Citation for published version (APA): Lutgers, H. L. (2008). Skin autofluorescence in diabetes mellitus Groningen: s.n.

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1 University of Groningen Skin autofluorescence in diabetes mellitus Lutgers, H.L. IMPORTANT NOTE: You are advised to consult the ublisher's version (ublisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2008 Link to ublication in University of Groningen/UMCG research database Citation for ublished version (APA): Lutgers, H. L. (2008). Skin autofluorescence in diabetes mellitus Groningen: s.n. Coyright Other than for strictly ersonal use, it is not ermitted to download or to forward/distribute the text or art of it without the consent of the author(s) and/or coyright holder(s), unless the work is under an oen content license (like Creative Commons). Take-down olicy If you believe that this document breaches coyright lease contact us roviding details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): htt:// For technical reasons the number of authors shown on this cover age is limited to 10 maximum. Download date:

2 CHAPTER 7 Skin autofluorescence: a tool to identify tye 2 diabetic atients at risk for develoing microvascular comlications E.G. Gerrits 1, H.L. Lutgers 2, N. Kleefstra 1,3, R. Graaff 4, K.H. Groenier 5, A.J. Smit 2,6 R.O. Gans 2,6, H.J. Bilo 1,2,6 1 Diabetes Centre, Isala Clinics, Zwolle, 2 Deartment of Medicine, University Medical Center Groningen, Groningen, 3 Langerhans Medical Research Grou, Zwolle, 4 Deartment of Biomedical Engineering, University Medical Center Groningen, Groningen, 5 Deartment of General Practice, University Medical Center Groningen and University of Groningen, Groningen, 6 University of Groningen, Groningen, the Netherlands Diabetes Care 2008; 31:517-21

3 Chater 7 ABSTRACT Introduction Skin autofluorescence (AF) is a noninvasive measure of the level of tissue accumulation of advanced glycation endroducts, reresenting cumulative glycemic and oxidative stress. Recent studies have already shown a relationshi between skin AF and diabetic comlications, and its redictive value for total and cardiovascular mortality in tye 2 diabetes mellitus. Our aim was to investigate the redictive value of skin AF for the develoment of microvascular comlications in tye 2 diabetes mellitus. Methods At baseline, skin AF of 973 well-controlled tye 2 diabetes atients was noninvasively measured with an AF reader. The aggregate clinical outcome was defined as the develoment of any diabetes associated microvascular comlication of 881 surviving atients which was assessed at baseline and at the end of follow-u. Single endoints were the develoment of diabetes associated retinoathy, neuroathy and (micro)albuminuria. Results After a mean follow-u eriod of 3.1 years, baseline skin AF was significantly higher in atients who develoed any microvascular comlication, neuroathy and (micro)albuminuria, but not in those who develoed retinoathy. Multivariate analyses showed skin AF as a redictor for the develoment of any microvascular comlication along with HbA1c; for the develoment of neuroathy along with smoking, and for the develoment of (micro)albuminuria together with gender, HbA1c and diabetes duration. Skin AF did not have redictive value for the develoment of retinoathy, albeit diabetes duration did. Conclusion Our study is the first observation of skin AF measurement as an indeendent redictor for the develoment of microvascular comlications in tye 2 diabetes mellitus. INTRODUCTION Hyerglycaemia, individual suscetibility and life-style are three key factors, which lay an imortant role in the develoment of microvascular disease in diabetes 150

4 Skin autofluorescence in microvascular comlications mellitus. One of the consequences of hyerglycaemia and attendantly increased generation of free radicals is the increased formation of advanced glycation endroducts (AGEs), besides the increased olyol and hexosamine fluxes, and activation of rotein kinase C, which all contribute to tissue damage in diabetes [1,2]. Those AGEs can be described as the final roducts of slowly occurring non-enzymatic glycation of roteins that form cross-links with long-lived roteins such as collagen (the so called Maillard reaction). They may also accumulate as a result of oxidative stress related glycoxidation and lioxidation athways. In the DCCT, intensive treatment as comared with conventional treatment showed that long term intensive treatment of hyerglycaemia in tye 1 diabetic atients imroved glycemic control, and delayed the rogression of microvascular comlications [3]. The UKPDS and other rosective studies have also shown an association between hyerglycaemia and increased risk of microvascular comlications in tye 2 diabetes [4-6]. The DCCT skin collagen ancillary study grou showed the association of long-term intensive treatment of hyerglycaemia, as comared with conventional treatment, with lower levels of AGEs in skin collagen and they showed that these AGE levels in skin biosies redicted the risk of develoment or rogression of microvascular disease in tye 1 diabetes mellitus, even after adjustment for HbA1c [7,8]. A newly described noninvasive method to assess tissue AGEs concerns skin autofluorescence (AF). This method is based on the secific fluorescence characteristics of AGEs and has been validated against secific AGE levels in skin biosies in atients with diabetes or on hemodialysis, and in healthy controls [9,10]. Recently, the relationshi between skin AF, reflecting AGE accumulation, and outcome has been studied in tye 2 diabetes. Besides its relation with chronic comlications of diabetes (in cross-sectional analyses), skin AF has also shown its indeendent redictive value for cardiovascular mortality and morbidity in atients with tye 2 diabetes, and in atients with end-stage renal disease undergoing hemodialysis [10-12]. 151

