause the lens fibres to swell and rupture their membranes. This hypothesis was tested by measuring galactitol levels in galactose-fed rats (Kinoshita et al, 1962). Like glucose, galactose is converted to its sugar alcohol, galactitol, by aldose reductase. Unlike sorbitol, galactitol is not metabolised by sorbitol dehydrogenase, and therefore galactitol accumulates to a greater degree than sorbitol. As a result sugar cataracts develop earlier in galactose-fed than in diabetic rats. Galactose feeding caused a rapid increase in galactitol levels. Furthermore the accumulation of galactitol led to a proportional increase in water content. Moreover the increase in water content was sufficient to cause rupturing of lens fibres and hence cataract development.
The changes that occur in the lens during sugar cataract development are summarised in Figure 1.8. As polyols accumulate, in response to elevated polyol pathway flux, a hypertonic condition is created within the lens. To maintain osmotic equilibrium water is drawn into the lens fibres leading to swelling. As a result of this swelling an increase in membrane permeability occurs. This is accompanied by changes in the intracellular levels of electrolytes, with intracellular sodium rapidly rising and potassium falling. In addition there is a decrease in cellular levels of reduced Normal Vacuolar stage Cortical cataract Nuclear cataract
Glucose
Glucose
Ln
Figure 1.8 The intracellular biochemical changes that accompany diabetic cataract formation. K denotes potassium; Na, sodium; Cl, chloride ion; AA, amino acids; ATP, adenosine triphosphate; GSH, reduced glutathione, (taken from Kador and Kinoshita,1985). glutathione, myo-inositol, ATP and free amino acids. Eventually the swollen lens fibres rupture forming vacuoles. As membane permeability increases, the intracellular level of sodium excedes that of potassium and there is a cessation of protein synthesis causing a loss of dry weight. At this stage the increasing number of vacuoles leads to cortical opacification. Progression to the final nuclear cataract stage is accompanied by complete loss of osmotic integrity, with electrolytes, amino acids and proteins being able to freely penetrate the highly permeable lens.
This osmotic theory is supported by a variety of studies. Lens incubated in vitro in media containing elevated levels of glucose or galactose show increased hydration and polyol accumulation. This results in membrane permeability changes, alterations in biochemical parameters, and opacification similar to that observed in vivo (Kador and Kinoshita, 1985). Moreover, these changes can be prevented by incubating the lenses in osmotically compensated media.
In the diabetic rat lens, aldose reductase activity is elevated and sorbitol dehydrogenase activity is decreased when compared to non diabetic rat lens (Varma and Kinoshita, 1974a). Both these changes favour sorbitol accumulation and hence cataract formation. The importance of aldose reductase in cataract formation is also supported by the fact that cataracts are not formed in lenses when aldose reductase levels are low (Kuck, 1970; Varma and Kinoshita, 1974b), and that cataracts develop faster when aldose reductase levels are high (Varma et al, 1977). Kuck (1970) and Varma and Kinoshita (1974b) found no evidence of cataracts in diabetic mice, and despite elevated plasma glucose levels, the lenses contained
54 only very low levels of sorbitol. In contrast, the degu, a rodent native to the Andes, rapidly develops cataract following the induction of diabetes. Aldose reductase in the lens of the degu was found to be three to four times higher than that seen in the rat lens (Varma et al, 1977). Aldose reductase activity is lower in the human lens than in a number of animal lenses (Jedziniak et al, 1981). However it is strictly compartmentalised to the epithelial cells (Akagi et al, 1984) giving rise to a localised accumulation of sorbitol. Such a localised accumulation is capable of producing an osmotic pressure of a magnitude that has been shown to cause cataract formation in rabbit lenses
(Chylack and Kinoshita, 1969).
The most convincing evidence implicating polyol pathway activity in sugar cataract formation has come from experiments using aldose reductase inhibitors. The prevention of cataract formation by aldose reductade inhibition was first demonstrated in vitro by Kinoshita et al (1968) using tetramethylene glutaric acid (TMG). TMG prevented galactitol formation in lenses incubated in medium high galactose levels. Moreover, secondary changes, such as osmotic swelling, electrolyte imbalance, and opacity formation were also prevented. Alrestatin was the first orally effective aldose reductase inhibitor, and demonstrated that aldose reductase inhibition could delay cataract formation in vivo (Dvornik et al, 1973). Rats fed on a diet containing 30% galactose and 0.7% alrestatin showed a markedly delayed onset of cataract when compared to control rats. Since alrestatin, most orally effective aldose reductase inhibitors have been shown to be capable of preventing or even reversing cataract development in galactosemic or streptozotocin diabetic rats (Peterson et al, 1979; Hu et al, 1973; Simard-
Duquesne et al, 1985; Beyer-Mears et al, 1986a; Stribling et al, 1985). 1.5.5 The role of the polyol pathway In other diabetic
complications
In addition to its role in the pathogenesis of sugar cataract, polyol pathway activity has been implicated in the pathogenesis of diabetic neuropathy, retinopathy and nephropathy.
Aldose reductase has been shown by a variety of techniques to be present in the nerve (McDonald et al, 1987; Akagi et al, 1984; Naeser et al, 1988), as has the accumulation of sorbitol and fructose (Stribling et al, 1985; Finegold et al, 1983; Gabbay, 1969; Naeser et al, 1988; Tomlinson and Mayer, 1985; Hotta et al, 1985; Walker-Griffin et al, 1987; Mayhew et al, 1983; Hale et al, 1987). Furthermore this accumulation can be prevented by aldose reductase inhibitor treatment (Gabbay, 1969; Finegold et al, 1983; Stribling et al, 1985; Naeser et al, 1988; Tomlinson and Mayer, 1985; Hotta et al, 1985; Walker-Griffin et al, 1987). Moreover aldose reductase inhibitor treatment has been shown to reverse defects in nerve function in diabetic patients and rats, such as decreased nerve conduction velocity (Hotta et al,
1985; Stribling et al, 1985; Tomlinson et al, 1984; Judzewitsch et al, 1983;
Greene et al, 1987a), impaired axonal transport (Tomlinson et al, 1984; Dahlin et al, 1987), and impaired sodium-potassium dependent ATPase activity (Greene and Lattimer, 1984a).
Polyol pathway activity has similarly been associated with retinopathy.
Aldose reductase has been identified in the retina (Vinores et al,1988; Akagi et al, 1983 and 1984; Naeser et al, 1988; Gabbay and Cathcart, 1974),
56 although initially thought to be localised in the Muller cells (Ludvigson and Sorenson, 1980), it has since been shown to be present in most regions of the retina (Vinores et al,1988; Akagi et al, 1984). In addition sorbitol accumulation and its reversal by aldose reductase inhibitors has also been demonstrated in the retina (MacGregor et al, 1986a; Naeser et al, 1988; Akagi et al, 1983; Nagata and Robison, 1987; Segawa et al, 1988). Moreover, as with neuropathy, aldose reductase inhibition prevents functional and structural changes, such as electro reti nog ram abnormalities
(Segawa et al, 1988), retinal capillary basement membrane thickening (Robison et al, 1986; Chandler et al, 1984), retinal inner limiting membrane thickening (Nagata and Robison, 1987), and blood-retinal barrier alterations
(Cunha-Vaz et al, 1986).
Aldose reductase has also been demonstrated immunohistochemically in human (Grimshaw and Mathur, 1989; Wirth and
Wermuth, 1985; Corder et al, 1979), dog (Kern and Engerman, 1982), and rat kidney (Ludvigson and Sorenson, 1980; Kikkawa et al, 1987; McDonald et al, 1987). It has also been shown in the renal medulla cells (Wirth and
Wermuth, 1985a; Bedford et al, 1987) and in the epithelial and mesangial cells of the glomerulus (Ludvigson and Sorenson, 1980; Kikkawa et al,
1987). Sorbitol accumulation has also been reported in the kidney, as has its prevention by aldose reductase inhibition (Maueret al, 1989; Beyer-
Mears et al, 1984; Kikkawa et al, 1987). Aldose reductase inhibition has been reported to prevent glomerular basement membrane thickening and increase mesangial volume (Mauer et al, 1989), diminish proteinuria (Beyer-
Mears et al, 1986b), and to reverse decreased glomerular sodium- potassium ATPase activity (Cohen et al, 1985). Thus primarily through aldose reductase inhibitor studies, polyol pathway activity has been implicated in the development of a number of the diabetic complications other than sugar cataract. It is however difficult to apply the osmotic theory directly to the pathogenesis of neuropathy, retinopathy and nephropathy. Whilst sorbitol has been shown to accumulate in nerve (Gabbay, 1969), retina (MacGregor et al, 1986a), and the glomerulus of the kidney (Beyer-Mears et al, 1984), it does not accumulate to the same extent as seen in the lens (Gabbay, 1973). Therefore sorbitol accumulation in these tissues is unlikely to produce a significant osmotic force unless highly localised (Clements, 1979; Ludvigson and Sorenson,
1980). Such localisation seems unlikely since aldose reductase activity (Vinores et al, 1988) and sorbitol accumulation (MacGregor et al, 1986a) has been demonstrated in most regions of the retina, and moreover increased tissue water content does not appear to be necessary for the impairment of nerve conduction (Greene, 1983). As osmotic effects are unlikely to give rise to the development of neuropathy, retinopathy and nephropathy a second mechanism, involving alterations in myo-inositol metabolism, has been proposed to link polyol pathway activity to the development of these complications (Greene et al, 1988).
1.5.6 Myo-inositol metabolism and the complications of diabetes
The link between altered myo-inositol metabolism and the development of neuropathy was first demonstrated in 1975. Greene et al
(1975) found that streptozotocin-diabetic rats had decreased motor nerve conduction velocities (MNCV) and decreased myo-inositol levels. Intensive
58 insulin therapy not only normalised MNCV but also corrected nerve myo inositol levels. Furthermore, when the diets of untreated rats where supplemented with 1% myo-inositol similar corrections to decreased MNCV and depleted myo-inositol levels where observed. Since myo-inositol treatment did not alter blood glucose levels, it was therefore proposed that the defect in MNCV seen in diabetes is mediated through abnormalities in myo-inositol metabolism (Winegrad and Greene, 1976). Similar decreases in nerve myo-inositol in the rat and its correction by myo-inositol treatment have been reported by a number of other groups (Stewart et al, 1967; Palmano et al, 1977; Yue et al, 1984; Gillon et al, 1983), and decreased myo-inositol levels have been reported in sciatic nerves, obtained post mortem, of diabetic patients when compared to normal subjects (Mayhew et al, 1983). However such findings have not been universally reported
(Jeffreys et al, 1978; Hale et al, 1987; Poulsom et al, 1983; Dyck et al, 1988; Cameron et al, 1986; Low et al, 1986).
The link between altered myo-inositol metabolism and decreased nerve conduction velocity is believed to be due to decreased membrane- bound sodium-potassium ATPase (Na-KATPase) activity (Greene et al,
1985). Decreased nerve Na-KATPase activity in diabetes was first demonstrated by Das et al (1976), and Clements (1979) speculated that the observed decrease in Na-KATPase might be due to decreased nerve myo inositol. Evidence has since been gathered that implicates myo-inositol depletion in the impairment of Na-KATPase activity. Simmons et al (1982) demonstrated that energy utilisation was diminished when tissue myo inositol was slightly depleted in in vitro incubations. In addition, decreased Na-KATPase activity in sciatic nerve homogenates from streptozotocin- diabetic rats was prevented by feeding the rats a diet supplemented with myo-inositol (Greene and Lattimer, 1983). Moreover, in untreated spontaneously diabetic BB rats there are parallel decreases in nerve myo inositol, Na-KATPase activity and nerve conduction velocity. Insulin treatment or myo-inositol supplementation that restores nerve myo-inositol levels also leads to an increase in both Na-KATPase activity and nerve conduction velocity (Greene, 1986b).
It has been speculated that myo-inositol modulates Na-KATPase activity through its incorporation into phosphatidylinositol and subsequent breakdown (Greene et al, 1985). Breakdown of phosphatidylinositol yields inositol triphosphate and diacylglycerol (Berridge, 1984) both of which, either directly or indirectly through calcium mobilisation, activate protein kinase C. As reduced Na-KATPase activity has been linked to decreased protein kinase C activity (Greene and Lattimer, 1986), myo-inositol metabolism may be linked to Na-KATPase activity through protein kinase C activity, modulated by alterations in phosphoinositide turnover.
In addition to its role in decreased nerve conduction velocity, reduced
Na-KATPase activity has also been implicated in decreased uptake of myo inositol (Greene and Lattimer, 1984b). The myo-inositol concentration of peripheral nerve is 90-100 fold greater than that found in plasma. This concentration gradient is believed to be maintained by a sodium-dependent carrier mediated transport system driven by the sodium gradient generated by the Na-KATPase pump (Greene and Lattimer, 1983). This led to the proposal that decreased Na-KATPase activity might cause a decrease in nerve myo-inositol uptake, which has been supported by the findings that
60 improvements in Na-KATPase activity correct sodium dependent myo inositol uptake in vitro (Greene and Lattimer, 1984b).
Evidence linking polyol pathway activity and nerve conduction defects mediated by altered myo-inositol metabolism has also come from studies involving aldose reductase inhibitors (ARIs). As described earlier, aldose reductase inhibition prevents the slowing of nerve conduction in diabetic rats. In addition aldose reductase inhibitors prevent myo-inositol depletion (Finegold et al, 1983; Greene et al, 1987a; Gillon et al, 1983; Tomlinson et al, 1984), and in some, but not all, studies ARIs have been shown to prevent the associated decrease in Na-KATPase activity (Greene and Lattimer, 1984a; Greene et al, 1987a; Lambourne et al, 1988). The mechanism linking polyol pathway activity and myo-inositol metabolism remains unclear. One study has shown that sorbitol accumulation in cultured neuroblastoma cells causes a decrease in sodium-dependent myo-inositol transport resulting in decreased myo-inositol levels, and moreover this is prevented by aldose reductase inhibition (Yorek et al, 1987). Whilst the precise mechanism remains uncertain, a self perpetuating relationship (Figure 1.9) has been proposed linking polyol pathway activity, myo-inositol metabolism,
Na-KATPase activity and nerve conduction defects (Greene et al, 1987b).
Defective nerve conduction velocity in diabetic rats may be explained by the accumulation of sodium in nerve fibres as a result of impaired Na-
KATPase activity. Na-KATPase activity has been shown to be localised at the nodes of Ranvier (Vorbrodt et al, 1982). This causes a localised accumulation of sodium that blocks nodal depolarisation and which may cause diminished nerve conduction velocity (Brismar and Sima, 1981; Hyperglycaemia
Competitive t Polyol Pathway Inhibition Activity + i Na*" -dependent Ml uptake
^Tissue I Na+/K+ATPase Ml \ctivity
-I Protein Kinase C i Phophoinositide Activity Metabolism
si Diacylglycerol Availability y
Figure 1.9 Proposed self-perpetuating metabolic defect involving polyol pathway activity, phosphoinositide metabolism, protein kinase C, and sodium-potassium-ATPase. Ml denotes myo-inositol; Na+/K+ATPase, sodium-potassium- ATPase; and Na+, sodium ion. (Adapted from Greene et al, 1987b). Greene et al, 1984). In addition persistent localised swelling at the node of Ranvier is believed to contribute to axo-glial dysjunction, one of the earliest structural changes seen in diabetic BB rats and insulin-dependent diabetic humans with neuropathy (Sima et al, 1988 and 1986).
An additional mechanism that may play a role in the development of diabetic neuropathy is altered axonal transport (Tomlinson and Mayer, 1984). A number of studies have shown slow anterograde transport of axonal components to be defective in diabetic animals (Jakobsen and Sidenius, 1980; Sidenius and Jakobsen, 1982; Mayer et al, 1984; Tomlinson et al, 1984 and 1986; Robinson et al, 1987; Willars et al, 1987). Moreover, in some cases, defective axonal transport may be prevented or reversed by myo-inositol or aldose reductase inhibitor treatment (Tomlinson et al, 1984; Mayer and Tomlinson, 1983). This has led to the proposal that altered myo inositol metabolism, as a result of elevated polyol pathway flux, may be responsible for certain of the axonal transport abnormalities seen in diabetic neuropathy.
There is a body of evidence implicating altered myo-inositol metabolism in the pathogenesis of the other complications of diabetes. Myo inositol depletion has been demonstrated in the retina (MacGregor et al,
1986a; MacGregor and Matschinsky, 1986), the kidney (Beyer-Mears et al, 1984), and in the blood vessel wall (Morrison, 1984). In addition, decreased
Na-KATPase activity has also been shown in these tissues (MacGregor et al,
1986b; Cohen, 1986; Simmons and Winegrad, 1989). As in the nerve, aldose reductase inhibition has been shown to prevent these defects (MacGregor and Matschinsky, 1985; Cohen et al, 1985). This has led to the proposal that altered myo-inositol metabolism and Na-KATPase activity, as a result of elevated polyol pathway activity, may provide a common mechanism linking hyperglycaemia to the development of the diabetic complications (Winegrad, 1987).
However there is a growing body of evidence to suggest that the relationship between myo-inositol metabolism, Na-KATPase activity, and the pathogenesis of the complications is not as straight forward as has been proposed. For example, myo-inositol depletion has not been found in all studies, particuarly in those involving humans (Jeffreys et al, 1978; Hale et al, 1987; Poulsom et al, 1983; Dyck et al, 1988; Cameron et al, 1986; Low et al, 1986). One explanation proposed for this discrepancy is that myo-inositol uptake into discreet pools, rather than total nerve myo-inositol, may control Na-KATPase activity, and this impaired uptake may not be reflected by a decrease in total nerve myo-inositol (Winegrad et al, 1989). Moreover any decrease in myo-inositol may be a transient effect, seen in acute but not chronic diabetes (Cameron et al, 1986).
