Skin Advanced Glycation Endproducts (AGEs) Glucosepane and. Methylglyoxal Hydroimidazolone are Independently Associated with Longterm

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1 Page 1 of 47 Skin Advanced Glycation Endproducts (AGEs) Glucosepane and Methylglyoxal Hydroimidazolone are Independently Associated with Longterm Microvascular Complication Progression of Type I diabetes Saul Genuth 1*, Wanjie Sun 2, Patricia Cleary 2, Xiaoyu Gao 2, David R Sell 3, John Lachin 2, the DCCT/EDIC Research Group, Vincent M. Monnier 3,4* Short Title: Collagen AGEs and Microvascular Complications Departments: From the 1 Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio; the 2 Biostatistics Center, George Washington University, Rockville, Maryland; the 3 Departments of Pathology and 4 Biochemistry, and Case Western Reserve University School of Medicine, Cleveland, Ohio. *Co-corresponding authors: Vincent Monnier MD, [phone]; [fax], vmm3@cwru.edu, and Saul Genuth MD, smg15@case.edu; [phone]; [fax] 1 Publish Ahead of Print, published online September 3, 2014

2 Page 2 of 47 Abbreviations: AER, albumin excretion rate; AGE, advanced glycation end product; CML, N ε - (carboxymethyl)-lysine; DCCT, Control and Complications Trial; EDIC, Epidemiology of Interventions and Complications; GSPNE, glucosepane; CEL, carboxyethyl-lysine; G-H1, glyoxal hydroimidazolone; MG-H1, methylglyoxal hydroimidazolone. EDTRS, Early Treatment of Diabetic Retinopathy Scale; LRT, likelihood ratio test. 2

3 Page 3 of 47 ABSTRACT Six skin collagen AGEs originally measured near Control and Complications Trial (DCCT) closeout in 1993 may contribute to the metabolic memory phenomenon reported in the follow-up EDIC complications study. We now investigated whether addition of 4 originally unavailable AGEs, i.e. glucosepane (GSPNE), hydroimidazolones of methylglyoxal (MG-H1) and glyoxal (G-H1), and carboxyethyl-lysine (CEL), improves associations with incident retinopathy, nephropathy, and neuropathy events during years post DCCT. The complete 10 AGE panel is associated with 3 step ETDRS worsening of retinopathy (p 0.002), independent of either mean DCCT or EDIC A1c. GSPNE and fructose-lysine (furosine, FUR) correlate with retinopathy progression, independently of A1c. The complete panel also correlates with microalbuminuria (p=0.008) and FUR with nephropathy independently of A1c (p 0.02). Neuropathy correlates with the complete panel despite adjustment for A1c (p 0.005). MG-H1 and FUR are dominant, independent of A1c (p<0.0001) whereas A1c loses significance after adjustment for the AGEs. Overall, the added set of four AGEs enhances the association of the original panel with progression risk of retinopathy and neuropathy (p<0.04) but not nephropathy, while GSPNE and MG-H1 emerge as the principal new risk factors. Skin AGEs are robust long-term markers of microvascular disease progression, emphasizing the importance of early and sustained implementation of intensive therapy. A large body of epidemiological evidence and animal experimentation underlay the hypothesis that hyperglycemia is a causative factor in the development of microvascular and neuropathic complications in diabetes 1. The demonstration by the Control and Complications Trial (DCCT) that intensive treatment which lowered mean blood glucose approximately 75mg/dl and 3

4 Page 4 of 47 mean HbA1c about 2.0%(21.9 mmol/mol), compared to conventional treatment, sharply and significantly reduced the development and progression of these complications 2 confirming this hypothesis. Nonetheless, the mechanistic connection of hyperglycemia to complications appears complex, containing multiple strands, even if glycemic exposure is primary 3. One path from hyperglycemia to complications is the formation of AGEs which both directly and by crosslinking can affect protein structure and function 4. We have previously demonstrated cross-sectional associations between a panel of 4 AGEs and 2 solubility abnormalities in collagen from a skin biopsy obtained prior to DCCT closeout and the presence of retinopathy, nephropathy and neuropathy in DCCT subjects 5. These included the early glycation product fructose-lysine (fructosamine) measured in acid hydrolysate as furosine, pentosidine and carboxymethyl lysine (CML). Furthermore, we subsequently demonstrated that these AGE abnormalities, especially furosine and CML, were highly correlated with the progression of retinopathy and nephropathy over the ensuing 10 years in the EDIC observational study of the DCCT cohort of patients 6. Using a simultaneously obtained skin biopsy near DCCT closeout, which was stored at -80 o C awaiting the discovery of AGEs not available in 1993, we have now tested the hypothesis that the previously not available AGEs glucosepane (GSPNE) 7, carboxyethyl-lysine (CEL) 8, and the hydroimidazolones of glyoxal (G-H1) and methylglyoxal (MG-H1) 9 determined by liquid chromatography mass spectrometry (LC/MS/MS) as described by Thornalley et al 10 are correlated with complication risk progression compared to the original set. In that regard we recently reported the association between these markers and concurrent progression of diabetic complications during the DCCT 7, which revealed that GSPNE was a particularly robust indicator of past cumulative glycemia that was strongly associated with 4

