Eye Complications and Markers of Morbidity and Mortality in Long‐term Type 1 Diabetes
2011; Wiley; Volume: 89; Issue: thesis1 Linguagem: Inglês
10.1111/j.1755-3768.2010.02105.x
ISSN1755-3768
Autores Tópico(s)Advanced Glycation End Products research
ResumoThe incidence of type 1 diabetes is rising all over the world. Furthermore, the increased life-expectancy of type 1 diabetic patients is likely to cause a higher number of diabetes-related micro- and macrovascular complications in the years to come. In order to examine the level of long-term complications in type 1 diabetes as well as potential markers of micro- and macroangiopathy, a population-based cohort of Danish type 1 diabetic patients was examined in order to achieve the following aims: To evaluate diabetic retinopathy as a long-term marker of all-cause mortality in type 1 diabetes (Paper I). To estimate the long-term incidence and associated risk factors of blindness (Paper II) and cataract surgery (Paper III) in type 1 diabetes. To use retinal vascular analyses in order to investigate the associations of long-term micro- and macrovascular complications and retinal vascular diameters (Paper IV) and retinal fractals (Paper V) in type 1 diabetes. To examine N-terminal pro brain natriuretic peptide (Paper VI) and osteoprotegerin (Paper VII) as non-invasive markers of micro- and macrovascular complications in type 1 diabetes. In Paper I it was a major finding that, despite a mean age of only 38.3 years at baseline, 44.7% of the patients died during the 25-year follow-up. Patients who had proliferative retinopathy as well as proteinuria at the baseline examination had a significantly higher mortality. For these, the 10-year survival was only 22.2%. As demonstrated in Paper II, blindness is an important issue in type 1 diabetes. The 25-year cumulative incidence of blindness was 7.5%. Glycaemic regulation and maculopathy at baseline were both identified as risk factors of blindness. Finally, mortality was higher in patients who went blind during the follow-up. Cataract surgery is quite common in type 1 diabetes. In Paper III a 25-year cumulative incidence of 20.8% was found. Adjusted for mortality, this was even higher (29.4%). As compared to patients without diabetes, cataract surgery takes place approximately 20 years earlier in type 1 diabetic patients. Age and maculopathy at baseline were both identified as predictors of cataract surgery. In Paper IV it was demonstrated that patients with retinal arteriolar narrowing were 2.17 and 3.17 times more likely to have nephropathy and macrovascular disease, respectively. This was an important finding that suggests that retinal fundus photos may be used in order to predict the risk of non-ophthalmological complications in type 1 diabetes. Retinal fractal analysis is another way to evaluate the vascular system of the retina. In Paper V we found associations between retinal fractal and microvascular – but not macrovascular – disease. For instance, patients with lower fractal dimensions were more likely to have proliferative retinopathy (OR 1.45, 95% CI 1.04–2.03) and neuropathy (OR 1.42, 95% CI 1.01–2.01). NT-proBNP is likely to be a future predictor of diabetes-related complications. In Paper VI higher levels of NT-proBNP were related to nephropathy (OR 5.03, 95% CI 1.77–14.25), neuropathy (OR 4.08, 95% CI 1.52–10.97) and macrovascular disease (OR 5.84, 95% CI 1.65–20.74). These associations should be confirmed in future prospective studies. As opposed to NT-proBNP, osteoprotegerin is less likely to be a predictor of either micro- or macrovascular disease in type 1 diabetes. As demonstrated in Paper VII, even though association between higher levels of OPG and nephropathy were found in an age- and sex-adjusted model (OR 2.54, 95% CI 1.09–5.90), this was no longer statistically significant when other factors were taken into account. Overall, it was demonstrated that various complications such as mortality, blindness and cataract surgery were high in type 1 diabetes. Furthermore, retinal arteriolar narrowing, decreased retinal fractals and plasma NT-proBNP were associated with various micro- and macrovascular complications. If confirmed by prospective studies, these modalities may be used in order to identify patients at risk of diabetes-related