In addition, the role of Na-KATPase as a link between myo-inositol metabolism and nerve conduction defects has been questioned. Whilst
Greene and Lattimer (1983 and 1984a) have reported that aldose reductase inhibition prevents decreased Na-KATPase activity in diabetic rats,
Lambourne et al (1988) found that aldose reductase inhibition had no effect on Na-KATPase activity in the nerves of streptozotocin-diabetic rats.
Furthermore whilst ganglioside treatment prevented the fall in Na-KATPase activity in diabetic rat nerve, it had no effect on either nerve conduction velocity or myo-inositol levels (Calcutt et al, 1988). In the kidney while Na-
64 KATPase activity is decreased in acute diabetic rats (Cohen, 1986) it is elevated when diabetes is present for greater than 3 weeks (Cohen and Klepser, 1988). Moreover the decrease in Na-KATPase activity seen in the acutely diabetic rat preceeds any detectable decrease in tissue myo-inositol (Cohen, 1990).
These findings argue against the proposed relationships linking myo inositol, Na-KATPase activity, and the pathogenesis of the diabetic complications and suggest that the mechanism or mechanisms through which myo-inositol metabolism has its effect remain to be resolved.
1.5.7 Polyol pathway activity and altered cellular redox state
A third potential mechanism linking elevated polyol pathway activity and the pathogenesis of the diabetic complications is altered cellular redox state. Increased flux through the polyol pathway leads to alterations in the ratios of reduced and oxidised nucleotides, the conversion of glucose to sorbitol affecting the NADPH/NADP+ ratio and the conversion of sorbitol to fructose affecting the NAD+/NADH ratio. Altered cellular redox state has been implicated primarily in caractogenesis in the lens. Incubations of rat lenses in high glucose concentrations leads to an increase in NADH and a fall in NAD+ levels, resulting in an increased NADH/NAD+ ratio (Cheng and
Gonzalez, 1986). Such an increase in the NADH/NAD+ ratio might potentially result in impaired glycolytic flux because of the reduced availability of NAD+ for glyceraldehyde-3-phosphate dehydrogenase. In addition an increased NADH/NAD ratio might also inhibit respiration and moreover decreased ATP levels have been observed in lenses incubated in high glucose concentrations (Gonzalez et al, 1984a). There is indirect evidence for a similar increase in the NADH/NAD+ ratio in human diabetic lenses (Cheng and Gonzalez, 1986). The increase in NADH/NAD+ ratio is linked to the polyol pathway since addition of the aldose reductase inhibitor sorbinil to the incubation medium leads to a normalisation of the ratio.
Similarly the NADPH/NADP+ ratio is altered in the lenses of diabetic and galactosemic rats (Varma and Kinoshita, 1974a; Lee et al, 1985). Moreover as a result of increased aldose reductase activity, the turnover of
NADPH to NADP+ is increased by 3000% in rabbit lenses incubated in 35.5mM glucose (Gonzalez et al, 1984b), and by 900% in the human lens incubated in similar conditions (Jedziniak et al, 1981). This excessive use of NADPH by aldose reductase may hinder the lens's ability to maintain its sulphydryl state through the NADPH-dependent glutathione reductase/peroxidase system (Giblin et al, 1981), and therefore reduce its resistance to oxidative stress. Hydrogen peroxide detoxification is inhibited in lenses incubated in 35.5mM glucose and this inhibition is prevented by sorbinil (Cheng and Gonzalez, 1986). In addition NADPH utilisation by aldose reductase may also effect other NADPH-requiring mechanisms, such as fatty acid and cholesterol synthesis. Similar alterations in redox state have also been demonstrated in the erythrocyte incubated in 50mM glucose (Travis et al, 1971), and may well occur in other tissues, although in the nerve glutathione metabolism does not appear to be altered by polyol pathway activity (Carroll et al, 1986).
66 1.5.8 The erythrocyte polyol pathway
The human erythrocyte has been shown to contain sorbitol and fructose at concentrations that exceed those found in the plasma (Morrison et al, 1970). Furthermore intracellular sorbitol and fructose concentrations increased with increasing extracellular concentrations (Morrison et al, 1970). These observations led to the proposal that the polyol pathway is active in the human erythrocyte and that its activity is increased by increasing extracellular glucose concentrations.
However the presence of the polyol pathway in the erythrocyte remained controversial since the identity of the sorbitol producing enzyme was unresolved. Two members of the aldo-keto reductase family, aldose reductase and L-hexonate dehydrogenase, have been shown to be capable of reducing glucose to sorbitol. Since the Km of L-hexonate dehydrogenase for glucose is very high, Travis and co-workers (1971) proposed that it was unlikely that glucose was a substrate for it under physiological conditions, and that the presence of sorbitol in erythrocytes from non-diabetic humans indicated that the enzyme responsible for its formation was aldose reductase. However whilst two groups were able to purify L-hexonate dehydrogenase from human erythrocytes neither group was able to purify aldose reductase (Beutler and Guinto, 1973; Gabbay and Cathcart, 1974), and they concluded therefore that aldose reductase was not present in the erythrocyte. It has since been shown that both enzymes can be purified from the erythrocyte (Srivastava et al, 1984; Nakayama et al, 1989). Further evidence implicating aldose reductase in the production of erythrocyte sorbitol has come from studies involving specific inhibitors of aldose reductase. The aldose reductase inhibitor TMG was shown to prevent the accumulation of sorbitol in erythrocytes incubated in vitro with high glucose concentrations (Malone et al, 1980), a finding that has been subsequently corroborated with a variety of other aldose reductase inhibitors (Malone et al, 1984; Popp-Snijders et al, 1984). In addition to aldose reductase the presence of sorbitol dehydrogenase has also been demonstrated in the human erythrocyte (Barretto and Beutler, 1975). As is the case in other tissues the physiological role of the polyol pathway in the erythrocyte is unknown.
In the streptozotocin-induced diabetic rat erythrocyte sorbitol levels have been shown to be closely correlated with both sciatic nerve and lens sorbitol levels. This was also true following treatment with the aldose reductase inhibitor Sorbinil, moreover the dose of Sorbinil required to produce a 50% reduction in erythrocyte sorbitol was similar to that required to produce the same reduction in both nerve and lens (Malone et al, 1984). On the basis of these observations Malone and co-workers proposed that measurements of erythrocyte sorbitol might provide a measure of polyol pathway activity in inaccessible tissues, such as the nerve, lens and kidney, in which direct measurements of polyol pathway activity are impossible
(Malone et al, 1984). In addition the measurement of erythrocyte sorbitol levels has been used by pharmaceutical companies as an indicator of the efficacy of their aldose reductase inhibitors in humans. A variety of inhibitors have been demonstrated to be potent inhibitors of erythrocyte sorbitol accumulation in vivo following periods of treatment ranging from 2 to 12 weeks (Malone et al, 1984; Popp-Snijders et al, 1984; Hotta et al, 1983).
This lowering of erythrocyte sorbitol levels reflects the availability of inhibitor
68 in the blood, a fact demonstrated by the finding that Tolrestat normalises erythrocyte sorbitol levels when steady state plasma concentrations are attained (Raskin et al, 1985). 1.6 Aims of project
The aims of the work described in this Thesis were threefold. The diabetic complications do not develop inexorably in all diabetic patients, which may suggest that other factors, in addition to hyperglycaemia, are involved in their development. Elevated polyol pathway activity is one biochemical abnormality that has been implicated in the pathogenesis of the diabetic complications. Given its proposed role in the development of the complications and the variability in their onset and severity, the question that arises is how polyol pathway activity varies in diabetic and non-diabetic individuals. Accordingly the first aim was to determine to what extent the activities of the component enzymes of the polyol pathway, namely aldose reductase and sorbitol dehydrogenase, vary in the erythrocytes of diabetic and non-diabetic subjects. Whilst the erythrocyte might not be the ideal tissue to use, since it is not a primary site for the development of complications, it is the only tissue available in sufficient quantities for this type of study.
Erythrocyte sorbitol determination has been used in a number of studies as a measure of polyol pathway activity. In particular sorbitol accumulation has been taken to indicate chronic polyol pathway activity.
However it remains unclear how well sorbitol accumulation, or fructose accumulation for that matter, reflects aldose reductase or sorbitol dehydrogenase activities. Therefore a second aim of this study was to examine the relationship between erythrocyte sorbitol and fructose concentrations and the activities of the polyol pathway enzymes. In the streptozotocin-induced diabetic rat there is a good correlation between eythrocyte sorbitol concentration and sciatic nerve and lens sorbitol concentrations. In the light of this finding it has been proposed that erythrocyte polyol pathway activity might provide a good model for polyol pathway activity in less accessible tissues, such as the nerve, lens, and kidney. Measurements of erythrocyte polyol pathway activity might therefore prove valuable in elucidating the role of the pathway in the development of complications in these less accessible tissues. For this to be valid there should be a demonstrable relationship between quantitative measurements of the various complications and erythrocyte polyol pathway activity. Thus the third aim of this Thesis was to determine the relationship between the activities of erythrocyte aldose reductase and sorbitol dehydrogenase and quantitative measurements of autonomic and peripheral neuropathy, retinopathy, and nephropathy in diabetic patients. Chapter Two
Materials and Methods 2.1 Materials
Hexamethyldisilazane (HMDS) and trimethylchlorosilane (TMCS) were obtained from Pierce and Warriner (Chester, UK). Electran grade hydrolysed (Smithies) potato starch was obtained from BDH Chemicals Ltd (Poole, UK), and Agar Noble was obtained from Difco Laboratories (Detroit, Michigan, USA). Glucose oxidase was supplied by the Boehringer Corporation (London) Ltd (Lewes, UK). AH-Sepharose 4B and Matrex Gel Orange A were supplied by LKB-Pharmacia Ltd (Milton Keynes, UK) and Amicon Ltd (Stonehouse, UK) respectively. Goat anti-rabbit IgG-horse raddish peroxidase conjugate was obtained from Sigma (London) Chemical
Company (Poole, UK). All other chemicals and enzymes were supplied by either BDH Chemicals Ltd (Poole, UK) or Sigma (London) Chemical Company (Poole, UK). [1-14C]mannitol was obtained from NEN, Du Pont (UK) Ltd (Stevenage, UK) and n-[1-14C]hexadecane from Amersham International Ltd (Little Chalfont, UK). Nitrocellulose, sold as Hybond C, was supplied by Amersham International Ltd. Ultra pure water was prepared by reverse osmosis followed by deionisation using an Elgastat (Elga, High
Wycombe, UK). 2.2 Methods
The methods described in this Chapter are those methods used throughout the three results Chapters. Specific clinical procedures are described in the Experimental section of the relevant Chapter.
2.2.1 Krebs-Ringer Bicarbonate Buffer Krebs-Ringer bicarbonate buffer, pH 7.4 was made up as follows: Solution A
g in 5 L
NaCI 41.48 KCI 2.12 NaHCOa 1.79
MgS04.7H20 1.76
KH2 PO4 0.96 Solution B 1.3% NaHC03
Solution C 0.11 mol/l CaCl 2 87 ml of solution A were mixed with 14 ml of solution B and the
mixture gassed with O 2/CO2 in a 37°C water bath until equilibrated (approx
20 minutes). 2.4 ml of solution C was then added and the solution was gassed at 37°C until required.
2.2.2 Erythrocyte sorbitol dehydrogenase activity
Erythrocyte sorbitol dehydrogenase activity was measured using the
2-step spectrophotometric assay of Barretto and Beutler (1975). Principle SDH Sorbitol + NAD + Fructose + NADH
Hexokinase Fructose + ATP Fructose-6-Phosphate + ADP
Phosphoglucose Fructose-6-Phosphate Glucose-6-Phosphate Isomerase I + Glucose-6-Phosphate Glucose-6-Phosphate + NAD 6-Phosphogluconate + NADH Dehydrogenase
Procedure Whole blood was collected in EDTA and stored at 4°C. The red cells were washed twice in cold physiological saline (NaCI, 9%w/v), and the supernatant aspirated away. The red cells were then haemolysed by freeze thawing. For the first step of the assay the following reaction mixture was used:
Tube
1 mol/l Tris-HCI pH 8.0 100 pi 50 mmol/l NAD+ 100 pi
200 mmol/l MgCl2 100 pi
1 mol/l sorbitol 100 pi water 400 pi
A control tube with the 1 mol/l sorbitol replaced by water was prepared for each sample. The reaction was started by the addition of 200 pi
of haemolysate to each tube. The mixtures were incubated at 37°C with shaking for 45 minutes, and the reaction was stopped by the addition of 1 ml
7 5 of 7% perchloric acid (PCA). The tubes were then centrifuged at 1200 g for 10 minutes, and 1 ml of the supernatant transferred to a second preweighed tube.The supernatant was then neutralised by the addition of 2 mol/l potassium carbonate and the volume brought to 1.5 ml with water. The fructose content of the supernatant was then assayed using the following system:
Cuvette
1 mol/l Tris-HCI pH 8.0 100 pi
10 mmol/l MgCl2 100 pi 20 mmol/l ATP 100 pi
10 mmol/l NADP+ 50 pi PCA extract 300 pi
water 350pi
glucose-6-phosphate dehydrogenase (300 U/ml) 5 pi hexokinase (2500 U/ml) 5 pi The cuvettes were then incubated at 37°C for 10 minutes, and a baseline reading at 340 nm was taken using a Pye Unicam SP8-400 UV/VIS spectrophotometer. The reaction was started by the addition of phosphoglucose isomerase (5 pi, 100 U/ml). The reduction of NADP to
NADPH was followed at 340 nm, and a final reading taken.
Calculation of specific activity
Sorbitol dehydrogenase specific activities were calculated in the following manner: Spec Act _ ODsamp - ODcont ^ y Dilution factor mg/protein 6.22 45
Extinction Coeffiecent Time of Assay of NADP+ Specific activity expressed as mllnits/mg protein, where 1 unit of activity = 1 pMole of NADP+ reduced per minute.
2.2.3 Measurement of erythrocyte monosaccharide and polyol
concentrations
Erythrocyte monosaccharide and polyol concentrations were measured by gas-liquid-chromatography (GLC) according to the method of Stribling et al (1985).
Reagents 1) a-Methyl-D-mannoside solution 166.5 pi of stock solution (12 mg/ml in
0.25% benzoic acid) was made upto 500 ml with water. 2) Sugar standards Stock solutions (10 mg/ml in 0.25%
benzoic acid) of fructose, glucose, galactose, myo-inositol, and sorbitol were prepared. 40 pi of each sugar
was pipetted into a flask and 31.25 ml
of the diluted mannoside solution added. This gives a 64 pg/5ml sugar
standard solution. 32, 16, 8, and 4
pg/5ml sugar standards were
prepared by diluting this stock solution.
77 3) Silylation Mixture Pyridine: hexamethyldisiiazane (HMDS): trimethylchlorosilane
(TMCS) in the ratio 10:2:1.
Procedure 2 ml of washed erythrocytes were added to 50 ml polycarbonate centrifuge tubes (BDH, Poole, UK) stored on ice, 5 ml of diluted mannoside solution was then added to each tube. This lyses the erythrocytes and the a- methyl-D-mannoside acts as an internal standard. In addition a standard curve was prepared by pipetting 5 ml of each of the sugar standards into polycarbonate tubes. 5 ml of dilute mannoside solution acts as a blank. The polycarbonate tubes were then placed in a boiling water bath for 25 minutes to denature the blood proteins. The tubes were then removed onto ice and 5 ml of water added to each. The denatured protein was broken up with a glass rod and the polycarbonate tubes centrifuged at 1200 g for 15 minutes. The supernatants were then decanted into glass vials and shell-frozen prior to freeze-drying overnight. The contents of each vial were then silylated by adding 0.5 ml of silylation mixture to each. Complete silylation was achieved by leaving the samples at room temperature for 36 hours. After 36 hours the samples were extracted by the addition of 4 ml of water to each vial. The contents of each vial were then transferred to centrifuge tubes and 300 pi of cyclohexane added to each. The tubes were then vortexed and centrifuged at 1200 g for 10 minutes. 200 pi of the organic layer from each tube was transferred into an autosampler vial prior to sugar determination by capillary
GLC. Chromatography was performed on a Varian 3300 gas chromatograph (Varian Instrument Group, Walnut Creek, California, USA) fitted with a Chrompack WCOT fused silica CPSil 8 column (25 m x 0.32 mm)
78 (Chrompack, Middleburg, The Netherlands). The injector temperature was 200°C and the initial column temperature was 100°C. After injection of the sample there was an initial hold period of 2 minutes. The column temperature was then raised at a rate of 2°C/minute for 33 minutes, giving a total running time for each sample of 35 minutes. Sugar peaks were detected by flame ionisation detection (detector temperature 300°C) and the peaks were integrated by an attached Varian 4290 integrator. Sugar peaks were identified by their retention time relative to the internal standard, a- methyl-D-mannoside (Table 2. 1). The relative retention times were determined by running single sugar standards. The ratio of area under the sugar peak to area under the internal standard peak was calculated for each sugar. A graph of standard sugar ratio versus standard sugar concentration
(pg/ml) was plotted and the unknown monosaccharide and polyol concentrations read directly off the standard curve. Concentrations were expressed in pg/ml of erythrocytes.
Validation Experiment
To determine the effect of washing erythrocytes with saline, intracellular glucose, fructose, sorbitol, and myo-inositol concentrations were measured in washed and unwashed erythrocytes using the procedure described above. In the case of unwashed erythroctes an allowance must be made for the contribution of plasma monosaccharides and polyols. Plasma concentrations of glucose, fructose, sorbitol, and myo-inositol were measured as described in Section 2.2.5. The volume of plasma in the erythrocyte pellet was determined by measuring the relative amounts of [1- 14C]mannitol in a sample of plasma and in a sample of erythrocytes. To take account of any quenching all samples were spiked with 10000 dpm of n-[1- Table 2.1 Relative retention times of sugars and polyols separated by gas- liquid-chromatography.