5 Chater 7 In this study, we analyzed whether skin AF, as a marker of AGE accumulation, can redict the develoment of microvascular comlications in a tye 2 diabetes oulation. METHODS Patients. Between May 2001 and May 2002, 973 rimary care tye 2 diabetes atients were included in the study cohort and had a skin AF measurement. The included atients were all articiating in a shared-care roject of the Zwolle Outatient Diabetes roject Integrating Available Care (ZODIAC)-study and have also been described elsewhere [11]. During follow-u, data of 967 atients were analyzed for this study (6 atients were lost to follow-u). Eighty-six atients died before the end of follow-u and this subgrou will be addressed searately from the surviving 881 atients. Patients with a Fitzatrick class V-VI skin tye were excluded, because of the limitation of the autofluorescence reader (AFR) to measure accurately in dark skin tyes [13-15]. All articiating atients visited the outatient clinic at least once a year. Follow-u ended at January All of the included atients had given their informed consent, and aroval by the local ethical committee had been obtained. Skin autofluorescence. The AFR (rototye of the current AGE Reader; DiagnOtics BV, Groningen, the Netherlands), which measures skin AF, illuminates a skin surface of ~4 cm 2, guarded against surrounding light, with an excitation light source with eak intensity at ~370 nm. Emission light and reflected excitation light from the skin are measured with a sectrometer in the nm range, using a glass fiber. AF was comuted by dividing the average light intensity of the emission sectrum nm by the average light intensity of the excitation sectrum nm, multilied by hundred and exressed in arbitrary units (a.u.). Skin AF of all atients was assessed at the volar side of the arm, 10 cm below the elbow fold. Six diabetes secialist nurses did the AF measurements with 2 identical AFR devices. The AFR has been validated and more extensively been described in revious studies [9,11]. 152

6 Skin autofluorescence in microvascular comlications Data collection. Clinical data and laboratory results were obtained at the time of the baseline skin AF measurement. Serum creatinine, nonfasting liids (total cholesterol, LDL cholesterol, HDL cholesterol, triglycerides), and urinary albumin and creatinine were measured according to the standard laboratory rocedures. HbA1c was measured with a Primus CLC-385 using boronate affinity chromatograhy and high-erformance liquid chromatograhy (reference value %). Blood ressure measurement was a single measurement obtained after 5 minutes rest, with the atient in seated osition, and using an aneroid device. At each visit to the outatient clinic, and at the end of follow-u, the absence or resence of retinoathy, neuroathy and (micro)albuminuria were assessed. Clinical endoints. The aggregate clinical endoint was the develoment of any diabetes associated microvascular comlication, which was defined as the resence of at least one of the following diabetic comlications: retinoathy, neuroathy and/or (micro)albuminuria, according the ADA Diabetes definitions [16]. The single clinical endoints were described as the develoment of retinoathy, neuroathy or (micro)albuminuria. Retinoathy was determined by an ohthalmologist based on retinal hotograhy. Presence of at least background retinoathy was assumed to imly retinoathy. Neuroathy was examined using a 5.07/10-g Semmes-Weinstein monofilament, alied on the dorsum of both feet at three different, non-callused areas (first toe, and first and fifth distal metatarsal bone). Neuroathy was considered in case of diminished sensibility, which was defined in case of at least two incorrect resonses after 3 alications at each area (2 real and 1 false alication) [17,18]. (Micro)albuminuria at baseline was defined as an albumin-to-creatinine ratio >2.5 mg/mmol for men and >3.5 mg/mmol for women in 2 subsequent urine samles or once in the year before baseline while using an ACE-inhibitor at baseline [19]. Newly develoed (micro)albuminuria at follow-u was defined as an albumin-to-creatinine ratio >2.5 mg/mmol for men and >3.5 mg/mmol for women in 2 urine samles (one in the year before and one at the moment during follow-u) or an abnormal level of the albumin-to-creatinine ratio in the year before the end of follow-u whilst using an ACE-inhibitor at follow-u. 153