complications. This could, ultimately, lead to decreased mortality and morbidity in type 1 diabetic patients. Acta Ophthalmologica Thesishttp://www.actaophthalmologica.com Jakob Grauslund Faculty of Health ScienceUniversity of Southern DenmarkDepartment of OphthalmologyOdense University Hospital, Denmark To Vilma, my wonderful daughter This thesis is based on the following papers which will be referred to by their Roman numerals: I Grauslund J, Green A, Sjølie AK (2008). Proliferative retinopathy and proteinuria predict mortality rate in type 1 diabetic patients from Fyn County, Denmark. Diabetologia 51: 583–588. II Grauslund J, Green A, Sjølie AK (2009). Blindness in a 25-year follow-up of a population-based cohort of Danish type 1 diabetic patients. Ophthalmology 116: 2170–4. III Grauslund J, Green A, Sjølie AK (2009). Cataract surgery in a population-based cohort of type 1 diabetic patients: long-term incidence and risk factors. Acta Ophthalmologica Scandinavica. doi: 10.1111/j.1755-3768.2009.01619.x IV Grauslund J, Hodgson L, Kawasaki R, Green A, Sjølie AK, Wong TY (2009). Retinal vessel calibre and micro- and macrovascular complications in type 1 diabetes. Diabetologia 52: 2213–2217. V Grauslund J, Green A, Kawasaki R, Hodgson L, Sjølie AK, Wong TY (2010). Retinal vascular fractals and micro- and macro-vascular complications in type 1 diabetes. Ophthalmology 117: 1400–1405. VI Grauslund J, Nybo M, Green A, Sjølie AK (2010). N-terminal pro brain natriuretic peptide reflects long-term complications in type 1 diabetes. Scand J Clin Lab Invest 70: 392–398. VII Grauslund J, Rasmussen LM, Green A, Sjølie AK (2010). Does osteoprotegerin relate to micro- and macrovascular complications in long-term type 1 diabetes? Scand J Clin Lab Invest 70: 188–193. The work presented in this doctoral thesis was carried out at the Department of Ophthalmology, Odense University Hospital in 2006–2009. It is a successor to the PhD Thesis Long-term mortality and retinopathy in type 1 diabetes that was accepted in 2010. I would like to express my sincere gratitude to the following, without whom this work would not have been possible. Firstly I would like to express my thankfulness to my friend and mentor Anne Katrin Sjølie who inspired and guided me throughout this work. Her dedication to this project helped and inspired me all the way. Likewise Anders Green was always able to find time for guidance and fruitful discussions. I am very thankful for this. A part of this thesis was done at the Centre for Eye Research Australia in Melbourne, Australia. I am very grateful to Tien Wong for giving me this opportunity and for his help throughout the study. I am also very indebted to my friend and mentor Ryo Kawasaki who supervised my stay in Melbourne. Likewise I would not have been able to learn how to grade vascular diameters and fractals without the tremendous help from Lauren Hodgson, Annie McAuley, Yumiko Kawasaki and Ignatios Koukouras. I am very grateful for your friendliness and, in particularly, for the nice weather you arranged for the entire month I was in Melbourne! At the Ocular Epidemiology Reading Center in Madison, Wisconsin, USA, I had the great pleasure of receiving good advice and invaluable suggestions from Ron Klein, Barbara Klein and Stacy Meuer. This was a huge help for which I am very grateful. Back home again, Lars Melholt Rasmussen and Mads Nybo from the Department of Biochemistry, Pharmacology and Genetics, Odense University Hospital were always very helpful. Thanks. From the same department I would also like to thank Charlotte Olsen, Lone Hansen, Kirsten Ulla Bahrt, Anette Tyrsted Mikkelsen and Gitte Primdahl Nielsen who all helped me to sample and store the blood throughout the study. I am indebted to Kelvin Kamp Mortensen, the head of the Department of Ophthalmology, Odense University Hospital, who gave me the time and opportunity to do this work at the department. Likewise, I would like to give my thanks to my colleagues in the daily work. Among these, a special credit has to be given to my friends and colleagues at the department's Research Unit: Karen Bjerg Pedersen, Birgitte Justesen, Majbrit Lind and Flemming Møller were always very helpful and supportive. Thanks. My family and friends provided moral support through all the years. I am very grateful