Sugar Relative Retention Time
a-Methyl-mannoside 1 .0 0 a-Fructose 1.07 p-Fructose 1.09
a-Galactose 1 .2 0 a-Glucose 1.26 p-Galactose 1.30 Sorbitol 1.36 Galactitol 1.38 p-Glucose 1.51
Myo-inositol 1 .6 8
80 14C]hexadecane and recounted. Monosaccharide and polyol concentrations measured in unwashed erythrocytes were then corrected by subtracting the contribution due to plasma monosaccharides and polyols. The concentrations of glucose, fructose, sorbitol, and myo-inositol present in washed and unwashed erythrocytes are shown in Table 2 .2 . The washing of erythrocytes with saline prior to the determination of intracellular sugar concentrations leads to a loss of both glucose and fructose. Sorbitol and myo-inositol concentrations, however, are unaffected by the washing procedure. Therefore whilst this technique gives an accurate measure of both intracellular sorbitol and myo-inositol concentrations, it underestimates the concentrations of both glucose and fructose.
2.2.4 Measurement of erythrocyte aldose reductase activity
Aldose reductase activity was measured as galactitol accumulation rate, with the galactitol being measured by GLC according to the method of Stribling et al (1985). Galactose is converted by aldose reductase to its corresponding polyol galactitol. However galactitol, unlike sorbitol, is not metabolised by sorbitol dehydrogenase and so it accumulates in the erythrocyte.
Reagents
1) Krebs-galactose solution Krebs-Ringer bicarbonate buffer was prepared as described earlier. 0.624 g
galactose was added per 1 0 0 ml of buffer. Table 2.2 Monosaccharide and polyol concentrations in washed and
unwashed erythrocytes.
Sugar Washed erythrocytes Unwashed erythrocytes (ng/ml) (gg/ml)
Glucose 95.9 ± 21 a447.3 ± 8.0 Fructose 4.0 ±0.4 a7.2 + 0.4
Sorbitol 3.5 ±0.6 3.2 ±0.5 Myo-inositol 6.4 ±0.8 5.9 ±0.9
Data expressed as mean ± SEM of 5 determinations.
Statistically significant differences denoted by a) p< 0 .0 0 1
82 2) Sugar standards As described earlier in measurement of erythrocyte monosaccharides and polyols
with the sorbitol replaced by galactitol.
Procedure Whole blood was collected into heparin and centrifuged at 800 g for 7 minutes at 0-4°C. The plasma was aspirated away and the red cells resuspended in cold physiological saline (NaCI, 9% w/v). The cells were centrifuged again as before and the supernatant aspirated away. This washing was repeated once more. Washed erythrocytes were stored on ice before use. 8 ml of Krebs-galactose solution was pipetted into 25 ml siliconised glass flasks. 2 ml of washed erythrocytes was added to each flask, giving a final galactose concentration of 27.75 mmol/l (5 mg/ml). Immediately after the addition of washed erythrocytes, each flask was flushed with O 2/CO2 (19:1) for 30 seconds and sealed with laboratory film. Each flask was transferred to a 37°C shaking incubator for exactly one hour.
At the end of the incubation period, the contents of each flask were transferred to a 50 ml polycarbonate centrifuge tube standing on ice. The flask was washed with 1 0 ml of cold saline and the washings added to the polycarbonate tube. The tubes were then centrifuged at 800 g for 7 minutes at 4°C and the resulting supernatant aspirated away. The cells were then washed again and the supernatant aspirated away once more. Erythrocyte galactitol was then measured as described earlier (see measurement of erythrocyte monosaccharide and polyol concentrations).
Calculation of specific activity Erythrocyte galactitol concentrations were determined from a standard curve and expressed in pg/ml red blood cells. Aldose reductase specific activity was then calculated as follows. Spec Act = GalactiJ0' (ff/ml RBC) x _ L - x _1 mg/ml protein 180.2 60
Specific activity was expressed as pU/mg protein, where 1 Unit = 1 pmole of galactose reduced per minute. The coefficient of variation of the assay was < 10%.
2.2.5 Determination of plasma monosaccharides and polyols by gas liquid chromatography
Reagents
1 ) a-methyl mannoside solution 1 ml of stock solution (1 2 mg/ml in
0.25% benzoic acid) was made up to 30 ml with water.
2 ) Sugar standards Stock solutions of glucose, fructose,
sorbitol, and myo-inositol (1 0 mg/ml in 0.25% benzoic acid) were prepared.
0.5 ml of each standard was made up to 250 ml with water to give a 20 pg/ml
stock solution. This was then diluted to give 15, 10, and 5 fig/ml standards.
3) Silylation Mixture Pyridine: hexamethyldisilazane
(HMDS): trimethylchlorosilane
(TMCS) in the ratio 1 0 :2 :1 .
Procedure
1 ml samples of plasma and standards were taken and mixed with 2 ml of 99.9% ethanol in centrifuge tubes. 20 pi (8 pg) of the a-methyl mannoside solution was added to each tube and the tubes were again
84 mixed. The a-methyl mannoside acts as an internal standard. The samples were allowed to stand for 60 minutes before being centrifuged at 16000.g for 20 minutes. The supernatants were then transferred into glass tubes and evaporated under nitrogen. The samples were then silylated by adding 500 pi of silylation mix to each sample and allowing to stand at room temperature overnight. The samples were extracted in the following manner. 4 ml of water was added to each sample followed by 300 pi of cyclohexane. The samples were mixed and centrifuged at 1 2 0 0 g for 1 0 minutes. 2 0 0 pi of the organic layer from each tube was transferred into an autosampler vial prior to sugar determination by capillary GLC. Chromatography was performed on a Hewlett-Packard 5890 gas chromatograph (Hewlett-Packard Ltd, Wokingham, UK). The injector temperature was 240°C and the initial column temperature 200°C. After injection of the sample the column temperature was kept at 200°C for 1 2 minutes and then raised by 20°C/minute for until a temperature of 260°C was attained. Sugar peaks were detected by flame ionisation detection (detector temperature 240°C). Sugar concentrations were expressed as pg/ml of plasma.
2.2.6 In V itro erythrocyte incubations
Reagents 1) Krebs-glucose solution Krebs-Ringer bicarbonate buffer was
prepared as described earlier. 80 mg of
glucose was added to 1 0 0 ml of buffer. Procedure
Whole blood was collected into heparin and centrifuged at 800 g for 7 minutes at 0-4°C. The plasma was aspirated away and the red cells
85 resuspended in cold physiological saline (NaCI, 9% w/v). The cells were centrifuged again and the supernatant aspirated away. This washing was repeated once more. Washed erythrocytes were stored on ice before use. 10 ml of Krebs-glucose solution was pipetted into 25 ml siliconised glass flasks. 5 ml of washed erythrocytes was added to each flask, giving a final glucose concentration of 30 mmol/l. Immediately after the addition of washed erythrocytes, each flask was flushed with O 2/CO2 (19:1) for 30 seconds and sealed with laboratory film. Each flask was transferred to a 37°C shaking incubator for 30 minutes. At the end of the incubation period, the contents of each flask were transferred to a 50 ml polycarbonate centrifuge tube standing on ice. The flask was washed with 10 ml of cold saline and the washings added to the polycarbonate tube. The tubes were then centrifuged at 800 g for 7 minutes at 4°C and the resulting supernatant aspirated away. The cells were then washed again and the supernatant aspirated away. Erythrocyte monosaccharide and polyol levels, and aldose reductase activity were then measured as described earlier.
2.2.7 Starch gel electrophoresis of haemolysates and staining
for sorbitol dehydrogenase activity
Horizontal starch gel electrophoresis was performed using the modified method of Donald et al (1980). Reagents
1) Bridge buffer 0.3 mol/l Tris-phosphate, pH 8 .6
2 ) Gel buffer 1/20 dilution of bridge buffer in water
3) Activity stain 250 mg sorbitol, 40 mg NAD+, 100 mg sodium
pyruvate, 1 0 0 mg pyrazole, 2 ml phenazine
86 methosulphate (PMS) (5 mg/ml), and 2 ml MTT (3- [4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide; Thiazolyl blue) (5 mg/ml) in 100 ml 0.5
mol/l Tris-HCI, pH 8.0 Procedure 11% starch gels were prepared in the following manner. 44g of starch (see Materials) was added to 400 ml of gel buffer. The mixture was then boiled, being stirred continuously, until clear, and then degassed. Prior to pouring 100 mg of NAD+ was added to the mixture. After the gel had been poured it was covered and allowed to set at 0-4°C for about 45 minutes. Fresh whole blood was collected into EDTA and haemolysates were prepared as for the sorbitol dehydrogenase activity assay. Samples were soaked into Whatman No 17 paper, blotted, and inserted into the gel. Electrophoresis was performed in an LKB H4 horizontal gel electrophoresis tank (LKB-Pharmacia Ltd, Milton Keynes, UK) at 0-4°C, 3 V/cm for 17 hours. 400 ml of bridge buffer was used in each electrode compartment, and 50 mg NAD+ was added to the cathodal compartment prior to electrophoresis.
Wicks were made of Whatman 3MM paper. Following electrophoresis the gel was sliced in half horizontally, and the cut surfaces were stained for sorbitol dehydrogenase activity in the following manner. 1 0 0 ml activity stain was mixed with 100 ml 2 % Agar Noble (see Materials) and allowed to cool to
60°C. The mixture was then poured over the cut surface of the gel and allowed to set. The gel was then incubated at 37°C in the dark for about 2 hours. Sorbitol dehydrogenase activity is visualised by the appearance of purple bands, formed as a result of the reduction of MTT by NADH, which in turn is produced by the oxidation of sorbitol to fructose by sorbitol
87 dehydrogenase. Sodium pyruvate and pyrazole are added to the stain to inhibit lactate dehydrogenase and alcohol dehydrogenase respectively.
2.2.8 Protein determination
Protein concentrations were determined by the method of Bradford (1976) using a Bio-Rad protein determination kit (Bio-Rad Laboratories Ltd,
Watford, UK).
Reagents 1) Dye reagent Dilute 1 volume of Dye Reagent Concentrate with 4 volumes water. Filter through Whatman No 1
paper, and dilute reagent stored at room
temperature for upto 2 weeks.
2 ) Protein standard Bovine serum albumin 1 mg/ml. Procedure
The protein standard was diluted to give a standard curve ranging from 0 .2 to 1 .0 mg/ml. 0.1 ml of standards and appropriately diluted samples were placed into test tubes, and 5.0 ml of dilute dye reagent was added to each tube. A blank tube was prepared by adding 5.0 ml of dilute dye reagent to 0.1 ml of sample buffer. The tubes were mixed by vortexing and after a period of from 5 minutes to 1 hour the OD 595nm, versus the reagent blank, was measured using a Pye Unicam SP8-400 UV/Vis spectrophotometer. A standard curve was then plotted and the unknown values read from the standard curve.
88 2.2.9 Measurement of plasma glucose concentrations
Patients attending outpatient clinics at Charing Cross Hospital had their plasma glucose concentrations determined by the Department of Chemical Pathology at the hospital. Glucose levels were determined using a Yellow Springs Instruments Model 23AM Glucose Analyser (Yellow Springs Instrumentation Co Inc, Yellow Springs, Ohio, USA). In all other instances plasma glucose concentrations were determined by the glucose oxidase method of Fleming and Pegler (1963) as modified by Lloyd and Whelan (1969).
Principle Glucose Oxidase Glucose + H2O + O2 Gluconate + H 2O2
Peroxidase 2O2 + o-dianisidine — Oxidised o-dianisidineH
Reagents
1) Assay mixture 1.5 mg glucose oxidase, 1 mg peroxidase, 10 mg a-dianisidine HCI in 50 ml 100 mmol/l sodium
acetate pH 4.8
2 ) Glucose standard 3 mmol/l glucose Procedure
0.5 ml whole blood was added to 2 ml perchloric acid (PCA) in a preweighed test tube. The tubes were centrifuged at 1200 g for 10 minutes and the supernatant transferred to a second preweighed test tube. The supernatant was then neutralised with KOH and centrifuged again. The
89 neutralised supernatant was removed and stored at - 2 0 °C prior to glucose determination. The glucose standard was diluted to give a standard curve ranging from 0 to 600 nmol/ml. 50 pi of the diluted standards and appropriately diluted samples were pipetted in triplicate onto a 96 well microtitre plate (Sterillin, Feltham, UK). 100 pi of assay mixture was then added to each well and the contents of each well were mixed thoroughly. The plate was then covered with parafilm and incubated at 37°C for a minimum of 1 hour. After which 100 pi of 9 mol/l sulphuric acid was added to each well and the well contents mixed. The plate was then read at 540 nm
(filter 6 ) on a Multiskan MCC/340 Platereader (Labsystems, Uxbridge, UK). Unknown glucose concentrations were then determined from the standard curve.
2.2.10 Determination of glycosylated haemoglobin
Glycosylated haemoglobin (HbAi) levels in the blood from diabetic patients were measured by the Department of Chemical Pathology at Charing Cross Hospital. HbAi levels were determined by ion exchange chromatography using a Bio-Rad Haemoglobin A1 by Column Test kit (Bio-
Rad Laboratories Ltd, Watford, UK).
2.2.11 Screening for aldose reductase isoenzymes in the
erythrocyte
Introduction
As described in Section 1.6 one aim of the project was to look for electrophoretic variants of erythrocyte aldose reductase. One approach that
90 has been successfully used to look for isoenzymes in erythrocytes is gel electrophoresis followed by activity staining to visualise the enzyme bands
(Harris and Hopkinson, 1976). Accordingly, as described below, our initial approach was to separate haemolysates by native-polyacrylamide gel electrophoresis (PAGE) and then to stain for aldose reductase activity. A second approach that was also employed was to probe Western blots with an antibody raised against purified human aldose reductase. Experimental Separation of isoenzymes bv native-PAGE and detection bv activity staining
Proteins were separated by native-PAGE on an 8-25% gradient
Phastgel (LKB-Pharmacia Ltd, Milton Keynes, UK) using a 3 step procedure that runs for 280 accumulated volt hours. 1 pl of sample was loaded per lane and electrophoresis took approximately 1 hour. Enzyme bands were visualised by activity staining for either lactate dehydrogenase or aldose reductase. The stain used for lactate dehydrogenase was as follows: 0.05 mol/l Tris-HCI, pH 7.5 containing 0.8 mg/ml NAD+, 0.4 mg/ml MTT, 0.2 mg/ml
PMS, and 25 mmol/l sodium lactate. The stain for aldose reductase was as follows: 63 mmol/l sodium phosphate, pH 7.0 containing 10 mg/ml nitrobenzyl alcohol, 0.25 mg/ml NADP+, 0.1 mg/ml MTT, 0.1 mg/ml PMS.
Gels were incubated in the stain at 37°C in the dark for 1 hour. Enzyme activity is visualised by the appearance of purple bands.
Purification of human placental aldose reductase
Aldose reductase was purified from human placentas using the rapid purification method of Kador et al (1981). All steps were performed at 4°C. Prior to use placentas were stored frozen at -20°C. Thawed placentas were homogenised in a Waring blender with 2 vol (w/v) of 0.1 mol/l Na, K- phosphate buffer, pH 6 .8 . The homogenate was filtered and centrifuged for 30 minutes at 1200 g. The supernatant was decanted and solid ammonium sulphate (170g/l) was added with stirring to give a final saturation of 30%.
The saturated solution was allowed to stand for 1 hour before centrifuging at 15000 g for 30 minutes. The supernatant was decanted and solid ammonium sulphate (270g/l) was added to give a final saturation of 70%. This was allowed to stand overnight before being centrifuged at 15000 g for 30 minutes. The precipitate was then resuspended in a minimal volume of water. This was then dialysed against 2 x 50 vol of 0.1 mol/l Na, K- phosphate buffer, pH 6.2, containing 1 mmol/l mercaptoethanol. 50 ml aliquots of the dialysate were stored at -70°C.
1 0 0 ml of the dialysate was loaded onto a column containing 2 0 0 ml of AH-Sepharose 4B coupled to 4-carboxybenzaldehyde (prepared according to the method of Kador et al, 1979). Protein was then eluted with 0.1 mmol/l Na, K-phosphate buffer, pH 6.2 containing 5 mmol/l mercaptoethanol and 0 .0 2 % (w/v) sodium azide at a rate of 28 drops/minute.
2 0 0 drop fractions were collected and assayed for protein and aldose reductase activity. A sample elution profile is shown in Figure 2.1. Fractions containing aldose reductase activity were pooled and concentrated in an Amicon column-eluant concentrator (Amicon Ltd, Stonehouse, UK) fitted with a PM10 membrane to a final volume of approx 30 ml. Glycerol was added to give a 5% (v/v) solution.
92 Aldose Aldose reductase activity (U/min/ml)
Figure 2.1 Elution profile obtained on carboxybenzaldehyde coupled AH-
Sepharose 4B. (o) denotes protein concentration (OD 280 nm),
and (•) denotes aldose reductase activity (U/min/ml).
93 This concentrate was then added to a column containing 50 ml of Matrex Gel Orange A and allowed to equilibrate for 1 hour. The column was then eluted with 0.1 mmol/l Na, K-phosphate buffer, pH 6 .2 containing 5 mmol/l mercaptoethanol, 0.02% (w/v) sodium azide, and 5% (v/v) glycerol at a rate of 28 drops/minute and 200 drop fractions were collected. After 20 fractions had been collected 50 ml of buffer containing 0.6 mmol/l NAD+, a further 20 fractions were then eluted. This was followed by the addition of 50 ml of buffer containing 0.5 mmol/l digitonin and the elution of a further 20 fractions. 50 ml of buffer containing 0.1 mmol/l NADPH was added to the column and eluted with buffer. Fractions were collected and assayed for aldose reductase activity. Fractions containing aldose reductase activity were combined and concentrated as before to give a final volume of approx 15 ml. A sample elution profile is shown in Figure 2.2. Enzvme analysis Enzyme activity in the fractions was assayed by measuring the oxidation of NADPH spectrophotometrically at 340 nm. The assay system comprised 0.03 mmol/l NADPH, 0.4 mmol/l ammonium sulphate, 10 mmol/l
DL-glyceraldehyde, and 0.04 mol/l HEPES (N-[ 2 -hydroxyethyl]piperazine-N-
[2-ethanesulphonic acid]) buffer. The protein content of the fractions was determined by measuring the optical density at 280 nm. Protein concentration at each stage of the purification was determined as previously described (Section 2.2.8). Enzyme purity was determined by SDS-PAGE using a 12% acrylamide separating gel (Laemmli, 1970).