7 Chater 7 Statistical analysis Oneway ANOVA using osthoc multile comarisons (with Bonferroni correction) was used to comare mean skin AF between subgrous of microvascular comlications in the 881 surviving atients. Subgrous are: A. no microvascular comlication at baseline nor at follow-u. B. no microvascular comlication at baseline, but a microvascular comlication at follow-u. C. a microvascular comlication at baseline and at follow-u. Univariate and multivariate multinominal regression analyses were erformed to determine the relationshi of skin AF to the resence or develoment of microvascular disease. Patients without signs of microvascular comlications at baseline nor at follow u were the reference categories in these calculations. In the multivariate analyses we controlled for otential confounding risk factors for the develoment of microvascular comlications which were derived from the UKPDS findings, thereby including: gender, diabetes duration, HbA1c, current smoking, systolic blood ressure, HDL cholesterol, LDL cholesterol and triglycerides with the addition of BMI [4]. Odds-ratios (OR) and confidence intervals (95%) for skin AF were calculated in the univariate and in the multivariate analyses; -values <0.05 were considered to be statistically significant. RESULTS The baseline characteristics of the surviving study oulation including mean skin AF of the total grou are shown in Table 1. Mean age of our study oulation was 66 years, 46% male, with a relatively short median diabetes duration of 4.0 years and an interquartile range between 1.5 and 8.1 years. Eighty-five ercent of this wellcontrolled diabetic study oulation was on a diet and/or oral agents; the other 15% of atients received insulin or combined insulin/oral agent treatment. In the 881 survivors, the revalence of retinoathy, neuroathy and (micro)albuminuria at baseline was 19%, 24% and 24% resectively, resulting in an overall ercentage of atients with a diabetes associated microvascular comlication of 50%. 154

8 Table 1. Characteristics of the tye 2 diabetic atients Skin autofluorescence in microvascular comlications Characteristic n=881 Age in years 66 (11) Gender (M/F) 406/475 Smoking (%) 19 BMI (kg/m²) 29.4 (4.8) Systolic blood ressure (mmhg) 146 (20) Diabetes duration (years) 4.0 ( )* HbA1c (%) 6.6 ( )* Creatinine (µmol/l) 95 (19) Creatinine clearance (Cockcroft-formula) (ml/min) 77 (27) Urinary albumin-to-creatinine ratio 1.41 ( )* Total cholesterol (mmol/l) 5.2 (1.0) HDL cholesterol (mmol/l) 1.3 (0.3) LDL cholesterol (mmol/l) 2.9 (0.9) Triglycerides (mmol/l) 2.1 ( )* Microvascular disease (%) Retinoathy (%) Neuroathy (%) (Micro)albuminuria (%) Macrovascular disease (%) 37 Skin autofluorescence (total grou) (a.u.) 2.74 (0.7) Values are exressed as mean (SD). *Median and Interquartile range Table 2 shows the mean baseline skin AF of the 881 survivors subdivided in grous with continued absence or resence or the develoment of microvascular comlications at follow-u. During a median follow-u eriod of 3.1 years, 61 atients (7.0%) develoed retinoathy; their baseline skin AF did not differ from skin AF levels of atients who did not show or already had retinoathy at baseline. However, skin AF was higher in the atient grous who develoed neuroathy or (micro)albuminuria comared to those without these comlications. At follow-u newly develoed neuroathy was diagnosed in 7.5% and newly develoed (micro)albuminuria was found in 10.1%; 12.5% of the oulation develoed at least one microvascular comlication. Skin AF at baseline was also significantly higher in the atient grous who develoed any microvascular comlication or who already had a microvascular comlication at baseline, comared to those atients who did not develo any microvascular disease at all. 155