for this. And finally, without the never-ending love and support from my dear wife Julie and our lovely daughter Vilma, I could not have made this thesis. Thank you. I am very grateful for the financial support provided for this study. With regard to this I would like to thank Velux Foundation, Danish Eye Health Society, Institute of Clinical Research at University of Southern Denmark, Sehested Hansen s Foundation, Danish Diabetes Association, Synoptik Foundation, The A.P. Møller Foundation for the Advancement of Medical Science, The Danish Society of Ophthalmology, The Legacy of Teacher Karen Svankjær Yde, The Legacy of Engineer August Frederik Wedel Erichsen, The Legacy of A. and J. Rasmussen and Odense University Hospital. And, of course, I would like to acknowledge each and every patient who participated in this study. I thank you all. Jakob Grauslund, December 2010 Conflicts of interest: None. ANP: Atrial natriuretic peptide AVR: Arteriolar venular ratio BNP: Brain natriuretic peptide CI: Confidence interval CRAE: Central retinal arteriolar equivalent CRVE: Central retinal vein equivalent CWS: Cotton wool spots DCCT: Diabetes Control and Complications Trial Df: Fractal dimension DME: Diabetic macular oedema DR: Diabetic retinopathy DRS: Diabetic Retinopathy Study ETDRS: Early Treatment Diabetic Retinopathy Study HbA1: Haemoglobin A1 HbA1c: Haemoglobin A1c HR: Hazard ratio IHD: Ischaemic heart disease IRMA: Intraretinal microvascular abnormalities IRIS: International Retinal Imaging Software NPDR: Non-proliferative diabetic retinopathy NT-proBNP: N-terminal pro brain natriuretic peptide OPG: Osteoprotegerin OR: Odds ratio PDR: Proliferative diabetic retinopathy WESDR: Wisconsin Epidemiologic Study of Diabetic Retinopathy Denne afhandling er den 9. november 2010 af Akademisk Ra˚d, Det Sundhedsvidenskabelige Fakultet, Syddansk Universitet antaget til forsvar for den medicinske doktorgrad. Ole Skøtt h.a.dec. Forsvaret finder sted d. 25. marts 2011 kl. 14.00 i Auditoriet, Winsløwparken 25, 5000 Odense C. This thesis has been accepted for defence for the medical doctoral degree on November 9 2010 by the Academic Council, Faculty of Health Science, University of Southern Denmark. Ole Skøtt Dean The defence will take place on March 25 2011 at 14.00 in Auditoriet, Winsløwparken 25, DK 5000 Odense C, Denmark. Members of the evaluation commitee: Professor, MD, PhD, Massimo Porta, Unit of Internal Medicine 1, University of Turin, Italy. Professor, dr.med., Toke Bek, Dept. of Ophthalmology, Aarhus Hospital, Aarhus, Denmark. Professor, dr.med. Henning Beck-Nielsen, Department of Endocrinology, Odense University Hospital and Clinical Institute, University of Southern Denmark (chairman), Odense, Denmark. Type 1 diabetes is an autoimmune disease caused by the destruction of the beta cells of the pancreas that leads to insulin deficiency and chronic hyperglycaemia. Mortality within a few years after the onset of diabetes was inevitable prior to the introduction of insulin by Banting and Best in 1922. Since then, the life expectancy of patients with type 1 diabetes has increased, but early mortality still remains an important issue (Moss et al. 1991; Soedamah-Muthu et al. 2006a). The prolonged life expectancy for patients with type 1 diabetes raises another important issue. The chronic load of hyperglycaemia may lead to various micro- and macrovascular complications. Among the former, microvascular changes in the eyes, kidneys and nerves lead to retinopathy, nephropathy and neuropathy, respectively. For the larger arteries, macrovascular complications like ischaemic heart disease (IHD) and stroke have a significant impact on morbidity and mortality (Moss et al. 1991; Laing et al. 2003; Soedamah-Muthu et al. 2004, 2006b). Diabetic retinopathy affects almost all patients with type 1 diabetes with duration of diabetes of 15 years or more (Klein et al. 1984c; Grauslund et al. 2009). Diabetic retinopathy is characterized by lesions of the retinal microvasculature. Although the exact mechanisms that lead to DR are not completely clear, it seems evident that chronic hyperglycaemia has a pivotal role in the pathogenic alterations of the retinal microvasculature. This was demonstrated in the Diabetes Control and Complications Trial in which intensive insulin therapy