Preparation of antibodies to placental aldose reductase Female English rabbits (approx 2 Kg body weight) were injected subcutaneously with 1 0 0 pg of purified aldose reductase mixed 1:1 with complete Freunds adjuvant. The rabbits were boosted at 2 - and 4-week
94 iue 2.2 Figure
Protein (OD 280 nm) reductase activity (U/min/ml).reductaseactivity aldose denotes (•) and nm), 280 (OD concentration protein lto poie band n arx e Oag . (o)denotes A. Orange Gel Matrex on obtained profile Elution
Aldose reductase activity (U/min/ml) intervals with a further 100 jig of enzyme mixed 1:1 with complete Freunds adjuvant. Reactivity of the antibody was determined on Ouchterlony plates and dot-blots. Dot-blots Dot-blots were performed as follows. A nitrocellulose strip was soaked in Tris-buffered saline (TBS) for 1 0 minutes and then blotted dry on filter paper. 1 pl samples of antigen diluted in TBS were dotted onto the nitrocellulose and allowed to soak in. The nitrocellulose was then washed twice in TBS for 10 minutes. It was then blocked by placing in blocking solution (TBS + 3% non-fat freeze-dried milk (Marvel)) for 60 minutes at room temperature with shaking. After washing twice more in TBS the nitrocellulose strip was incubated with primary antibody (either anti-aldose reductase antibody or rabbit pre-immune serum) diluted 1/10 in TBS + 1% Marvel at room temperature overnight. The strip was then washed briefly in water, twice in TTBS (TBS + 0.05% Tween 20) for 10 minutes and then incubated with goat anti-rabbit IgG horse raddish peroxidase conjugate
(diluted 1/1000 in TBS + 1 % Marvel) for 3 hours at room temperature with shaking. After washing briefly with water and twice in TTBS, the strip was immersed in colour reagent (30 mg 4-chloro-napthol in 10 ml ice cold methanol + 30 jil 30% H2O2 in 50 ml TBS) for 15 minutes. The reaction was stopped with water.
Isoelectric focusing of haemolvsates
Isoelectric focusing was performed on ultrathin-layer polyacrylamide gels. Polyacrylamide gels (5% (w/v) acrylamide, 0.15% (w/v) bis-acrylamide, 0.45mg% (w/v) riboflavin, 11% (w/v) sucrose, 2.4% (v/v) ampholyte (pH 3-
10), 125 x 260 x 0.4 mm) were cast using the flap technique, and allowed to set overnight under a fluorescent lamp. The cathode and anode solutions
96 were 1 mol/l NaOH and 1 mol/l H 3 PO4 respectively. 1 cm wide strips of Whatman 17 paper were soaked in the electrolyte solutions, blotted dry and
laid carefully along the edges of the gel. 2 0 pi samples of haemolysates were applied to the gels 1 cm from the cathode using Whatman 3mm paper tabs (3x4 mm). Isoelectric focusing was performed on a Pharmacia FBE- 3000 horizontal electrophoresis system (LKB-Pharmacia Ltd, Milton Keynes, UK) at 4°C using the following conditions. The gels were prefocused at 3 W for 500 volt hours. The samples were then applied and the gels run at 6 W for 2 0 0 0 volt hours. Western Blotting Following isoelectric focusing gels were soaked in 20 mmol/l Tris/ 150
mmol/l glycine/ 20% (v/v) methanol buffer, pH 8.5 (TGM) for 1 0 minutes. Proteins were then blotted onto nitrocellulose in the following manner. The gel was covered with a nitrocellulose membrane that had previously been soaked in TGM. On top of this was placed one sheet of Whatman 3mm and two sheets of Whatman 17 paper (cut a few cm larger than the gel and soaked in TGM). This construct was then sandwiched between two larger glass plates and covered and sealed with cling film. The blot was then left at room temperature for 3 hours. After transfer the nitrocellulose was blocked, incubated with antibodies and immersed in colour reagent as previously described for the Dot-blots.
Results and discussion
The applicability of using electrophoresis on the Pharmacia Phastsystem (LKB-Pharmacia Ltd, Milton Keynes, UK) followed by activity staining to screen for the presence of isoenzymes was tested by resolving lactate dehydrogenase isoenzymes in serum samples. As shown in Figure
97 2.3, lactate dehydrogenase isoenzymes could be succesfully separated and visualised in serum samples using native-PAGE on the Phastsystem followed by activity staining. However attempts to look for aldose reductase isoenzymes using this technique were unsuccessful. This inability to visualise aldose reductase by activity staining most probably reflects the lability of the erythrocyte enzyme (Nakayama et al, 1989). Given the lability of the enzyme it was decided that Western blotting might provide a more viable approach, since this technique does not require the presence of active enzyme.
In order to raise an antibody to aldose reductase it was first necessary to purify a quantity of the human enzyme. As summarised in Table 2.3, using the procedure of kador et al (1981), aldose reductase from human placenta was obtained with an average 843-fold purification and 8 .6 % yield. The enzyme preparation gave a single band with an apparent molecular weight of 37 Kd on SDS-electrophoresis (Figure 2.4A). This value is in good agreement with previously published values (Clements and Winegrad, 1972). A second band could occasionally be made out just below this band and this was most probably a proteolysis product. Antibodies raised against this purified enzyme cross-reacted with it on Ouchterlony plates and on both
Dot- and Western blots (Figure 2.4A and B), whilst pre-immune serum did not.
Whilst it was possible to detect purified placental aldose reductase on
Western blots, aldose reductase could not be detected on blots of haemolysates separated by either SDS-electrophoresis or isoelectric focusing. A number of possible explanations exist for this inability to detect
98 a b c b a
Figure 2.3 Separation and visualisation of lactate dehydrogenase (LDH)
isoenzymes by native-PAGE followed by activity staining. Lane a) LDH markers; b) LDH isoenzymes in serum from a healthy
patient; and c) LDH isoenzymes in serum from a patient with a
myocardial infarction.
9 9 Table 2.3 Purification of human placental reductase
Purification Total proteinTotal activity Spec activity Purification Yield (mg) (U/min/ml) (U/min/mg) <%) Dialysate 3070 4.27 0.0015 1 1 0 0 .0 AH-Sepharose 454 1.79 0.0125 5.5 38.5
Matrex gel 0.37 0.47 1 .2 1 843 8 .6
Mean of three determinations based on 100 ml of dialysate. a b 1 2
A B
1 2
66K
35K
14K
Figure 2.4 A: a)SDS-polyacrylamide gel of purified human placental
aldose reductase. Lane 1) molecular weight markers, and
2) aldose reductase, b) Western blot of purified placental
aldose reductase. B: Dot-blots of a) pre-immune serum and b) anti-aldose
reductase antibody against 1) Tris-buffered saline and 2) purified aldose reductase (a) 50ng/dot and b)100ng/dot).
101 erythrocyte aldose reductase. One possible explanation is that erythrocyte aldose reductase is immunologically distinct from placental aldose reductase. However Nakayama and co-workers (1989) have recently shown that purified erythrocyte aldose reductase will cross-react with antibodies to the placental enzyme. A more likely explanation for the inability to detect aldose reductase in erythrocytes is its low concentration. On Dot-blots it was possible to detect down to about 50 ng of the purified placental enzyme. In their recent paper Nakayama and co-workers (1989) reported that 34 g of total erythrocyte protein yielded a mere 95 pg of aldose reductase protein. On this basis 20 pi of haemolysate (amount loaded onto gels) will contain only 14 ng of aldose reductase, an amount that is below the lower detection limit of the technique. Using a similar approach Grimshaw and Mathur (1989) have also been unable to detect aldose reductase in extracts of human erythrocytes, the lower detection limit of their assay being2 0 ng of enzyme protein. Therefore whilst these techniques can be successfully used to look for isoenzymes in other tissues they are at present not sensitive enough to look for isoenzymes in the erythrocyte.
2.2.12 Statistical Methods
All data are expressed as mean ± SEM. Where appropriate statistical analysis was performed using the Students t-test for unpaired data. Where there was any doubt about the validity of using a parametric test, for example when the sample group was small, both parametric and non-parametric analysis were carried out. If it is valid to use a parametric test the Null
Hypothesis probability will be smaller than that from the non-parametric test.
If use of a parametric test is inappropriate the Null Hypothesis probability for
102 the non-parametric test would be lower, as departures from the assumptions underlying the use of a parametric test make the test less powerful. In such cases non-parametric analysis was performed using either the Wilcoxon signed rank or the Mann-Whitney rank-sum test as indicated in the text. Linear regression was calculated by the least squares method and checked using the non-parametric Spearmans correlation coefficient. For all analyses the level of significance was taken as p<0.05.
103 Chapter Three
Erythrocyte sorbitol dehydrogenase activity in diabetic and
non-diabetic subjects
104 3.1 Introduction
Over the last thirty years a great deal of attention has been focused on the role of the polyol pathway in the development of the diabetic complications. Whilst the majority of this attention has focused on aldose reductase, the first enzyme of the pathway, little work has been done on the second enzyme, namely sorbitol dehydrogenase. Variations in sorbitol dehydrogenase activity may be equally as important as alterations in aldose reductase activity. For example a reduction in sorbitol dehydrogenase activity could lead to increased sorbitol accumulation in tissues susceptible to the development of complications. Moreover inherited variations in sorbitol dehydrogenase activity may affect the onset of the complications.
Two studies in the literature have reported sorbitol dehydrogenase deficiencies in the erythrocytes of families with congenital cataract (Vaca et al, 1982; Shin et al, 1984). Shin et al (1984) measured sorbitol dehydrogenase activity in the father, mother, son and daughter of one family.
Low activity was found in the father and son both of whom had cataracts. The results of the second study were less clear, since although low sorbitol dehydrogenase activity was found in the erythrocytes of five individuals, all of whom were male, one of these individuals did not have cataracts. Moreover a granddaughter with a near normal sorbitol dehydrogenase activity had cataract (Vaca et al, 1982). The clearest example of a genetically determined sorbitol dehydrogenase deficiency comes from a study on the strain of mice C57BL/UA (Holmes et al, 1982). Using cellulose acetate electrophoresis, no sorbitol dehydrogenase activity was detected in
105 extracts of several tissues taken from this strain of mice, which were normal in all other respects.
In addition reduced levels of sorbitol dehydrogenase activity have been reported in the erythrocytes of insulin-dependent diabetic patients when compared to non-diabetic controls (Barretto et al, 1985; Medina et al,
1987). Barretto et al (1985) found that erythrocyte sorbitol dehydrogenase activity was significantly lower (p<0.001) in 34 diabetic patients (mean ± SD:
0.219 ± 0.085 U min'1 g Hb'1) than in 27 normal blood donors (mean ± SD: 0.34 ± 0.094 U min'1 g Hb'1). Similar findings were reported by Medina et al (1987), who found erythrocyte sorbitol dehydrogenase activity to be significantly lower in diabetic patients than in professional blood donors (mean ± SD: 0.246 ± 0.08 versus 0.292 ± 0.097 IU g Hb'1, p<0.001). The significance of these findings is unclear since other studies have reported different results. Crabbe et al (1980) found that diabetic patients with retinopathy had a higher sorbitol dehydrogenase activity than non-diabetic controls, but that diabetic patients with retinopathy and cataract had lower sorbitol dehydrogenase activities than the control patients. Furthermore
Vaca et al (1982) found higher sorbitol dehydrogenase activities in diabetic patients than in controls. Further to these activity variants, electrophoretic variants have also been reported in two mammalian species, namely the pig
(Op’t Hof, 1969) and the horse (Porter and McGuinness, 1987). However in a study of 665 normal subjects Charlesworth (1972) found no evidence for polymorphism at the sorbitol dehydrogenase locus, although one rare electrophoretic variant was found in a number of members of one family.
These studies suggest that variations in erythrocyte sorbitol dehydrogenase
106 activity may exist, and that such variations may arise secondary to diabetes or be genetically conferred.
Based on this premiss the aim of the work presented in this chapter was to measure sorbitol dehydrogenase activity in the erythrocytes of insulin-dependent and non-insulin-dependent diabetic patients and normal controls, and to look for activity and electrophoretic variants.
107 3.2 Experimental
Erythrocytes from 102 diabetic patients and 30 non-diabetic controls
were screened for sorbitol dehydrogenase activity and electrophoretic variants using the methods described in Chapter 2. The diabetic cohort was recruited from patients attending the Diabetic Clinic at Charing Cross Hospital and comprised 47 insulin-dependent patients and 55 non-insulin- dependent diabetic patients. The 47 insulin-dependent patients consisted of 26 males and 21 females with ages ranging from 14-78 years. The group
had a mean duration of disease (determined from clinical records) of 13 years (range 2 months to 42 years). Blood glucose levels ranged from 2.4- 22.4 mmol/l (mean ± SEM: 9.0 ± 0.4 mmol/l), and glycosylated haemoglobin (HbAi) levels ranged from 3.7-13.7% (mean ± SEM: 8.3 ± 0.2%). The 55 non-insulin-dependent patients comprised 28 males and 27 females. Ages ranged from 40-83 years, and known duration of disease was between 1 and 20 years. The non-insulin-dependent-diabetic patients had blood glucose levels ranging from 4.5-13.8 mmol/l (mean ± SEM: 10.0 ± 1.0 mmol/l), and HbAi levels between 5.0 and 12.4% (mean ± SEM: 8.2 ± 0.3%). The non-diabetic control group was made up of non-diabetic patients attending Charing Cross Hospital, and healthy volunteers from the
Department of Biochemistry. The group consisted of 13 males and 17 females with ages ranging from 22-80 years.
108 3.3 Results
Red cell sorbitol dehydrogenase activities in the three groups are shown in Figure 3.1. The mean sorbitol dehydrogenase activity in the insulin-dependent diabetic patient group was 0.161 ± 0.01 mU/mg protein (± SEM), with individual values ranging from 0.081 to 0.3 mU/mg protein, and the non-insulin-dependent-diabetic group had a mean sorbitol dehydrogenase activity of 0.168 ± 0.01 mU/mg protein (range 0.076 to 0.365 mU/mg protein). The non-diabetic controls had enzyme activities ranging between 0.095 and 0.306 mU/mg protein with a mean value of 0.160 ± 0.01 mU/mg protein (± SEM). There was no statistically significant difference in
sorbitol dehydrogenase activity between either the insulin-dependent or the non-insulin-dependent diabetic patients when compared to the non-diabetic controls (p = 0.931 and p = 0.45 respectively), nor when compared to each other (p = 0.47).
A wide variation in sorbitol dehydrogenase activity was observed in both diabetic and normal subjects, with the coefficient of variation being 42,
44, and 34% for the insulin-dependent diabetic, non-insulin-dependent diabetic and control patients respectively. Whilst there was such a wide variation in activity, individual activities did not vary considerably with time. In a group of 19 diabetic patients fresh blood samples were taken and sorbitol dehydrogenase activities determined one month after the initial measurements were made. As shown in Figure 3.2 there was little variation in enzyme activity over this time period, with the average coefficient of variation between the two measurements being 13%.
109 SDH Activity (mU/mg protein) 0.0 0.2 0.3 0.4 0.1
u Figure 3.1 SDH activity in insulin-dependent (IDDM) and non-insulin- non-insulin- and (IDDM) insulin-dependent in activity SDH 3.1Figure • • • • DM ID CONTROLS NIDDM IDDM controls. non-diabetic and patients diabetic (NiDDM) dependent t t 110 • • • • • • t • *!*• i i Figure 3.2 The variation of erythrocyte sorbitol activity with time. Repeat activities were determined approximately one month after the initial measurements (r=0.88, p<0.01).
1 1 1 Age differences between patients in each group do not appear to be responsible for the wide variations in enzyme activity seen, since there was no correlation between sorbitol dehydrogenase activity and age in any of the three groups (Figure 3.3). In the non-diabetic group, however, sorbitol dehydrogenase activity was statistically significantly greater (0.176 ± 0.01 versus 0.139 ± 0.01 mU/mg protein, p < 0.05) in females than in males (Figure 3.4). No sex effect was found in either of the two diabetic cohorts (Figure 3.4). Moreover there was no difference in enzyme activities between either non-diabetic and diabetic males or females.
The influence of duration of diabetes and blood glucose control, both acute and chronic, on erythrocyte sorbitol dehydrogenase activity was examined in both diabetic groups. In neither group of diabetic patients, as shown in Figure 3.5, did duration of disease have any influence on erythrocyte enzyme activity. In addition to being independent of duration sorbitol dehydrogenase activity is also independent of the quality of blood glucose control. Long term control was determined by measuring glycosylated haemoglobin (HbAi) levels, and as shown in Figure 3.6 there was no correlation between HbAi levels and red cell enzyme activity in either diabetic group. Moreover sorbitol dehydrogenase activity does not appear to be acutely dependent on blood glucose levels, since in neither group was there any relationship between plasma glucose levels and sorbitol dehydrogenase activity (Figure 3.7).
Whilst the wide variation in sorbitol dehydrogenase activities did not allow patients to be classified into subgroups on the basis of activity (ie high versus low), patients were identified with sorbitol dehydrogenase activities
1 1 2 B 0.4
c CD 0 Q_1 0.3 cn E 3 0.2
> • • o < X 0.1 GOQ
0.0 _ j______i______i______i 0 20 40 60 80
Age (Years)
Figure 3.3 The effect of age on erythrocyte sorbitol dehydrogenase activity in A) insulin-dependent (o) and non-insulin-dependent diabetic patients (•), and B) non-diabetic controls.