9 Chater 7 Table 2. Mean skin AF (SD) at baseline and mean differences between grous A= B= C= B vs. A C vs. A C vs. B Microvascular comlication t 0 : absent t fu : absent t 0 : absent t fu : resent t 0 : resent t fu : resent Retinoathy AF n 2.69 (0.73) (0.74) (0.72) 169 AF 95% CI Neuroathy AF n 2.67 (0.72) (0.75) (0.75) 215 AF 95% CI (Micro)albuminuria AF n 2.62 (0.68) (0.67) (0.83) 207 AF 95% CI < Any AF n 2.52 (0.69) (0.66) (0.75) 441 AF 95% CI < < Data are means (SD) of skin AF in arbitrary units within the grou, and mean differences ( AF) between grous calculated with ANOVA (95% Confidence interval) with Bonferroni correction; t 0, baseline; t fu, follow-u Multinominal logistic regression analysis showed that skin AF was a strong redictor of the develoment of the aggregate of microvascular comlications (OR 2.05 [ ], < Skin AF was significantly associated with the develoment of retinoathy (OR 1.42 [ ], =0.042), neuroathy (OR 1.59 [ ], =0.005), and (micro)albuminuria (OR 1.73 [ ], <0.001). After correction for the confounding risk factors, baseline skin AF still aeared to be significantly associated with the develoment of these endoints, excet for retinoathy (OR 1.21 [ ], =0.32), Table 3. Diabetes duration at baseline was the only significant indeendent variable for the develoment of retinoathy in this multivariate analysis (OR 1.10 [ ], <0.001). Surviving smokers develoed less often neuroathy comared to non-smokers. In the non-surviving grou (86 atients) 70% had a microvascular comlication at baseline; there were 23 non-surviving smokers. Seventy ercent of the non-surviving smokers already had a microvascular comlication at 156

10 Skin autofluorescence in microvascular comlications baseline and 13% of the non-surviving smokers develoed a microvascular comlication before they died. When baseline skin AF levels are categorized in subgrous of ractically feasible levels of skin AF (3 categories in rounded tertiles: skin AF < 2.35 a.u.; 2.35 skin AF < 3.00 a.u.; skin AF 3.00 a.u.), atients in the category skin AF 3.00 a.u do have a higher chance to develo a microvascular comlication comared to atients with a lower skin AF level (Table 4). Table 3. Variables related to the develoment of microvascular comlications in tye 2 diabetes mellitus by multinominal logistic regression analysis Any microvascular comlication Retinoathy Neuroathy (Micro)albuminuria Variables value OR (95%CI) value OR (95% CI) value OR 95% CI value OR 95% CI Skin AF < ( ) ( ) ( ) < ( ) Gender ( ) ( ) ( ) ( ) HbA1c ( ) ( ) ( ) ( ) Diabetes duration ( ) < ( ) ( ) ( ) Smoking ( ) ( ) ( ) ( ) Systolic BP ( ) ( ) ( ) ( ) LDL cholesterol ( ) ( ) ( ) ( ) HDL cholesterol ( ) ( ) ( ) ( ) Triglycerides ( ) ( ) ( ) ( ) BMI ( ) ( ) ( ) ( ) Table 4. Newly develoed microvascular comlications subdivided in 3 skin AF-grous n Microvascular comlication Skin AF <2.35 a.u Skin AF < 3.00 a.u. Skin AF 3.00 a.u 708 Retinoathy 15/241 (6.2) 18/251 (7.2) 28/216 (13.0) 662 Neuroathy 11/219 (5.0) 27/247 (10.9) 28/196 (14.3) 65 (Micro)albuminuria 18/225 (8.0) 31/253 (12.3) 38/179 (21.2) 431 Any 23/161 (14.3) 41/167 (24.6) 45/103 (43.7) Number (%) of newly develoed microvascular comlications of subgrous; n is number of atients who did not have the comlication at baseline 157

11 Chater 7 DISCUSSION Our study rovides the first evidence that skin AF is an indeendent redictor of the develoment of microvascular comlications in a well-controlled tye 2 diabetes oulation. Searately, this also holds for the develoment of neuroathy and (micro)albuminuria (and in univariate analysis for retinoathy). This noninvasive marker of tissue AGE accumulation may reflect the deleterious effects of long-term glycemic and oxidative stress. It was recently shown that skin AF is a redictor of 5- year coronary heart disease and mortality in diabetes [12]. The resent study showed that skin AF also has a redictive value for the develoment of microvascular comlications, which in the analysis of this study is suerior to that of many other commonly used risk redictors like diabetes duration and HbA1c in tye 2 diabetes. This conclusion is alicable for rimary care tye 2 diabetes atients, treated according to current standards, which is the large majority of tye 2 diabetes atients in the Netherlands. The DCCT/EDIC substudy already showed the redictive value for skin AGE levels obtained from skin biosies for the rogression of microvascular comlications in atients with tye 1 diabetes [8]. Our study oulation consisted of tye 2 diabetes atients, with skin AGE level assessment by means of a noninvasive, raid method. Another difference is that the DCCT-substudy investigated the develoment as well as the rogression of microvascular comlications. The limited follow-u eriod, the low rate of clearly classifiable rogression of the microvascular comlications, esecially retinoathy, and the confounding role of introduced medication made us decide to restrict our study to the evaluation of the develoment of microvascular comlications and not to address rogression of these diabetic comlications. In retinoathy, skin AF turned out to have no rognostic value in the multivariate analysis. Possible exlanations are the short follow-u eriod and the smaller amount of atients who develoed retinoathy comared to the develoment of the other comlications. Moreover the different athohysiologic mechanisms of microvascular damage in the different organs (retina, kidneys and neurons) could lay a role in the differences in incidence rates of outcomes. In articular, the athobiology of 158