led to a 76% decreased risk of incident DR (The Diabetes Control and Complications Trial Research Group 1993). Thickening of the basement membrane of the retinal endothelial cells as well as pericyte loss and increased capillary permeability are among the first physiological changes in the diabetic retina (Kohner 1993; Gardiner et al. 2007). This causes capillary closure and, hence, retinal nonperfusion. To compensate, other capillaries dilate, which leads to microaneurysms – the first type of visible lesion caused by DR. When retinal ischaemia progresses, other lesions like cotton wool spots (CWS), venous beading and intraretinal microvascular abnormalities (IRMA) might occur (Kohner 1993). These characteristics are commonly known as nonproliferative diabetic retinopathy (NPDR) as opposed to proliferative diabetic retinopathy (PDR) that may come secondary to further retinal ischaemia. In PDR, new vessels emerge from the retinal vessels. Visual loss may be caused by these vessels as a result of preretinal and vitreous haemorrhage followed by tractional retinal detachment. At any stage, vision can also be impaired by diabetic macular oedema (DME) caused by the breakdown of the blood–retinal barrier. Owing to the excess mortality in type 1 diabetes, it is important to be able to identify the high-risk patients. Proteinuria has been identified as an important risk factor (Borch-Johnsen et al. 1985; Borch-Johnsen & Kreiner 1987; Klein et al. 1989b; Rossing et al. 1996; Soedamah-Muthu et al. 2008), and a relative cardiovascular mortality of 37 and 4.2 when compared to the general population has been reported for type 1 diabetic patients with and without proteinuria, respectively (Borch-Johnsen & Kreiner 1987). Poor glycaemic regulation has also been recognized as a risk factor of mortality in type 1 diabetes in some (Moss et al. 1994; Shankar et al. 2007) but not all studies (Muhlhauser et al. 2000). For instance, in an 11-year follow-up of the Wisconsin Epidemiologic Study of Diabetic Retinopathy (WESDR), patients in the highest quartile of glycosylated haemoglobin had a relative risk of 2.42 and 3.28 for all-cause mortality and cardiovascular mortality, respectively, when compared to patients in the lower quartile (Shankar et al. 2007). It is still debatable whether DR is also associated with mortality in type 1 diabetes. Several studies have been performed (Klein et al. 1999; Rajala et al. 2000; Cusick et al. 2005; van Hecke et al. 2005) but long-term data from population-based studies have so far only been presented in a 16-year follow-up of the WESDR (Klein et al. 1999). Klein et al. found PDR to be associated with all-cause mortality in an age- and sex-adjusted model [hazard ratio (HR) 5.53, 95% confidence interval (CI) 3.06–9.99] but not in a multivariate model (HR 1.28, 95% CI 0.62–2.62). Furthermore, the study demonstrated a higher all-cause mortality as well as a greater risk of IHD for patients with PDR when compared to patients with NPDR. The study indicates an important confounding effect of other risk factors such as proteinuria, glycosylated haemoglobin, hypertension and smoking. It also suggests that PDR is more likely to be a risk factor of mortality in type 1 diabetes than NPDR. The higher risk of mortality for patients with the former may reflect more severe systemic morbidity among patients with PDR. Consequently, additional subgroup analyses are needed to determine this. However, the combined effect of retinopathy and other risk factors on mortality has not been investigated in previous studies. Diabetic retinopathy is the most common reason for visual impairment and blindness for the working-age population of the Western world (Klein & Klein 1995). It has been demonstrated by the Diabetic Retinopathy Study (DRS) that properly timed panretinal photocoagulation decreases the 2-year risk of severe visual loss from 7% to 3% in patients with PDR (The Diabetic Retinopathy Study Research Group 1981). Likewise, for patients with DME, it was shown in the Early Treatment Diabetic Retinopathy Study (ETDRS) that focal photocoagulation halves the 3-year risk of severe visual loss (Early Treatment Diabetic Retinopathy Study Research Group 1985). Despite the implementation of strict glycaemic control as well as the use of laser photocoagulation, visual