113 Figure 3.4 The influence of sex on erythrocyte sorbitol dehydrogenase dehydrogenase sorbitol erythrocyte on sex of influence The 3.4 Figure SDH Activity (mU/mg protein) 0.4 0.1 - 0.1 0.0 J 0.0 0.3- 0.2- 1 controls. non-diabetic and patients, diabetic (NIDDM) dependent ciiy n nui-eedn (DM ad non-insulin- and (IDDM) insulin-dependent in activity M • • • • • • • • • V • • • • • • • • • • • • • • • • • • • IDDM
o o o o°o ° o ° F o 8 Q 8 °0 o CCD 8 114 •+ •• •+ •1. * • • • M w t • •
NIDDM o o O O O 8°8 °§° o o o 0 o F ••• • • ••• CONTROLS M • 8 •
o o o ° 8°8 O o o o o o o Duration of diabetes (Years)
Figure 3.5 The effect of duration of diabetes on erythrocyte sorbitol dehydrogenase activity in insulin-dependent (o), and non-insulin-dependent (•) diabetic patients.
115 HbA1 (%)
Figure 3.6 The relationship between HbA1 and erythrocyte sorbitol dehydrogenase activity in insulin-dependent (o), and non-insulin-dependent (•) diabetic patients.
1 16 Figure 3.7 Correlation of erythrocyte sorbitol dehydrogenase activity and plasma glucose levels in insulin-dependent (o), and non-insulin-dependent (•) diabetic patients.
1 17 that were consistently 2 standard deviations above or below the mean of the control group. In 2 patients with low activity and in 3 with high activity the relationship between sorbitol dehydrogenase activity and the development of diabetic complications was examined. In this small number of patients examined no relationship was observed between low or high enzyme activity and the presence or absence of complications (ie cataract, retinopathy, neuropathy, and nephropathy). The presence or absence of complications in each patient was determined from hospital records. In addition no electrophoretic variants of sorbitol dehydrogenase were observed. Erythrocytes were screened for sorbitol dehydrogenase isoenzymes by running haemolysates on starch gels and staining for enzyme activity. Figure 3.8 shows a typical starch gel following staining for sorbitol dehydrogenase activity. Electrophoresis revealed the presence of only one, cathodically migrating, variant of sorbitol dehydrogenase in both the diabetic and control patients. Figure 3.8 Starch gel electrophoretic pattern of sorbitol dehydrogenase in the erythrocytes of insulin-dependent diabetic patients. Hb denotes haemoglobin band, SDH denotes the sorbitol dehydrogenase band.
1 1 9 3.4 Discussion
Sorbitol dehydrogenase activities determined in the three groups of subjects are in good agreement with previously reported values (Vaca et al, 1982; Barretto et al, 1985). Like Crabbe et al (1980) and Vaca et al (1982), but unlike Barretto et al (1985) and Medina et al (1987) no significant difference in activities was observed between diabetic patients and controls. These discrepancies in findings cannot be explained easily. They are unlikely to be methodological however, since like Barretto et al and Vaca et al, an indirect spectrophotometric assay was used. Although age-related changes in sorbitol dehydrogenase activity have been reported in a number of rat tissues (Cao Danh et al, 1985), no effect of age on erythrocyte enzyme activity was observed in either diabetic or control subjects. This is in agreement with Jedziniak et al (1981) who found no correlation between age and sorbitol dehydrogenase activity in the human lens. In contrast with an earlier study (Medina et al, 1987), where no sex effect was seen, non diabetic males had a significantly lower sorbitol dehydrogenase activity than females. Of interest is the observation that in a family with congenital cataracts, those subjects with low sorbitol dehydrogenase activities and cataract were male (Shin et al, 1984). Moreover in a second family with red cell sorbitol dehydrogenase deficiency, low enzyme activities were found exclusively in male members of the family (Vaca et al, 1982). Since no effect of sex was observed in either insulin-dependent or non-insulin-dependent diabetic patients, it is possible that sorbitol dehydrogenase activity is hormonally regulated and that this regulation is perturbed by diabetes.
120 The wide variation in sorbitol dehydrogenase activity seen in the three groups (coefficient of variation 34-44%) confirms other preliminary studies
(Crabbe et al, 1980; Vaca et al, 1982; Barretto et al, 1985; Medina et al, 1987). The coefficient of variation in these studies ranged from 23% to 47%. This variation is not attributable to differences in glycaemic control since there is no correlation between acute or chronic glycaemic control and enzyme activity, and furthermore glucose levels do not alter sorbitol dehydrogenase activity in vitro (Barretto et al, 1985). In agreement with Charlesworth (1972) starch gel electrophoresis revealed only one electrophoretic variant.
Since sorbitol dehydrogenase activities were similar when measured at a monthly interval it was possible to identify certain patients with consistently high and low activities (greater or less than 2 SD from the normal mean). Whilst not a primary aim of this study, the relationship between high and low sorbitol dehydrogenase activity and the presence of complications was examined in this small group of patients. No relationship was found between high and low activity and the presence or absence of complications. Similarly Medina et al (1987) found no significant differences in enzyme activity in diabetic patients with or without cataract. In addition
Crabbe et al (1980) found no differences between diabetic patients with or without retinopathy. On the basis of this study and the earlier study of
Charlesworth (1972) it appears that erythrocyte sorbitol dehydrogenase is encoded at a single locus, and that only one allele exists. Furthermore variations in sorbitol dehydrogenase activity, as measured in the erythrocyte, are not related to the presence or absence of the diabetic complications, at least in the small number of patients studied.
121 Chapter Four
Erythrocyte polyol pathway enzyme activities and the severity of
the diabetic complications
122 4.1 Introduction
As described in the Introduction clinically apparent complications do not develop in all diabetic patients. Regardless of the quality of their glycaemic control and duration of diabetes some 20-25% of all diabetic patients will not develop complications, whereas a small percentage (<5%) will develop severe complications after a short duration of disease, even with good glycaemic control (Raskin and Rosenstock, 1986). This suggests that factors other than hyperglycaemia and duration of disease are involved in the development.of the diabetic complications.
It has been proposed that increased polyol pathway activity plays a central role in the development of the diabetic complications. Given the variability in the onset and severity of the complications the question that arises is how does polyol pathway activity vary between individuals. As described in Chapter 3 wide variations exist in erythrocyte sorbitol dehydrogenase activity in both diabetic and non-diabetic subjects. In addition two controversial reports have suggested that variations exist in the activity of erythrocyte aldose reductase in diabetic patients (Crabbe et al,
1980; Srivastava et al, 1986). Accordingly one aim of this study was to determine to what extent the activities of the component enzymes of the polyol pathway vary in the erythrocytes of diabetic and non-diabetic subjects.
Sorbitol accumulation in the erythrocyte has been used in a variety of studies as an indicator of polyol pathway activity. In particular the reduction in erythrocyte sorbitol levels has been used to demonstrate the efficacy of a variety of aldose reductase inhibitors (Malone et al, 1984; Popp-Snijders et
123 al, 1984; Hotta et al, 1983). However it remains unclear how well sorbitol accumulation, or for that matter fructose accumulation, reflects aldose reductase or sorbitol dehydrogenase activities. Therefore a second aim was to examine the relationship between erythrocyte sorbitol and fructose concentrations and the activities of the polyol pathway enzymes.
Malone and co-workers (1984) have proposed that the erythrocyte polyol pathway might provide a good model of the polyol pathway in other less accessible tissues, such as the nerve, kidney, and the lens. Moreover they proposed that measurements of erythrocyte polyol pathway activity
might be valuable in elucidating the role of the pathway in the development of the complications in these less accessible tissues. For this to be true there should be a demonstrable relationship between quantitative measurements of various complications and erythrocyte polyol pathway activity. Therefore the third aim of the work described in this Chapter was to examine the relationship between quantitative measurements of the severity of neuropathy, both autonomic and peripheral, retinopathy and nephropathy and erythrocyte polyol pathway activity.in a group of long-duration insulin- dependent diabetic patients.
124 4.2 Experimental
4.2.1 Patient selection
Insulin-dependent diabetic patients on the registration list of the Diabetic Clinic at Charing Cross Hospital with a duration of 25 years or greater were invited to participate in the study. 34 patients agreed and were subsequently matched with a group of 22 age-matched controls. The diabetic cohort comprised 18 males and 16 females with a mean age of 51 ± 3 years (± SEM), and a mean duration of diabetes of 32 ± 1 years. The control group, made up of 12 males and 10 females, had a mean age of 45 ±
3 years.
4.2.2 Clinical evaluation
The subjects attended the investigation session non-fasted in the morning and all blood samples were taken and clinical procedures performed that morning. The severities of the various complications were assessed as described below.
Autonomic neuropathy
Autonomic neuropathy was assessed using four standard cardiovascular reflex tests: heart rate (HR) response to deep breathing, immediate HR response to standing, HR response to a Valsalva manoeuvre and blood pressure (BP) response to standing. These tests have been used extensively in the assessment of autonomic neuropathy (Thomas and Ward, 1975; Ewing and Clarke, 1986; Ewing et al, 1985). HR variation was monitored by electrocardiography (ECG) using a Cambridge Instruments Ltd
125 electrocardiograph, monitoring standard lead II. HR was determined by measuring the R-R intervals on the ECG trace. 11 HR response to deep breathing In the normal individual one would expect to observe HR variation during deep breathing due to alterations in vagal activity in phase with respiration. This results in an increased HR during inspiration and a decreased HR during expiration. However in diabetic patients with autonomic neuropathy there is a noticeable reduction and sometimes complete absence of HR variation. HR variation during deep breathing is expressed as the E:l ratio, that is the ratio of the HR during expiration to that during inspiration. The test was performed in the following manner. The patient lay quietly on the couch and then breathed deeply and evenly at six breaths per minute whilst the ECG was running. This was achieved by the investigator counting a ten second cycle in time with a stop-clock (ie ln-2-3-
4-5-Out-2-3-4-5), with the patient taking a deep breath in and out when indicated. This ten second cycle was repeated three times in succession and a mean of the three E:l ratios used. 21 HR and BP response to standing There is a characteristic rapid increase in HR during the change in posture from lying to standing. The increase is maximal around the 15th beat after standing. This is followed by a relative overshoot that is maximal around the 30th beat. In patients with neuropathy there is only a gradual or no increase in HR on standing. This HR response is expressed as the 30:15 ratio which is the ratio of the longest R-R interval around the 30th beat to the shortest around the 15th. Standing causes blood to pool in the legs, resulting in a drop in the BP. Normally this is rapidly corrected by reflex peripheral vasoconstriction which brings the BP back to normal. In patients
126 with neuropathy occurs much more slowly. Therefore a significant drop in systolic BP one minute after standing is indicative of autonomic damage. HR and BP responses to standing were measured as follows. Two resting BP readings were taken. From lying on the couch the patient was asked to stand unaided whilst the ECG was running. HR was monitored for about 40 seconds and BP was taken one minute after standing. 3) HR response to a Valsalva manoeuvre The Valsalva manoeuvre involves forced expiration against a fixed pressure. During the strain period in a normal individual, the HR rises due to increased intrathoracic pressure. After release the HR slows back to normal. With autonomic damage, however, there is little variation in HR throughout the manoeuvre. The HR response is expressed as the Valsalva ratio which is the ratio of the longest R-R interval after the manoeuvre (within about 20 beats) to the shortest during the manoeuvre. The Valsalva manoeuvre was performed as follows. The patient sat quietly and then blew into a mouthpiece attached to a mercury manometer at a pressure of 40 mm Hg for
15 seconds. HR was monitored throughout the manoeuvre and for about 30 seconds afterwards. A combination of the results of these four cardiovascular reflex tests was used to give an overall measure of the extent of autonomic neuropathy
(described in detail in Section 4.3.2).
Peripheral neuropathy
Peripheral neuropathy was assessed by vibrametry using a Somedic AB Vibrameter. Vibration perception threshold (VPT) was determined at the tip of both great toes. The patient lay on a couch and was given time to relax. At each site, the probe of the vibrameter was placed in contact with the skin, the application pressure being maintained constant throughout the
127 procedure. Initially there was no vibration. As the amplitude of vibration was increased the patient was asked to indicate when he or she could first detect vibration distinct from the pressure of the probe. This reading was the VPT (expressed in pM). VPT was recorded three times on each toe and a mean value taken. The mean of the readings on both toes was taken as the final
VPT value. Retinopathy
The severity of retinopathy was assessed from retinal photographs.
Photographs were taken through dilated pupils using a Kowa retinal camera (Kowa Company Inc, Tokyo, Japan). The retinal photographs were evaluated blind by a member of the Department of Ophthalmology at Charing Cross Hospital, and graded by comparison to standard photographs
(Oakley et al, 1967). Nephropathy The severity of nephropathy was determined by measuring urinary protein excretion. The excretion of three proteins, namely albumin, IgG, and
6 2 -microglobulin was measured. Albumin and IgG excretion are both increased in patients with microproteinuria (Viberti and Keen, 1984), and the elevated excretion of albumin, in particular, is strongly associated with the development of clinical diabetic nephropathy (Viberti et al, 1982b). The increased excretion of albumin and IgG is believed to be glomerular in origin
(Viberti et al, 1982b), and increased excretion of these proteins is indicative of changes in glomerular structure and function. Glomerular filtration of albumin and IgG is determined by different factors. Filtration of albumin, an anionic protein, is governed by the negative electrical charge of the glomerular basement membrane and intraglomerular pressure, whereas the filtration of IgG, an electrically neutral protein, is governed by pore
128 radius/number and intraglomerular pressure (Viberti and Wiseman, 1986). Therefore the relative excretion of the two proteins gives a more detailed picture of changes occurring in the glomerulus. The increased excretion of
6 2 -microglobulin is indicative of altered tubular function, since 6 2 - microglobulin excretion is an indicator of tubular reabsorptive capacity (Viberti et al, 1982a). Protein excretion was measured in two timed, overnight urine samples. Overnight urine samples were taken to eliminate other factors, such as exercise, that might affect protein excretion rate. Urinary protein excretion was measured by the Unit for Metabolic Medicine at Guys Hospital, London. Albumin excretion was measured using an immunoturbimetric assay (Teppo, 1982), and IgG and 6 2 -microglobulin excretion were measured by enzyme-linked immunoassays (ELISA) (Bjerrum and Birgens,1986; Li et al, 1986).
129 4.3 Results
4.3.1 Erythrocyte polyol pathway enzyme activity and metabolite concentrations: Diabetic patients versus controls
There was a wide variation in aldose reductase activity in both the insulin-dependent patients and the age-matched controls (coefficient of variation (CV) 44% and 42% respectively). Aldose reductase activity ranged from 1.8 to 8.4 pU/mg protein (mean 3.6 ± 0.3 pU/mg protein) in the diabetic cohort, and from 0.7 to 3.5 pU/mg protein (mean 2.1 ± 0.2 pU/mg protein) in the non-diabetic controls. When compared with controls erythrocyte aldose reductase activity was significantly elevated in the diabetic patients (p<0.001, Table 4.1, and Figure 4.1). Erythrocyte sorbitol dehydrogenase activities in the two groups were similar to those reported previously
(Chapter 3), and consistent with these previous findings there was no statistically significant difference between activities (0.166 ± 0 .0 1 versus 0.165 ± 0.01 mU/mg protein) in the two groups (Table 4.1). Erythrocyte fructose levels were found to be raised in the diabetic cohort (10.9 ± 1.2 versus 3.6 ± 0.5 pg/ml RBC, p<0.001) when compared to the control group. Moreover there was a positive correlation between fructose levels and aldose reductase activity in the erythrocyte (r=0.47, p<0.01, Figure 4.2). No such relationship existed between erythrocyte sorbitol dehydrogenase activities and fructose concentrations (Figure 4.3).
Red cell sorbitol concentrations varied considerably in both the insulin-dependent diabetic patients (CV 95%) and the controls (CV 81%), and in greater than 50% of the controls sorbitol concentrations were below
130 Table 4.1 Erythrocyte aldose reductase and sorbitol dehydrogenase activities, and fructose, sorbitol and myo-inositol concentrations
in insulin-dependent diabetic patients and age-matched
controls.
AR activity SDH activity Fructose Sorbitol myo-inositol jiU/mg mll/mg pg/ml pg/ml pg/ml
IDDM Patients(34) a3.6 ±0.3 0.166 ±0.01 a10.9 + 1.2 1.6 ±0.3 a8.9 ±0.3
Controls (22) 2.1 ±0.2 0.165 ±0.01 3.6 ±0.5 0.5 ±0.1 6.9 ±0.4
Results expressed as mean ± SEM. Numbers of patients in parentheses. Statistically significant differences between diabetic patients and controls denoted by; a) p<0.001.
131 iue . Eyhoye loe euts atvt i insulin-dependent in activity reductase aldose Erythrocyte 4.1 Figure
Aldose reductase activity (jiU/mg protein) ibtcptet (DM ad o-ibtccnrl. Horizontal controls. non-diabetic and (IDDM) patients diabetic bars denote mean values. mean denotebars OTOS IDDM CONTROLS 080 132 001 0 .0 0 < p o ° • • t t • • • • 8 o __ RBC fructose (jig/ml RBC) iue 4.2 Figure h creain ewe eyhoye loe euts activity reductase aldose erythrocyte between correlation The n rcoecnetain r04, p<0.01) (r=0.47, concentration fructose and 133 4 0
O(XI 30 - cc E O) =L Q) CO
ZJ
O DCCQ
10 -
0 L- i _j 0.0 0.1 0.2 0.3
Sorbitol dehydrogenase activity (mU/mg protein)
Figure 4.3 The correlation between erythrocyte sorbitol dehydrogenase activity and fructose concentration.