12 Skin autofluorescence in microvascular comlications retinoathy might be different from those in the kidney and neurologic system due to a different role of vascular endothelial growth factor (VEGF) as a ossible mediator for roliferation [20]. (Micro)albuminuria is an early clinical sign of diabetic nehroathy; when left untreated it redicts a high risk for the develoment of rogressive renal damage which will eventually may lead to end stage renal disease. Progressive renal disease is also associated with a vastly increased cardiovascular risk. This study defined (micro)albuminuria as a sign of microvascular comlication with the intention to reflect early stages of diabetic nehroathy. In the redictive analyses the non-surviving atients were excluded from the analyses. These non-survivors had markedly increased skin AF values, but they also had a very high revalence of microvascular comlications at baseline (70%), so this does not reduce the strength of the relation between skin AF and microvascular comlications at all. Ethnicity is one of the mentioned UKPDS confounding risk factors for the develoment of microvascular disease. Because of the limitation of measuring skin AF in dark skin tyes, with the rototye of the AGE reader used in the resent study, eole with dark skin had to be excluded. Over 95% of the articiants were Caucasian, therefore ethnicity was not taken into account in the analyses. Further develoments of the AGE reader may hoefully enable measurements in dark skin tye in future investigations. Lutgers et al. reviously described the other limitations of the AFR as a marker of tissue AGE accumulation: non-fluorescent AGEs will not be measured with the AFR, and other tissue comonents which fluoresce in the same range of wavelength might be confounders [11]. In conclusion, our study confirms skin autofluorescence as a helful clinical method to identify tye 2 diabetes atients who are at risk for (develoing) any microvascular comlication, neuroathy and (micro)albuminuria. Further investigation with longer follow-u needs to be done to assess whether or not skin autofluorescence is a factor in the develoment of diabetic retinoathy, and to assess the relationshi of skin AF and 159

13 Chater 7 the rogression of microvascular comlications. Its non-invasive and time-saving alication makes the AFR an easy clinical tool useful in the out-atient clinic in the risk assessment as well as for monitoring changes in accumulation of tissue AGEs reflecting long term glycemic stress. REFERENCES 1. Smith U, Laakso M, Eliasson B, Wesslau C, Borén J, Wiklund O, Attvall S. Pathogenesis and treatment of diabetic vascular disease illustrated by two cases. J Int Med 260: , Brownlee M. The athobiology of diabetec comlications. A unifying mechanism. Banting Lecture Diabetes 54: , The Diabetes Control and Comlications Trial Research Grou: The effect of intensive treatment of diabetes on the develoment and rogression of long-term comlications in insulin-deendent diabetes mellitus. N Engl J Med 329: , Stratton IM, Adler AI, Neil HA, Matthews DR, Manley SE, Cull CA, Hadden D, Turner R, Holman RR. Association of glycaemia with macrovascular and microvascular comlications of tye 2 diabetes (UKPDS 35): rosective observational study. BMJ 321: , Klein R, Klein BE, Moss SE. Relation of glycemic control to diabetic microvascular comlications in diabetes mellitus. Ann Intern Med 124: 90 96, Pirart J. Diabetes mellitus and its degenerative comlications: a rosective study of 4,400 atients observed between 1947 and 1973 (art 1). Diabetes Care 1: , Monnier VM, Bautista O, Kenny D, Sell DR, Fogarty J, Dahms W, Cleary PA, Lachin J, Genuth S: Skin collagen glycation, glycoxidation, and crosslinking are lower in subjects with long-term intensive versus conventional theray of tye 1 diabetes: relevance of glycated collagen roducts versus HbA1c as markers of diabetec comlications: DCCT Skin Collagen Ancillary Study Grou: Diabetes Control and Comlications Trial. Diabetes 48: ,

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