loss and blindness are still major concerns in type 1 diabetes. In a 14-year follow-up of the WESDR, Moss et al. (1998) found a 2.4% incidence of blindness. However, the WESDR results only included patients who had participated at the baseline examination as well as the 14-year follow-up. Consequently, data were not included for the patients who became blind and died prior to the follow-up. This is a concern because mortality has been found to be three times higher in blind patients with type 1 diabetes (Sjolie & Green 1987). Furthermore, it was not possible to identify risk factors of blindness in the WESDR 14-year follow-up. Updated long-term incidence data of blindness in type 1 diabetes that accounts for the competing risk of death and also identifies risk factors of blindness are called for. Cataract is another common cause of visual impairment in type 1 diabetes (Janghorbani et al. 2000; Esteves et al. 2008). Among patients with type 1 diabetes of the WESDR, cataract has been identified as the second most common reason for blindness (PDR was the most common) (Klein et al. 1984b). In type 1 diabetes, cataract has been associated with age (Nielsen & Vinding 1984; Klein et al. 1985, 1995; Di et al. 1999; Janghorbani et al. 2000), duration of diabetes (Nielsen & Vinding 1984; Klein et al. 1985; Janghorbani et al. 2000), glycaemic regulation (Klein et al. 1985; Di et al. 1999; Janghorbani et al. 2000; Kato et al. 2001) and DR (Nielsen & Vinding 1984; Klein et al. 1985, 1995; Janghorbani et al. 2000; Kato et al. 2001). However, long-term data on the incidence of cataract surgery as well as associated risk factors in type 1 diabetes are needed in order to facilitate handling of the increased burden of the disease caused by the rising number of patients with diabetes. The human vascular tree is available for direct in vivo inspection in the eye. This allows examinations of the retinal microvascular system in different ways. The retinal vessels are part of the systemic microvasculature. Given this and the improved methods of capturing retinal images, investigations into the appearance of the retinal vascular system may be valuable in order to predict diabetes-related complications in other organs. Vascular diameter analysis is the most common method of analysing the retinal vascular system (Fig. 1). In previous studies, generalized arteriolar narrowing has been associated with age and hypertension (Hubbard et al. 1999; Leung et al. 2003b; Wong et al. 2003). For patients with diabetes, associations have been found between retinal vascular diameters and retinopathy (Klein et al. 2004b, 2007; Alibrahim et al. 2006; Cheung et al. 2008; Rogers et al. 2008), nephropathy (Klein et al. 2003, 2007; Wong et al. 2004b) and cardiovascular disease (Klein et al. 2003, 2004a, 2007). For instance, in multivariate analyses of patients with type 2 diabetes from the WESDR, wider arterioles (fourth versus first quartile) were associated with an increased 10-year incidence of DR [odds ratio (OR) 1.78]. Likewise, wider venules were associated with increased 14-year incidence of diabetic nephropathy (OR 2.08) and increased 22-year stroke mortality (HR 1.71) (Cheung et al. 2007; Klein et al. 2007). Conversely, narrow arterioles (first versus fourth quartile) were associated with increased 14-year incidence of lower extremity amputation (OR 2.20), 22-year all-cause mortality (HR 1.18) and 22-year stroke mortality (HR 1.47). So far, most studies on retinal vascular diameters have been carried out in the general population or in patients with type 2 diabetes. Studies to associate retinal vascular calibres with long-term micro- and macrovascular complications in type 1 diabetes are still lacking. Left: Fundus photograph centred at the optic disk and used for vascular calibre analyses by the ivan program. Right: Identification of the largest arterioles (red) and venules (blue). See Methods–Retinal vascular calibre analysis for description of data analysis. Fractal analysis is another method of analysing the retinal vascular system. It is a more 'global' way of evaluating the retinal vasculature in which the features of the vascular tree are summarized into a single parameter. In general, fractal analysis is based on the concept of self-similarity. That is, the parts of a