134 the detection threshold of the GC. As shown in Table 4.1 there was no apparent difference between sorbitol concentrations in either group. There was no correlation between erythrocyte sorbitol and the activity of either aldose reductase (Figure 4.4) or sorbitol dehydrogenase (Figure 4.5). Increased polyol pathway activity has been linked to reduced myo-inositol levels in a number of tissues, notably the nerve. However in the diabetic group erythrocyte myo-inositol levels were found to be elevated (8.9 ± 0.3 versus 6.9 ± 0.4 pg/ml RBC, p<0.001, Table 4.1) when compared to controls.
The ratio of aldose reductase activity to sorbitol dehydrogenase activity might well provide a better measure of overall polyol pathway activity than the activities of the individual enzymes alone. This novel technique however revealed no differences between the diabetic patients and the non diabetic controls. A further finding was that the activities of the enzymes of the polyol pathway (Figures 4.6, 4.7, and 4.8), and erythrocyte sorbitol and fructose concentrations (not shown) were not influenced by age, duration of diabetes or HbAi levels. In agreement with the results obtained in Chapter 3 starch gel electrophoresis of haemolysates showed only one form of sorbitol dehydrogenase to be present in the erythrocytes of both groups.
4.3.2 Clinical status of the diabetic cohort
Plasma glucose levels in the diabetic patients ranged from 1.1 to 23.4 mmol/l (mean ± SEM: 11.5 ±1.1 mmol/l), and HbAi values were between 6.2 and 14.8% (9.0 ± 0.3%). The severity of autonomic neuropathy in each patient was assessed using the cardiovascular function tests described in Section 4.2.2. For each test, a normal value was assigned a score of zero, a
135 iue 4.4 Figure RBC sorbitol (pg/ml RBC) 10 8 4 0 L 0 2 " 6 - r 0 h creain ewe eyhoye loe euts activity reductase aldose erythrocyte between correlation The and sorbitol concentration. sorbitol and Aldose reductase activity (pll/mg protein) (pll/mg reductaseactivityAldose 136 _L 8 _i 10 Figure Figure RBC sorbitol (jug/ml RBC) 4.5 The correlation between erythrocyte sorbitol dehydrogenase dehydrogenase sorbitol erythrocyte between correlation The 4.5 activity and sorbitol concentration. sorbitol and activity Sorbitol dehydrogenase activity (mU/mgprotein)activity Sorbitoldehydrogenase 137 iue 4.6 Figure Aldose reductase activity (pll/mg protein) 10 - 4 8 0 L 0 6 2 - - - 0 h rltosi bten rtrct ads rdcae activity reductase aldose erythrocyte between relationship The n age. and —I 0 0 0 0 0 60 50 40 30 20 10 ______I ______I ______138 Age (Years)Age I ______I ______I _ _i 0 80 70 ______i iue 4.7 Figure Aldose reductase activity (|iU/mg protein) 10r 8 o L- o 20 h rltosi bten rtrct ads rdcae activity reductase aldose erythrocyte between relationship The and duration of diabetes. of durationand j _ 0 0 50 40 30 ______uain (Years)Duration 139 i ______I— _i 60 iue 4.8 Figure Aldose reductase activity (pU/mg protein) 10r o - 4 - 8 2 6 - - 5 3 The relationship between erythrocyte aldose reductase activity activity reductase aldose erythrocyte between relationship The n gyoyae heolbn ees (HbAi). levels haemoglobin glycosylated and 1 J 9 1 3 15 13 11 9 7 ______140 HbA1(%) I ______I ______I ______L_ borderline value a score of one, and an abnormal value was assigned a score of two. Normal, borderline and abnormal values for the Valsalva ratio, 30:15 ratio and BP response to standing were those used by Ewing and Clarke (1986), the E:l ratio being normalised for age using the normogram of Smith (1982). The results of the individual tests are detailed in the Appendix. Each patient was then assigned an overall autonomic neuropathy score using a modification of the method devised by Ewing and Clarke (1986). The overall autonomic neuropathy score was obtained as follows. The values obtained from all four tests were summed to give a combined total. This was then divided by the highest score obtainable by the patient to give an overall score between 0 and 1. This modification of the
Ewing and Clarke method allows patients to be included in the analysis who were unable to perform all the tests. There was a good correlation between the individual cardiovascular function tests, namely the heart rate response to a Valsalva manoeuvre (Figure 4.9), standing and deep-breathing (not shown), and the overall autonomic neuropathy score (r=0.63, p<0.001; r=0.6, p<0.001; r=0.52, p<0.01 respectively). On the basis of published normal and abnormal values for the cardiovascular function tests (Ewing and
Clarke,1986; Smith, 1982) 50% of the diabetic patients examined showed some degree of autonomic dysfunction.
Vibration perception threshold (VPT) was taken as a measure of peripheral sensory neuropathy. Although VPT provides a reproducible measure of nerve function it varies significantly with age, and therefore all values were age corrected. All values greater than 2 SD from an age- corrected mean being taken as abnormal (Goldberg and Linblom, 1979). Following correction for age 30% of the subjects had an abnormal VPT. Of
141 iue . Te orlto bten asla ai ad h overall the and ratio Valsalva between correlation The 4.9 Figure Valsalva ratio uooi nuoah cr (=.3 p<0.001). (r=0.63, score neuropathy autonomic Overall autonomic neuropathy score neuropathy Overallautonomic 142 the patients with abnormal VPT 60% additionally showed some degree of autonomic dysfunction. 32% of the diabetic cohort had clinical symptoms of neuropathy (clinical symptoms being defined as absence of deep tendon reflexes, presence of postural hypotension, or pain), and all but two of these patients had abnormal autonomic or peripheral function tests, whilst 14 patients had abnormal function tests but showed no signs of clinical neuropathy.
As described in Section 4.2.2 the severity of retinopathy was assessed by retinal photography . In the case of four patients in whom inadequate quality photographs were obtained , retinopathy status was evaluated from the Department of Ophthalmology case records. Furthermore in two patients the presence of cataract prevented any evaluation of the degree of retinopathy. Patients were placed into the following groups depending on severity (Oakley et al, 1967): (a) Normal: absence of retinal abnormalities; (b) Background retinopathy: presence of a small number of microaneurysms and/or hard exudate; (c) Pre-proliferative retinopathy: more extensive presence of background lesions and the appearance of 'cotton wool spots' and/or IRMA and venous beading; and (d) Proliferative retinopathy: the appearance of new vessels on the retina. The results of the retinopathy assessment are shown in the Appendix. The prevalence of retinopathy of all types in the group was 75%(67% background, 16% p re pro I iterative, and 16% proliferative).
The degree of nephropathy was evaluated by measuring urinary protein excretion. Albumin, IgG, and 6 2 -microglobulin excretion was measured and the individual results are presented in the Appendix. 24% of
143 the patients had an elevated albumin excretion rate (AER>30 mg/24 hours), and of these 50% showed proteinuria (AER > 300 mg/24 hours). One patient had end-stage renal failure and was classified as being proteinuric. 68% of the patient cohort had an elevated IgG excretion rate (>0.77 pg/min). There was a good correlation between AER and the excretion of IgG (r=0.97, p<0.001, results not shown). More than half (68%) of those patients with a normal AER had an elevated IgG excretion rate. Only 12% of the patients showed an elevated 32-microglobulin excretion rate (>176 ng/min). Moreover there was no correlation between 32-microglobulin and either IgG or albumin excretion (p>0.05 in both cases). In addition albumin and IgG excretion correlated significantly with HbA1 values (r=0.47 and r=0.55 respectively, p<0.01 in both cases). 9% of the whole study group (3 patients) were totally free of complications, whilst 21% had severe complications (severe complications being defined as either autonomic or peripheral neuropathy and retinopathy and abnormal AER). All of those patients who were free of retinopathy (25%) also had normal AER.
4.3.3 Relationship between polyol pathway enzyme activities
and metabolite concentrations and the severity of diabetic
complications.
Neuropathy
As described in Section 4.3.2 50% of the diabetic patients showed some degree of autonomic dysfunction. There was no relationship between erythrocyte aldose reductase (Figure 4.10) or sorbitol dehydrogenase (Figure 4.11) activities, or erythrocyte fructose and sorbitol concentrations (not shown) and the overall autonomic neuropathy score. Furthermore there
144 iue 4.10 Figure Overall autonomic neuropathy score 0.0 0.2 - 0.4 0.6 0.8 1.0
- 0 L h rltosi bten rtrct ads reductase aldose erythrocyte between relationship The activity and the overall autonomic neuropathy score neuropathy autonomic overall theand activity • • • • 4 8 6 4 2 »• »• • • • • Aldose reductase activity (|iU/mg) reductaseactivityAldose m m m m m m ------• • 145 i — • ------• ------l 10 iue 4.11 Figure Overall autonomic neuropathy score 0.0 L- 0.0 0.2 0.4 “ 0.4 0.8 0.6 1.0 U.O - - -
h rltosi bten rtrct sorbitol erythrocyte between relationship The dehydrogenase activity and the overall autonomic autonomic overall the and activity dehydrogenase erpty score neuropathy ------Sorbitol dehydrogenase activity (mll/mg) activitySorbitol dehydrogenase . 02 0.3 0.2 0.1 1 --- • • • •• • • • 1— 146 • • • • • • m • • • « — --- *— • • • • • • ------1 was no correlation between any of the indices of the polyol pathway and the individual cardiovascular function tests. In addition there was no evidence of a relationship between polyol pathway activity and the severity of peripheral sensory neuropathy, since there was no correlation (p<0.05 in all instances) between VPT and aldose reductase activity (Figure 4.12), sorbitol dehydrogenase activity (Figure 4.13), and erythrocyte fructose and sorbitol concentrations (not shown). Furthermore there was no correlation between the presence or absence of clinical symptoms of neuropathy and the activities of the polyol pathway enzymes.
Retinopathy
75% of the diabetic patients had some degree of retinopathy. When patients were subdivided on the basis of severity of retinopathy no relationship was found between the activities of aldose reductase and sorbitol dehydrogenase, erythrocyte fructose and sorbitol concentrations and the severity of disease (Table 4.2). Patients with preproliferative retinopathy had a significantly elevated sorbitol dehydrogenase activity (p<0.01), and those with proliferative retinopathy had a significantly decreased red cell sorbitol concentration (p<0.05) when compared to patients free of retinopathy.
Nephropathy
Analogous to the situation with retinopathy and neuropathy, no relationship was observed between the degree of albumin excretion and the activities of aldose reductase (Figure 4.14) and sorbitol dehydrogenase
(Figure 4.15) or the concentrations of erythrocyte fructose or sorbitol (not shown). However patients with proteinuria had significantly decreased
147 8
O) 7 - E 3 ^zi 6 “ > V-» o 03 CD (J) £ O ZJ ~Q 0) 4 CD (f) » O ]U » • 0 3 CD H — 1 tr >% o • _ o _n A 2 - LU • •
_i 50 100 150
Mean VPT (pM)
Figure 4.12 The correlation between erythrocyte aldose reductase activity and vibration perception threshold (VPT).
148 Figure 4.13 The correlation between erythrocyte sorbitol dehydrogenase dehydrogenase sorbitol erythrocyte between correlation The 4.13 Figure Erythrocyte sorbitol dehydrogenase activity (rnll/mg) 0.1 0.3r 0.0 • 0 ciiyadvbain ecpintrsod (VPT). threshold perception andvibration activity I ______l _ 0 0 150 100 50 149 Mean VPT (jaM)Mean VPT ------1 pg/ml 1.6 ±0.51.6 2.410.4 Sorbitol °0.4 + + 0.1 °0.4 ng/ml Fructose 9.6 ± 9.6 1.2 8.0 ±2.48.0 14.4 ± 3.7 14.4 14.3 ±2.614.3 ±0.5 1.6 mU/mg SDH SDH activity 0.16 ±0.010.16 pU/mg AR activity AR activity 4.2 ± 4.2 0.6 3.3 ±0.33.3 ±0.01 0.16 retinopathy. concentrations in insulin-dependent diabetic patients grouped on the basis of severity of Erythrocyte Erythrocyte aldose reductase and sorbitol dehydrogenase activities and fructose and sorbitol Retinopathy Grading Results Results expressed as ± mean SEM. of Numbers patients in parentheses. Statistically significant differences Normal Normal (8) between between function normal and differing degrees of retinopathy are denoted by; b) p<0.01, c) p<0.05. Mild Mild background (16) Preproliferative (4) Proliferative (4) ±1.5 4.1 ± ±0.9 4.3 0.01 b0.21 ±0.01 0.16 Table 4.2
150 iue .4 h rltosi bten rtrct ads reductase aldose erythrocyte between relationship The 4.14 Figure Albumin excretion rate (mg/24 hours) 0 0 0 0 1 0 0 0 1 0 0 1 0 1 1 - - - r o L ciiy n lui ecein ae Lg scale). (Log rate excretion albumin andactivity 2 Aldose reductase activity (pll/mg) reductaseAldoseactivity • • _L 4 151 6
8 10 Figure 4.15 The relationship between erythrocyte sorbitol dehydrogenase dehydrogenase sorbitol erythrocyte between relationship The 4.15 Figure Albumin excretion rate (mg/24 hours) ciiyadabmn xrto rt (o scale). (Log rate excretionalbumin and activity Sorbitol dehydrogenase activity (mU/mg) Sorbitolactivitydehydrogenase 152 erythrocyte sorbitol concentrations (p<0.01) when compared to patients with normal albumin excretion rates. IgG and 0 2 -microglobulin excretion was also independent of polyol pathway activity, since there was no correlation (p>0.05 in all cases) between the excretion rate of either protein and indices of pathway activity. When patients with normal albumin excretion rates were further subdivided into two groups on the basis of normal or abnormal IgG excretion, no significant differences were seen in enzyme activities and metabolite concentrations between the two groups.
153 4.4 Discussion
A major finding of this study is that erythrocyte aldose reductase activity is significantly elevated in diabetic patients when compared to age- matched controls. Two controversial studies have reported that erythrocyte aldose reductase activity is elevated in poorly-controlled diabetic patients (Srivastava et al, 1986) and in diabetic patients with retinopathy (Crabbe et al, 1980). In the present study however erythrocyte aldose reductase activities were found to be elevated in diabetic patients regardless of the quality of glycaemic control or complication status of the patient. The two previous studies used a direct spectrophotometric assay to measure erythrocyte enzyme activities, a technique that has been criticised by
Barretto et al (1985). The difference in methodology used to measure aldose reductase activity may well account for the different results obtained.
Immunohistochemical analysis indicates that aldose reductase levels are elevated in the retinas of diabetic patients (Vinores et al, 1988) and furthermore streptozotocin-induced diabetes in the rat is associated with elevated aldose reductase levels in the lens (Akagi et al, 1987), and in the kidney (Ghahary et al, 1989). The elevated aldose reductase levels in the kidney have been shown by Western blot analysis to be due to increased amounts of the enzyme protein rather than activation of the enzyme
(Ghahary et al, 1989). The finding of elevated aldose reductase mRNA levels in the kidney (Ghahary et al, 1989) implies that alterations in aldose reductase levels may be due to changes in gene expression or mRNA translation and stability rather than protein turnover. The increase in aldose reductase activity in the red cell of insulin-dependent-diabetic patients may
154 not be a function of increased enzyme levels, since the mature erythrocyte is unable to synthesise enzymede novo. Srivastava et al (1985) have demonstrated the presence of activated and unactivated forms of aldose reductase in the red cell, conversion of the unactivated to the activated form in vitro being promoted by glucose and its metabolites (Srivastava et al, 1985). In addition work in this laboratory (see Chapter 5) has shown that aldose reductase may be acutely activated in vivo by glucose. It is therefore possible that elevated aldose reductase levels in the erythrocytes of diabetic patients are due to posttranslational modifications of the enzyme, possibly via glycation. In agreement with the findings reported in Chapter 3 no difference was observed in sorbitol dehydrogenase activity between the diabetic patients and the age-matched controls.
Whilst washing erythrocytes prior to metabolite measurement leads to a significant reduction in fructose levels (see Chapter 2), it is still interesting to note that diabetic patients had elevated erythrocyte fructose levels compared to non-diabetic controls. Furthermore the degree to which fructose was elevated was dependent on the level of aldose reductase activity, although it cannot be ruled out that this is an artefact caused by the washing procedure. Similar elevations in fructose levels have been reported previously in a number of tissues susceptible to diabetic complications (Stribling et al, 1985; Poulsom and Heath, 1983). The values obtained for erythrocyte sorbitol levels in the non-diabetic and diabetic cohorts were in good agreement with previously published studies (Malone et al, 1980 and 1984; Popp-Snijders et al, 1984). Although red cell sorbitol was elevated in the diabetic group it did not reach statistical significance. The finding of elevated erythrocyte myo-inositol in the diabetic group has
155 been reported previously (Malone et al, 1980; Servo, 1977). This elevation contrasts with the depletion of myo-inositol in diabetic neuropathy (Greene et al, 1987b), and may be related to increased plasma myo-inositol concentrations in diabetes.
The finding of high sorbitol dehydrogenase activities relative to aldose reductase activities is in agreement with a previous study on the human lens (Jedziniak et al, 1981). This high level of sorbitol dehydrogenase activity relative to aldose reductase activity might account for the finding that fructose is the predominant polyol pathway metabolite found in the erythrocyte. As fructose and sorbitol concentrations are independent of sorbitol dehydrogenase activity this suggests that aldose reductase is the rate- limiting enzyme of the pathway in the erythrocyte. Furthermore the correlation between fructose concentration and aldose reductase activity suggests that red cell fructose, rather than sorbitol, might be a better indicator of erythrocyte polyol pathway activity.