pattern show the overall structure despite changes in magnification. Fractal patterns are common in nature and include snowflakes, leaves, trees and coastal lines. Fractal analyses have been known for some time (Mainster 1990; Daxer 1993a,b; Stosic & Stosic 2006). However, with a new method, International Retinal Imaging Software (IRIS-Fractal (National University of Singapore, Singapore; University of Sydney, Australia; and University of Melbourne, Australia), it is possible to grade each fundus photograph within 5 min when compared to up to 20 hr with earlier methods (Fig. 2) (Mainster 1990). In earlier studies, larger retinal fractals have been associated with early DR as well as retinal neovascularization (Daxer 1993b; Cheung et al. 2009). However, relations between retinal fractals and other micro- and macrovascular complications in type 1 diabetes have not yet been described so far. Such studies are important in order to evaluate retinal fractals as predictors of long-term complications in type 1 diabetes. Left: Cropped fundus photograph centred at the optic disk. Right: Line tracing provided by the International Retinal Imaging Software (iris). See Methods–Fractal analysis for description of data analysis. The burden of micro- and macrovascular complications is almost universal in type 1 diabetes. Besides a 97% prevalence of DR for patients with long-term type 1 diabetes (Grauslund et al. 2009), we were recently able to report a prevalence of neuropathy, nephropathy (micro- and macroalbuminuria) and macrovascular disease of 52.7%, 33.2% and 21.9%, respectively, among patients with type 1 diabetes from a population-based cohort with a median duration of diabetes of 43 years (Grauslund et al. 2009). Along with duration of diabetes, hyperglycaemia has been identified as the most important risk factor of microvascular complications (The Diabetes Control and Complications Trial Research Group 1993). Despite a strict glycaemic control, some patients will, inevitably, face diabetes-related complications. It is therefore vital to identify the patients in risk in order optimize treatment. Noninvasive markers of micro- and macrovascular complications are needed to accomplish this. Brain natriuretic peptide (BNP) is synthesized and secreted from the ventricular myocardium in response to myocyte stress and ischaemia (Levin et al. 1998; de Lemos et al. 2003). N-terminal proBNP (NT-proBNP) is an inactive fragment that is cleaved from BNP and is considered more stable for analysis (Downie et al. 1999). NT-proBNP has been associated with heart failure in non-diabetic patients (Hunt et al. 1997; McDonagh et al. 1998; Gardner et al. 2003), and with nephropathy (McKenna et al. 2001, 2005; Siebenhofer et al. 2003; Tarnow et al. 2005) and mortality (Hovind et al. 2003) in type 1 diabetes. However, relations to other diabetes-related complications have not been examined in type 1 diabetes. Osteoprotegerin (OPG) is another potential marker of micro- and macrovascular complications in type 1 diabetes. Osteoprotegerin is a known regulatory molecule in bone turnover (Simonet et al. 1997) and is also present in vascular smooth muscle cells (Zhang et al. 2002) and endothelial cells (Malyankar et al. 2000). Besides being associated with coronary heart disease (Jono et al. 2002; Omland et al. 2008), elevated levels of OPG have been described in type 1 diabetes (Galluzzi et al. 2005; Rasmussen et al. 2006). Furthermore, in type 1 diabetic patients with nephropathy, OPG has been associated with mortality (Jorsal et al. 2008), glycaemic regulation, systolic blood pressure, kidney function and cardiovascular disease (Rasmussen et al. 2006). However, relations to retinopathy and neuropathy have not been described. A population-based cohort of Danish patients with type 1 diabetes was examined in order to achieve the following aims: To evaluate DR as a long-term marker of all-cause mortality in type 1 diabetes (Paper I). To estimate the long-term incidence and associated risk factors of blindness (Paper II) and cataract surgery (Paper III) in type 1 diabetes. To use retinal vascular analyses in order to investigate the associations between long-term micro- and macrovascular complications and retinal vascular diameters (Paper IV) and retinal fractals (Paper V) in type 1 diabetes. To examine N-terminal pro-brain natriuretic peptide (Paper VI) and OPG (Paper VII) as noninvasive markers of micro- and macrovascular complications in type 1 diabetes. This thesis is based on the studies of a population-based cohort of patients with type 1 diabetes from Fyn County, Denmark. As of 1 July 1973, insulin prescriptions were used to identify all patients with type 1 diabetes from Fyn County with an onset of diabetes before the age of 30 (Green et al. 1981; Sjolie 1985). At that time, Fyn County had approximately 450 000 inhabitants and was considered a demographically representative 9% sample of the general Danish population. It was estimated that the patient material was more than 98% complete (Green et al. 1981). Seven hundred and twenty-seven patients were identified. Of these, 413 (56.8%) were men and 314 (43.2%) were women. All patients who were still alive were asked to participate in a first examination in 1981–1982 and a second examination in 2007–2008. The principles of the Declaration of Helsinki were followed, and written informed consents were obtained from all patients at the follow-up examination. Furthermore, approval was obtained from the local ethics committee. As of 1 June 1981, 627 of the 727 patients (86.2%) were still alive and living in Denmark. Prior to the examination, 96 patients had died and four had emigrated. The patients still available were invited to a clinical examination that took place between 1 June 1981 and 1 June 1982. A total of 577 patients (92.0%) chose to participate. Data were later lost on four patients, which left data available for 573 patients (321 men and 251 women). All examinations were performed by a single examiner [Anne Katrin Sjølie (AKS), Odense University Hospital, Odense, Denmark]. The first examination provides baseline data for the prospective studies of Papers I–III. For these papers, patients were followed from the date of the baseline examination until 6 November 2006 or censoring, whichever came first. Patients could be censored owing to death, emigration, unwillingness to provide data to scientific projects or if the event of interest had occurred (Paper I: death, Paper II: blindness, Paper III: cataract surgery). A structured interview was performed as well as a clinical examination, blood and urine sampling and a full ophthalmological examination. Patients were asked about their smoking habits. Current and former smokers were considered to be smokers for the upcoming analyses. Body mass index (BMI) was defined as the body weight divided by the square of the height and expressed in kg/m2. Blood pressure was measured by an Erkameter sphygmomanometer (Morton Medical Ltd, London, UK) on one arm with the patient in sitting position after 10 min of rest. Blood measurements included haemoglobin A1 (HbA1) made as total HbA1 with resin 70 (Bio-Rad, Hercules, CA, USA) at 20°C and pH 6.70. Urine protein was measured from a single-spot urine, and proteinuria was considered present if protein was ≥0.5 g/l. The best-corrected visual acuity was measured for both eyes. Both pupils were dilated using tropicamide 1%, and a slit lamp examination was performed (Haag-Streit, Wedel, Germany). Ophthalmoscopy was performed and retinopathy was classified (no retinopathy, NPDR or PDR) by a single trained grader (AKS). The level of DR was determined by the worse eye. NPDR was defined as one or more of the following characteristics: microaneurysms, haemorrhages, hard exudates, CWS, venous beading or IRMA. Proliferative DR was present for patients who had newly formed vessels in addition to the aforementioned. Finally, maculopathy was defined as retinal thickening and/or hard exudates in the macular area. As of 1 March 2007, 320 of the original 727 patients (44.0%) were still alive and living in Denmark. Of these, 208 (65.0%) agreed to participate in a clinical examination carried out between 1 March 2007 and 1 March 2008. Patients who had died prior to the second examination had a higher age in 1973 than patients who were still alive. The second examination provides data for the cross-sectional studies of Papers IV–VII. Participation in the first examination was not needed to participate in the second examination. All examinations were perform
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