If measurements of erythrocyte polyol pathway activity are to be of any use in elucidating the role of the pathway in the pathogenesis of the complications in inaccessible tissues, there should be a demonstrable relationship between polyol pathway enzyme activity and severity. To look for such a relationship quantitative measurements of the severity of the complications are required. The methods used in the present study to assess the severity of autonomic and peripheral neuropathy, retinopathy, and nephropathy are currently the most commonly used. Using these techniques the prevalences of autonomic and peripheral neuropathy, retinopathy and nephropathy found in the diabetic cohort in this study were
156 in reasonable agreement with previous studies (Pirart, 1978; Ewing et al, 1984; Klein et al, 1984a; Knowles, 1974; Decked et al, 1978). The finding of elevated IgG excretion in some patients with normal albumin excretion is in agreement with Decked et al (1988). This finding is explained by Decked et al as being the result of an increased negative pore charge in combination with either increased pore size or impaired tubular reabsorption. Impaired tubular reabsorption is unlikely to be a factor in the present study since 6 2 - microglobulin excretion (a marker of tubular reabsorption) was not elevated in these patients. The finding that AER and IgG excretion is significantly correlated with HbAi corroborates an earlier finding of Vibedi et al (1983b), and presumably relates to hypediltration associated with raised glomerular pressure.
There was no correlation between the overall autonomic neuropathy score or the individual cardiovascular function tests and the activities of aldose reductase and sorbitol dehydrogenase in the erythrocyte. The severity of peripheral neuropathy was assessed by measuring VPT at the tip of both great toes since VPT is altered in many neuropathic syndromes
(Ward,1988). As with autonomic neuropathy no correlation was observed between VPT and polyol pathway enzyme activities. Therefore differences in the degree of neuropathy, both peripheral and autonomic, do not appear to be related to differences in the activities of the enzymes of the polyol pathway, at least as measured in the red cell. These findings corroborate those of Broadstone and Pfeifer (1985) who found no correlation between erythrocyte sorbitol concentration and neuropathy, and those of Dyck et al
(1980 and 1988) who found no correlation between sural nerve sorbitol and fructose levels and the degree of neuropathy.
157 In addition the results suggest that differences in the severity of retinopathy and nephropathy are not related to variations in the activities of aldose reductase or sorbitol dehydrogenase. An unexpected finding was that those patients with proteinuria or proliferative retinopathy had significantly decreased erythrocyte sorbitol levels. This may reflect altered erythrocyte redox state. In the advanced stages of retinopathy and nephropathy the red cell may be unable to maintain membrane integrity through glutathione reductase. Thus although sorbitol is not normally permeable through the erythrocyte membrane (Wick and Drury, 1951), it is possible that in the advanced stages of retinopathy and nephropathy sorbitol is lost by leakage as a result of reduced membrane integrity.
On the basis of these results it is unlikely that measurements of erythrocyte polyol pathway enzyme activities will be of any use in elucidating the role of the polyol pathway in the development of complications in less accessible tissues. In particular measurements of erythrocyte sorbitol will be of less use since sorbitol accumulation is not related to either aldose reductase or sorbitol dehydrogenase activity.
A number of studies have shown that the microvascular complications, retinopathy and nephropathy, approach their maximum prevalence in patients with a disease duration of 25 years. A recent study also suggests that the prevalence of autonomic neuropathy peaks after 25 years duration (O'Brien and Corrall, 1988), although the basis for this has been questioned (Krowleski, 1988). Therefore a group of long-duration (>25 years) diabetic patients were recruited for the study since these patients
158 were unlikely to develop further complications. A major disadvantage of this selection procedure, however, is that the group of patients recruited is unlikely to contain anyone with aggressive early-onset complications. Thus the question of whether variations in polyol pathway enzyme activities are related to the early onset of complications cannot be addressed in this group of patients. A better approach might have been to recruit two distinct groups of patients, one with early onset complications, the other with a long duration of disease and no complications (see general discussion). A second limitation of the study was the size of the diabetic patient group. To increase the numbers it might have been better to have approached a number of other diabetic clinics and recruited long-duration patients from their registers.
159 Chapter Five
Activation of aldose reductase
160 5.1 Introduction
One interesting finding reported in Chapter 4 was that erythrocyte aldose reductase activity in insulin-dependent diabetic patients was significantly greater than in age-matched controls (3.6 ± 0.3 versus 2.1 ± 0.2 pll/mg protein, p<0.001). Similar findings have been reported by Srivastava et al (1986) and by Crabbe et al (1980). Srivastava et al (1986) reported that aldose reductase activity was 4-6 fold higher in diabetic patients than controls when blood sugar levels were above 400 mg% (>22 mmol/l). However in diabetic patients with normal blood sugar levels there was no significant difference between aldose reductase activities. Crabbe et al
(1980) found that aldose reductase activity was significantly elevated in erythrocytes from diabetic patients with retinopathy and no cataract and patients with retinopathy and cataract when compared to controls (p<0.01 and p<0.001 respectively). Moreover elevated levels of aldose reductase activity have been demonstrated immunohistochemically in the lenses and kidneys of streptozotocin-diabetic rats (Akagi et al, 1987; Ghahary et al, 1989). Increased aldose reductase activities could be explained either by an increase in the amount of enzyme protein or by an activation of the existing enzyme. Western blot analysis shows that increased aldose reductase activity in the kidney of streptozotocin-diabetic rats is associated with increased amounts of enzyme protein (Ghahary et al, 1989).
Furthermore the increased amounts of protein are due tode novo synthesis of enzyme rather than alterations in protein turnover, since Northern blot analysis reveals increased aldose reductase mRNA levels (Ghahary et al,
1989). Elevated aldose reductase activity in the lens is believed to have a similar basis (Akagi et al, 1987). Activation of the erythrocyte enzyme may
161 well occur via a different mechanism since the mature erythrocyte is unable to synthesise proteinde novo. Srivastava et al (1986) demonstrated that erythrocyte aldose reductase exists in two forms, a native or unactivated form and an activated form. The activated form shows different kinetic properties from the native form, having a Km for glucose that is 1/1 Oth that of the native enzyme and a Vmax that is much higher. Moreover the native form can be converted to the activated form in vitro by incubating with glucose and various glycolytic intermediates (Srivastava et al, 1985). Furthermore in diabetic patients with blood glucose levels greater than 15 mmol/l erythrocyte aldose reductase exists predominantly in the activated form (Srivastava et al, 1986). Further reports indicate that bovine lens aldose reductase can also exist in native and activated forms (Del Corso et al, 1987 and 1989). The activated form can be generated by incubating the native enzymein vitro with systems that generate oxygen radical species. Of relevance to the treatment of the diabetic complications is the fact that the activated form of aldose reductase is less sensitive to inhibition by aldose reductase inhibitors than the native form (Srivastava et al, 1986; Del Corso et al, 1989). Furthermore when cells are grown in culture in the presence of high glucose concentrations the inhibition of sorbitol production by Sorbinil is impaired (Lorenzi et al, 1987). The aim of the work presented in this
Chapter was to study the activation of erythrocyte aldose reductase in vivo in response to glycaemic challenge.
162 5.2 Experimental
Glucose tolerance tests were performed on 8 healthy non-diabetic subjects, all of whom were members of the Department of Biochemistry at Charing Cross and Westminster Medical School. The 8 volunteers comprised 6 males and 2 females, and they had a mean age of 28 ± 3 years (± SEM). Glucose tolerance tests were carried out as follows. Subjects fasted overnight and were then given 75 g of glucose orally. Blood samples were taken prior to the glucose challenge and at 20, 30, 60, 90, and 120 minutes thereafter for the measurement of plasma glucose, aldose reductase and sorbitol dehydrogenase activities, and erythrocyte fructose and sorbitol concentrations (assayed as detailed in Chapter 2). In addition erythrocytes were incubated in vitro with varying concentrations of glucose and aldose reductase activity and intracellular sorbitol and fructose concentrations measured.
163 5.3 Results
In response to a 75 g oral glucose challenge there was a transient rise in erythrocyte aldose reductase activity in the 8 subjects, that peaked at 30 minutes (Figure 5.1). The rise in aldose reductase activity mirrored the rise in plasma glucose concentration. The rise in activity ranged from 15 to 117% with a mean rise of 76% at 30 minutes (p<0.01 when compared to time zero). Furthermore there was a linear correlation between the percentage rise in erythrocyte aldose reductase activity and the rise in plasma glucose concentration (r= 0.53, p<0.05, Figure 5.2). There was no change in erythrocyte sorbitol dehydrogenase activity in response to acute glucose challenge. In addition to the rise in aldose reductase activity there was a transient rise in erythrocyte fructose concentration (Figure 5.3) that peaked at 60 minutes. No similar rise was seen in erythrocyte sorbitol concentration. Erythrocytes from 3 subjects were incubated with either no substrate or 30 mmol/l glucose as described in Chapter 2. Incubation in 30 mmol/l glucose led to a 53% rise in aldose reductase activity, whilst fructose and sorbitol concentrations rose by 1200% and 300% respectively.
164 a
o E E CD if) O O J3 O) CO E iD _C0 CL Erythrocyte Erythrocyte aldose reductase activity (pll/mg)
Figure 5.1 The response of erythrocyte aldose reductase activity (o) to changing plasma glucose concentration (•) following a glucose tolerance test. Statistical analysis was performed using the Wilcoxon signed-rank test. Statistically significant differences with respect to time zero are denoted by; a) pcO.001, b) p<0.01.
165 >
Increase in plasma glucose (mmol/l)
Figure 5.2 The relationship between the increase in erythrocyte aldose reductase activity and the rise in plasma glucose concentration (r=0.53, p<0.05).
166 o E E" CD CD O O JD CD 0 3 E CD 05 0. Erythrocyte fructose (pg/ml RBC) Erythrocyte
Figure 5.3 The response of erythrocyte fructose concentration (•) to changing plasma glucose concentrations (o) following a glucose tolerance test.
167 5.4 Discussion
The major finding of this chapter is the in vivo transient rise in erythrocyte aldose reductase activity (by 76%) following oral glucose challenge. Srivastava et al (1986) have shown previously that erythrocyte aldose reductase activity is increased several-fold when erythrocytes are incubated in vitro with 30-50 mmol/l glucose, a finding confirmed in our present study. Srivastava et al proposed that the increase in activity was due to activation of the enzyme and not due to increased amounts of enzyme protein, since the mature erythrocyte is unable to synthesise proteinde novo. Quantitation of enzyme protein by ELISA also supports this proposal
(Srivastava et al, 1986). This proposal is further supported by the finding that partially purified aldose reductase from erythrocytes can be activated by incubation in vitro with glucose and intermediates of the glycolytic pathway, such as glucose-6-phosphate and fructose-6-phosphate (Das and Srivastava, 1985b). On the basis of these findings Srivastava and co workers proposed that the mechanism of activation of aldose reductase might be via the direct interaction of glucose with the enzyme protein, that is via nonenzymic glycation (Srivastava et al, 1985). Such a mechanism may well explain the transient rise in aldose reductase activity seen in vivo, since the rise in enzyme activity parallels the rise in plasma glucose concentrations and, moreover, there is a linear correlation between the increase in enzyme activity and the rise in plasma glucose concentrations. Furthermore the time course of activation is similarin vivo and in vitro. Maximum activation in vitro occurs after 15 minutes of incubation with the activating system (Das and Srivastava, 1985b), whereas activation in vivo is maximal 30 minutes after the glucose challenge. A second potential
168 mechanism that might explain the rise in activity is increased production of NADPH (a cofactor in the aldose reductase reaction) as a result of increased glucose metabolism via the hexose monophosphate shunt. Such a mechanism, however, cannot explain the increase in activity seen when partially purified enzyme is incubated in vitro with glucose or other glycolytic intermediates.
The observation of a rise in red cell fructose concentration supports the suggestion made in Chapter 4 that red cell fructose might be a better indicator of erythrocyte polyol pathway activity than sorbitol. The reduction in fructose levels 60 minutes after glucose challenge may well be due to leakage of fructose into the extracellular medium rather than as a result of increased metabolism of fructose. Excess fructose is unlikely to enter the glycolytic pathway since fructose is a poor substrate for hexokinase and the enzyme fructokinase is not found in the erythrocyte. Moreover fructose could not enter the glycolytic pathway through conversion to glucose via a reversal of the polyol pathway since, although fructose is a good substrate for sorbitol dehydrogenase (Jedziniak et al, 1981), sorbitol is a poor substrate for aldose reductase. Incubation of erythrocytes with glucose in vitro however leads to a rise in both intracellular and extracellular fructose (Morrison et al, 1970), a finding that supports the proposal that fructose is lost from the erythrocyte by leakage to the extracellular medium. It must be pointed out that metabolite levels were measured in washed erythrocytes. Since the washing procedure leads to a loss of fructose it cannot be ruled out that these changes are artefactual, a consequence of the washing procedure. Unlike fructose no trend was seen in the response of sorbitol to glucose challenge. A similar finding was reported by Nagasaka et al (1988) following glucose
169 tolerance tests on 2 non-diabetic subjects. The observation that erythrocyte sorbitol and fructose concentrations alter rapidly in response to changes in
plasma glucose concentration argue against the proposal that measurements of sorbitol accumulation provide an indicator of chronic
polyol pathway activity. The rapid changes in concentration suggest that fructose and sorbitol concentrations provide no more than an a 'snapshot' of erythrocyte polyol pathway activity.
The finding that aldose reductase may be activated by glucose in vivo suggests that the basis for the elevated aldose reductase activities seen in insulin-dependent diabetic patients when compared to non-diabetic controls (see Chapter 4) may be posttranslational modification of the enzyme, possibly glycation.
170 Chapter Six
General Discussion
171 As described in the Introduction the aims of this Thesis were threefold. The first aim was to determine whether activity or electrophoretic variants of the enzymes aldose reductase and sorbitol dehydrogenase exist in the erythrocytes of diabetic and non-diabetic subjects. As described in Chapters
3 and 4 no difference was observed in sorbitol dehydrogenase activities between diabetic patients (both insulin-dependent and non-insulin- dependent) and non-diabetic controls. However in confirmation of earlier preliminary studies (Crabbe et al, 1980; Vaca et al, 1982; Barretto et al, 1985; Medina et al, 1987), we found a wide variation in activity between individuals. The basis for this wide variation in activity remains unclear. It does not appear to be a consequence of diabetes, since there was no correlation between sorbitol dehydrogenase activity and indices of diabetic control, namely plasma glucose concentrations and glycosylated haemoglobin levels. It is possible that sorbitol dehydrogenase is hormonally regulated since its activity was significantly lower in non-diabetic males than females, and that this may contribute to the variations in activity seen.
Two controversial studies have reported that erythrocyte aldose reductase activity is elevated in poorly-controlled diabetic patients (Srivastava et al, 1986) and in diabetic patients with retinopathy (Crabbe et al, 1980). In the present study however erythrocyte aldose reductase activities were found to be elevated in diabetic patients regardless of the quality of glycaemic control or complication status of the patient. As described in Chapter 4 these differences may arise from the different methodologies used to measure aldose reductase activity. In the previous studies aldose reductase activity was measured by following the oxidation of
NADPH spectrophotometrically, a method that has been criticised by
172 Barretto and co-workers (Barretto et al, 1985) since the presence of other enzymes, such as glutathione reductase and methaemoglobin reductase in the haemolysate will interfere with the assay. Such problems do not arise with the method used in the present study, since the conversion of galactose to galactitol by aldose reductase is measured directly by GLC. The presence of alternative substrates for aldose reductase such as glucose in the erythrocytes of diabetic patients may interfere with the assay method used in these studies. However it would be predicted that this would lead to an underestimation of enzyme activity.
The finding of only one form of sorbitol dehydrogenase in the erythrocyte confirms an earlier study by Charlesworth (1972) in non-diabetic subjects. For the reasons outlined in Chapter 2 , namely the lability of the erythrocyte enzyme and its low abundance, it was impossible to determine whether multiple isoenzymes of aldose reductase exist in the human erythrocyte.
The second aim of the Thesis was to examine the relationship between erythrocyte polyol pathway enzyme activities and the concentrations of polyol pathway metabolites. Measurement of erythrocyte sorbitol concentrations have been used by a number of groups as an indicator of polyol pathway activity, particularly when used to study the effect of treatment with aldose reductase inhibitors. However, the manner in which sorbitol concentrations are related to either aldose reductase or sorbitol dehydrogenase activity has not been examined extensively. Whilst there was no relationship between erythrocyte sorbitol concentration and the activity of either enzyme, there was a linear relationship between fructose
173 concentration and aldose reductase activity in washed erythrocytes. Whilst this relationship might be an artefact, a product of the washing procedure, it does suggest that fructose concentration might provide a more quantitative indicator of polyol pathway activity than sorbitol concentration. This might be predicted since fructose production is the end point of flux through the polyol pathway, while sorbitol concentration reflects both its production by aldose reductase and its breakdown by sorbitol dehydrogenase. However single measurements of erythrocyte fructose and sorbitol concentrations will provide no more than a 'snapshot' of polyol pathway activity, since as demonstrated in Chapter 5 erythrocyte sorbitol and fructose concentrations alter rapidly with changing plasma glucose concentrations.
A consistent finding was that sorbitol dehydrogenase activities were higher than those of aldose reductase and this may account for the fact that fructose rather than sorbitol is the predominant polyol pathway metabolite found in the human erythrocyte. This difference may in part be a function of the method of enzyme assay. Sorbitol dehydrogenase activity was measured at 1 mol/l sorbitol (Km 1.45 mmol/l), whereas aldose reductase was measured at 27.75 mmol/l galactose (Km 50 mmol/l). Thus sorbitol dehydrogenase activity approximates to Vmax, whereas the activity of aldose reductase is limited by substrate availability. However even if it is extrapolated that aldose reductase activity at approaching Vmax is 10 times greater than that measured, the activity of aldose reductase would still remain approximately 100 times less than that of sorbitol dehydrogenase.
The implication is that aldose reductase is the rate-limiting enzyme of the pathway in this tissue. This corroborates the earlier finding of Jedziniak and co-workers (1981) who found a similar relationship in the human lens, and is
174 in contrast to the situation in the rat where aldose reductase activity is greater than sorbitol dehydrogenase activity (Varma and Kinoshita, 1974a;
Naeser et al, 1988). This reversal in activity ratio may well account for the observation that sorbitol accumulation is far greater in diabetic rats than in diabetic humans (Malone et al, 1980; Popp-Snijders et al, 1984; Sommers et al, 1982; Stribling et al, 1985; Poulsom and Heath, 1983; Naeser et al, 1988). These observations raise the question of whether the rat is a good model for polyol pathway activity in man.
The third aim was to determine the relationship between the activities of erythrocyte aldose reductase and sorbitol dehydrogenase and autonomic and peripheral neuropathy, retinopathy, and nephropathy, since it has been proposed that measuring erythrocyte polyol pathway activity might prove valuable in elucidating the role of the polyol pathway in the development of complications in tissues such as the nerve, lens, and kidney (Malone et al, 1984). As described in Chapter 4 no relationship was observed between the activities of the polyol pathway enzymes and quantitative measurements of the severity of neuropathy, retinopathy, and nephropathy in a group of long- duration (>25 years) insulin-dependent diabetic patients. On the basis of these results it appears unlikely that measurements of erythrocyte polyol pathway activity will provide any information on the role of the pathway in the development of complications in susceptible tissues. Since it was not possible to screen for the presence of aldose reductase isoenzymes in the erythrocyte, it is impossible to exclude a role for any specific isoenzyme in the development of the diabetic complications. Grimshaw and Mathur
(1989) have reported the presence of only one form of aldose reductase in a
175 number of human tissues and thus it would appear unlikely that multiple isoforms exist in the erythrocyte.
Patients with a duration of disease of at least 25 years were chosen for this study on the basis that they were unlikely to develop further complications. A major limitation of this selection strategy is that it selects against patients with aggressive, early onset complications, since they are less likely to survive 25 years after initial diagnosis. Thus in the patient group studied, it is impossible to draw any conclusions about the role of the polyol pathway in the development of complications in this specific subgroup of patients. A far better approach to studying the role of the polyol pathway in the development of the complications would have been to recruit two groups of patients, one group comprising patients with early onset, aggressive complications, the other group comprising patients with a long- duration of disease but free of complications. A second major limitation was the small number of patients recruited for the study. Although all the insulin- dependent diabetic patients attending the Diabetic Clinic at Charing Cross Hospital with a duration of disease in excess of 25 years were invited to participate in the study only 34 consented to take part. To increase the number of patients participating in the study it might have been better to approach a number of other diabetic clinics and recruit patients from their registers. Indeed to recruit a large enough group of patients with early onset, aggressive complications to make any study meaningful it would have been essential to carry out a multi-centre study, since this subgroup represents a very small fraction of diabetic patients.
176 One question raised by the study concerns the mechanism that leads to elevated aldose reductase activity in the erythrocyte in diabetes. It is possible to envisage a variety of mechanisms that would result in elevated enzyme activity. At the DNA level increased gene expression may lead to elevated levels of protein and hence increased activity. There is a growing body of evidence that implicates increased gene expression with elevated aldose reductase levels in diabetes in a number of human and rat tissues. Akagi and co-workers (1987) have immunohistochemically demonstrated increased aldose reductase levels in diabetic lenses, whilst Vinores and co workers (1988) have demonstrated elevated levels in human diabetic retina. Similarly Ghahary and co-workers (1989) have shown by Western blot analysis that aldose reductase levels are raised in the kidneys of streptozotocin-induced diabetic rats, and by Northern blot analysis have shown that these elevated levels are associated with increased mRNA levels. Increased expression of the aldose reductase gene has also been shown to occur in cultured rabbit renal inner medulla cells when grown in hyperosmotic media (Garcia-Perez et al, 1989). Growth in hyperosmotic media is associated with an increase in aldose reductase protein (Bedford et al, 1987), which corresponds to an increase in mRNA levels (Garcia-Perez et al, 1989). A second potential mechanism that might account for elevated aldose reductase activity is gene polymorphism. Although at present the question of polymorphism at the aldose reductase gene is unresolved, it is not inconceivable that a polymorphism generating a more active form of aldose reductase might exist. At the RNA level increased mRNA stability or translation might also lead to elevated protein levels and hence activity. However to date there is no evidence to suggest that either process occurs.
177 Since the erythrocyte is anucleate none of the mechanisms outlined above are likely to account for the increased activity seen in the studies reported. In the case of the erythrocyte enzyme the increase in activity is most likely due to some form of posttranslational modification. Del Corso and co-workers (1989) identified two forms of bovine lens aldose reductase which they termed the unactivated and the activated forms. Furthermore they demonstrated that incubating the unactivated form with oxygen radical generating systems gave rise to the activated form. In addition Srivastava and co-workers (1985) demonstrated that erythrocyte aldose reductase exists in unactivated and activated forms. They also demonstrated that the unactivated form can be activated in vitro by incubating with glucose and various glycolytic intermediates.
As described in Chapter 5 erythrocyte aldose reductase is transiently activated in vivo by acute glycaemic challenge, a finding that corroborates the earlierin vitro studies of Del Corso et al (1989) and Srivastava et al (1985). For the experiments described in Chapter 5 the glycaemic challenge was provided by an oral glucose tolerance test. There are a number of problems associated with the use of a glucose tolerance test. One is that basal plasma glucose concentrations vary between individuals as does the maximum plasma glucose concentration attained. Thus the degree of glycaemic challenge will vary between individuals. Furthermore an individual's response to glucose challenge varies from day to day. These considerations may account for the variability seen in the response of erythrocyte aldose reductase activity to glucose challenge. A more reproducible method of examining the response of erythrocyte aldose reductase activity to glycaemic challenge might have been to use glucose
178 clamping techniques. Using this approach the degree of glycaemic challenge would have been kept constant between individuals, possibly giving more reproducible results. In addition the response of aldose reductase activity in the erythrocytes of diabetic patients could have been examined using this technique.
On the basis of Srivastava and co-workers (1985) in vitro studies and the in vivo studies described in this Thesis, it appears that erythrocyte aldose reductase may be activated through the direct interaction of glucose with the enzyme molecule, ie via glycation. Of interest is the observation that the formation of a Schiff base between pyridoxal phosphate and a lysine residue at the active site of aldose reductase from human psoas muscle, a situation analogous to glycation, gives rise to an enzyme form with an increased Vmax (Morjana and Flynn, 1989b). On the basis of the experiments performed it is impossible to exclude the possibility that activation is due to increased NADPH production as a result of increased flux through the hexose monophosphate shunt. However this could not account for activation seen in vitro. One way to eliminate this possibility would be to incubate erythrocytes in vitro with a sugar that is not metabolised by the cell but is a good substrate for the glycation reaction.
The finding that aldose reductase activity may be acutely modulated by glucose concentrations in vivo may have important consequences on the development of the diabetic complications. This finding suggests that hyperglycaemia may give rise to increased polyol pathway activity by more than one mechanism. Since aldose reductase has a high Km for glucose and shows first order kinetics elevated glucose concentrations may give rise
179 to increased activity and hence increased flux through the polyol pathway. In addition elevated glucose concentrations may also activate aldose reductase by glycation, giving rise to an even greater rise in polyol pathway activity, and possibly accelerating the development of complications.
Diabetes has also been shown to cause an increase in aldose reductase protein levels (Vinores et al, 1988; Ghahary et al, 1989; Akagi et al, 1987), which in turn will lead to an increase in polyol pathway activity. This raises the question of how does diabetes regulate aldose reductase levels? An understanding of the mechanism might provide an alternative avenue for therapeutic intervention. A second consequence of the activation of aldose reductase concerns the development of aldose reductase inhibitors. Since the activated form of aldose reductase has been shown to be less susceptible to inhibition (Srivastava et al, 1986; Del Corso et al, 1989), it is implied that during periods of hyperglycaemia, when the enzyme is in its activated form, the efficacy of aldose reductase inhibitors may be severely reduced. Therefore in order to improve efficacy in patients with recurrent hyperglycaemia potent inhibitors of the activated form of aldose reductase may need to be developed.
A question that remains unresolved is whether polymorphisms exist at the aldose reductase and sorbitol dehydrogenase gene loci, and what role, if any, possession of a specific aldose reductase or sorbitol dehydrogenase polymorphism plays on the development of the diabetic complications. To address this question cDNA clones for human aldose reductase and sorbitol dehydrogenase are required. Over the last few months cDNA sequences for human placental (Bohren et al, 1989; Chung and LaMendola, 1989) and foetal liver (Graham et al, 1989) aldose reductases have been published.
180 No cDNA sequence data is currently available for human sorbitol dehydrogenase, although the complete amino acid sequence of human liver enzyme has recently been published (Karlsson et al, 1989). One approach that has recently been used to clone the yeast TFIID gene when only partial amino acid sequence was known, was to make degenerate oligonucleotides complementary to the derived peptide sequences and use these as primers for polymerase chain reaction (PCR) amplification (Horikoshi et al, 1989). Since the whole amino acid sequence is known, using such as approach it should be possible to isolate cDNA clones for human sorbitol dehydrogenase by PCR amplification from a human cDNA library.
Detailed interpretation of the blotting data of Bohren et al (1989) in conjunction with analysis of the restriction map derived from the cDNA sequences (Bohren et al, 1989; Graham et al, 1989; Chung and LaMendola, 1989) show that it is likely that the gene for human aldose reductase contains introns. Further these blots show little evidence for multiple aldose reductase genes. No such data is available for the sorbitol dehydrogenase gene. Once obtained the future strategy will be to use cDNA clones to search for gene polymorphisms. This will be undertaken initially in DNA samples from randomly selected volunteers to enable the frequency at which any polymorphisms occur to be determined. DNA samples will be digested using specific restriction endonucleases, including Msp I, Taq I, Hind III, Bgl
II, Eco Rl, Bam HI and Pst I which detect polymorphisms at high frequency (Sholnick et al, 1984). Restriction digests will be analysed by Southern blot hybridisation for restriction fragment length polymorphisms using the aldose reductase and sorbitol dehydrogenase cDNA probes.
181 The next stage will be to determine the relationship between specific gene polymorphisms and the development of diabetic complications. Polymorphisms will be identified by Southern blot hybridisation using the aldose reductase and sorbitol dehydrogenase cDNA probes in restriction digests of DNA samples from diabetic patients. Two groups at either end of the clinical spectrum will be screened. As discussed earlier one group will comprise long duration diabetic patients without major complications, whereas the second group will comprise patients with complications despite a short duration of disease. To ensure adequate sized groups patients will be drawn from a large population survey in Western Australia (see Welborn et al, 1984), to where this laboratory will shortly move. The question to be addressed is whether any specific aldose reductase or sorbitol dehydrogenase polymorphism is disproportionately represented in either group of patients. Were a polymorphism to segregate in this manner routine screening would identify susceptible patients. Since no satisfactory treatment for the diabetic complications is currently available the early detection of susceptible patients may have a major impact on prognosis and management.
182 Chapter Seven
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231 Appendix
232 Table A1 Individual cardiovascular function test results
Hosp No 30/15 ratio Valsalva ratio E:l ratio BP resfDonse ANS to star iding (mm H9) 248602 1.00 2 1.00 2 -- 20 1 0.83 035667 1.06 0 -- 1.19 0 25 1 0.17 115248 1.00 2 1.69 0 1.04 2 6 0 0.50 193205 1.06 0 -- 1.19 0 7 0 0.00 556140 1.28 0 2.00 0 1.61 0 18 1 0.13 508780 1.14 0 1.33 0 1.08 2 10 0 0.25 209571 1.12 0 1.36 0 1.15 0 10 0 0.00 328524 1.12 0 1.31 0 1.07 2 3 0 0.25 025163 1.17 0 1.63 0 1.19 0 25 1 0.13 257559 1.11 0 1.08 2 1.18 0 8 0 0.25 048211 1.03 2 0.94 2 1.02 2 5 0 0.75 050151 0.97 2 1.15 2 1.07 2 6 0 0.75 643629 1.08 0 1.60 0 1.10 0 3 0 0.00 086639 1.10 0 1.43 0 1.17 0 4 0 0.00 144002 1.14 0 1.53 0 -- 10 0 0.00 028712 1.00 2 1.03 2 1.04 2 17 1 0.88 101296 0.96 2 0.96 2 1.01 2 7 0 0.75 240200 1.40 0 1.51 0 1.37 0 5 0 0.00 191234 1.28 0 1.59 0 1.10 2 5 0 0.25 418041 1.06 0 1.29 0 1.06 1 10 0 0.13 276014 1.03 1 1.22 0 1.18 0 2 0 0.13 511151 1.40 0 1.29 0 1.28 0 2 0 0.00 179381 1.20 0 1.17 2 1.30 0 + 16 0 0.25 287162 1.05 0 1.05 2 1.03 2 9 0 0.50 035801 1.08 0 1.25 0 1.12 0 0 0 0.00 081287 1.22 0 1.71 0 1.45 0 6 0 0.00 502807 1.02 1 1.22 0 1.33 0 18 1 0.25 187010 1.23 0 1.60 0 1.34 0 8 0 0.00 155916 1.17 0 1.73 0 1.11 2 10 0 0.13 081289 1.00 2 1.13 2 1.10 0 18 1 0.63 664615 - --- 1.08 1 - - 0.50 523353 1.09 0 1.22 0 1.16 0 10 0 0.00 223445 1.09 0 1.53 0 1.23 0 0 0 0.00 068366 1.00 2 1.11 2 1.09 1 4 0 0.63 For each test the first column is the actual value obtained, whilst the second column is the assigned score, where 0 denotes a normal value, 1 a borderline value, and 2 an abnormal value. ANS denotes overall autonomic neuropathy score.
2 3 3 Table A2 Individual vibration perception threshold results
Hosp No Mean VPT Score3 (UM) 248602 1.4 0 035667 1.7 0 115248 100.0 2 193205 2.8 0 556140 0.2 0 508780 1.5 0 209571 1.3 0 328524 0.4 0 025163 1.8 0 257559 129.1 2 048211 16.4 0 050151 1.7 0 643629 28.0 2 086639 4.5 2 144002 8.9 2 028712 62.0 2 101296 91.7 2 240200 0.9 0 191234 0.7 0 418041 5.9 0 276014 1.8 0 511151 1.0 0 179381 -- 287162 1.5 0 035801 42.9 2 081287 1.0 0 502807 5.0 0 187010 2.0 0 155916 1.5 0 081289 15.5 0 664615 84.6 2 523353 16.2 0 223445 2.1 0 068366 38.3 2 a) 0 denotes normal value, 2 abnormal value
2 3 4 Table A3 Individual retinopathy gradings
Hosp No MA c w s HE IRMA NV Grade3 248602 ----- A 035667 - - --- A 115248 + + - + - C 193205 ----- A 556140 - - --- A 508780 + ---- B 209571 + ---- B
328524 + --- - B 025613 ND ND ND ND ND bB 257559 ND ND ND ND ND bB 048211 + - + -- C 050151 + + --- C 643620 + ---- B 086639 + - + -- B
144002 + ---- B 028712 ND ND ND ND ND CND 101296 + ---- B
240200 + - + -- B
191234 ---- + D
418041 + - - -- B
276014 ----- dC 511151 ----- A
179381 + - - -- B 287162 ND ND ND ND ND bD
035801 + - + - + D
081287 + - --- B 502807 - ---- A
187010 + - - -- B 155916 - ---- A
081289 + - + -- B 664615 ND ND ND ND ND bD 523353 ND ND ND ND ND CND
223447 + ---- B
068366 - - --- A a) A denotes no retinopathy, B, background retinopathy; C, preproliferative retinopathy; D, proliferative retinopathy. b) Graded on the basis of clinical records. c) Cataract prevented assessment. d) Photocoagulation. ND denotes not determined due to poor photographs. + present, - absent
2 3 5 Table A4 Individual urinary protein excretion results
Hosp No Albumin igG 62 -microglobulin excretion excretion excretion (mg/24 hr)a (pg/min)b (ng/min)b 248602 6.9 N 0.18 0 34.43 0 035667 6 .0 N 0.55 0 84.20 0 115248 1267.8 P 16.86 1 87.33 0 193205 5.2 N 0.91 1 108.96 0 556140 4.8 N 0.24 0 25.12 0 508780 168.6 M 0.74 0 38.19 0 209571 4.9 N 0.63 0 31.60 0 328524 2 .0 N 0.98 1 226.23 1 025613 2.3 N 1.58 1 126.6 0 257559 24.5 N 2.41 1 28.71 0 048211 10.4 N 2 .0 2 1 59.08 0 050151 23.9 N 2.16 1 79.37 0 643620 26.5 N 2 .2 2 1 93.00 0 086639 71.6 M 1.59 1 421.68 1 144002 1 2 .6 N 3.11 1 52.88 0 028712 14.4 N 0.79 1 44.33 0 101296 7.0 N 1 .8 8 1 300.31 1 240200 365.5 P 2.99 1 29.83 0 191234 7.8 N 0.63 0 55.70 0 418041 1 2 1 0 .6 P 16.49 1 33.75 0 276014 44.1 M 1.71 1 119.27 0 511151 11.7 N 1.14 1 38.59 0 179381 1 1 .8 N 0.84 1 38.57 0 287162 - cP ---- 035801 16.1 N 1.01 1 3.23 0 081287 8.5 N 0.26 0 10.03 0 502807 12.3 N 1.19 1 98.04 0 187010 8.7 N 0.46 0 88.38 0 155916 8.4 N 0.72 0 25.55 0 081289 9.3 N 0.96 1 32.49 0 664615 ------523353 1 1 .6 N 1.64 1 125.93 0 223447 78.0 M 3.99 1 154.98 0 068366 18.2 N 1.80 1 287.29 1 - No urine sample provided a) N denotes normal excretion rate; M, microalbuminuria; P, albuminuria b) 0 denotes normal excretion rate,1 denotes abnormal excretion rate c) Patient with end-stage renal failure
2 3 6