5 Page 5 of 47 presence of nephropathic, retinopathic and neuropathic outcomes at the time of the biopsy 7. The present study was consequently undertaken to determine whether GSPNE or any of the added AGEs were also associated with the long-term prospective risk of retinopathy, nephropathy and neuropathy progression, individually or in combination with the originally measured AGEs and solubility markers during the 17 years of follow-up of this cohort during EDIC. RESEARCH DESIGN AND METHODS Subjects. 216 participants in the DCCT study from 8 clinics volunteered for a skin biopsy as previously described years prior to DCCT closeout. 123 were from the primary cohort (1-5 years diabetes duration and no retinopathy on fundus photography) and 93 were in the secondary cohort (1-15 years diabetes duration and at least one retinal microaneurysm on fundus photography). Retinopathy, nephropathy and neuropathy data at years of EDIC follow-up were available in 196 of those originally biopsied. During the early EDIC period mean HbA1c levels of the DCCT intensive and conventional treatment groups began to converge and became indistinguishable at approximately 7.9% by 5 years into EDIC 11,12. Outcomes Retinopathy. During EDIC, retinopathy was assessed by standardized seven-field fundus photography in one quarter of the cohort each year and in the entire cohort at EDIC years 4 and 10. All photographs were graded centrally, with graders masked to the therapy assignment, using the final ETDRS grading scale 13 and DCCT methods 14. Progression of retinopathy during the EDIC study was defined as a three-step or greater worsening at any time from the DCCT closeout up to the EDIC year 16 on the final EDTRS scale of retinopathy severity 13 or initial scatter photocoagulation therapy during this interval, among those free of scatter laser in DCCT. 5

6 Page 6 of 47 Nephropathy. Albumin excretion rate (AER) 15 was measured every two years in 1399 participants during the EDIC study. Progression of nephropathy was defined as the initial development of microalbuminuria (AER 40mg/24 h, n=50), or dialysis(n=3) or kidney transplant(n=3) at any time between the DCCT close out and the EDIC study year 15 or 16, among those free of microalbuminuria in DCCT. Since microalbuminuria is known to regress 16, a sustained microalbuminuria (AER 30mg/24 h) at two or more consecutive visits was used in a sensitivity analysis (reported in supplementary materials) to verify the results of a single AER 40mg/24 h. Neuropathy. Clinical and nerve conduction studies performed during the DCCT were repeated in EDIC during years Likewise, cardiovascular autonomic neuropathy (CAN) performed in DCCT was re-assessed in EDIC, first during EDIC years , and repeated in years Progression of neuropathy was defined as the initial development of confirmed clinical neuropathy or cardiac autonomic neuropathy in EDIC, among those free of both complications in DCCT. Confirmed clinical neuropathy was defined as the presence of both definite clinical neuropathy (the presence of signs and symptoms consistent with distal symmetrical polyneuropathy based on examination by a board-certified neurologist) and abnormal nerve conduction (one or more abnormal attributes in at least two anatomically distinct nerves among the sural, peroneal or median nerves) 17. Cardiac autonomic neuropathy was defined as either a R-R variation <15 or an R-R variation between 15 and 19.9 in combination 6

7 Page 7 of 47 with a Valsalva ratio 1.5 or a decrease of > 10mm Hg in diastolic blood pressure on standing 17. Biomedical Evaluations. Demographics were assessed by annual questionnaires. Blood pressure and hemoglobin A1c (HbA1c) were measured annually in EDIC. Plasma lipid concentration (cholesterol, and triglycerides)- was measured biannually during the EDIC study. Advanced glycation products (AGEs) in skin biopsies. The AGE measurements used for this prospective longitudinal study have been previously described in detail 7. Briefly recapitulated, the skin biopsy used in these studies was stored since 1993 at -80 C under argon. Insoluble collagen was prepared and enzymatically digested to release the free glycation products that were quantitated by liquid chromatography mass spectrometry using isotope dilution assays for glucosepane (GSPNE), carboxyethyl-lysine (CEL), glyoxal hydroimidazolone (G-H1), and methylglyoxal hydroimidazolone (MG-H1). This set of AGEs is referred to as the added set (n=4) in contrast to the original set of 6 AGEs/markers that included collagen fluorescence, acid solubility, pepsin digestibility, carboxymethyl-lysine and pentosidine and furosine 5,6. Although native fructose-lysine (FL) was automatically included in the new analytical system, we here report only the values of the originally measured acid hydrolytic conversion product furosine (FUR), but also provide some details on how furosine vs. fructose-lysine behave as prospective risk markers in the study below. 7

8 Page 8 of 47 Statistical methods. Univariate differences between groups were assessed using Wilcoxon ranksum test for quantitative or ordinal variables and Chi-square test for categorical variables. Skin collagen variables were adjusted for age and diabetes duration using residuals from a simple linear regression model. Spearman correlation was used to evaluate a monotone relationship among skin collagen variables and between HbA1c and skin collagens. Multivariable logistic regression models were used to assess the effect of skin collagen AGE variables on the odds of each complication in the EDIC. Significance of effects of specific covariate blocks, unadjusted as well as adjusted for other covariates, were evaluated using likelihood ratio test (LRT) 19. Backward elimination was used starting with all 10 AGEs (the original and new sets combined) to select the subset of skin collagen variables nominally significant for the risk of complications at the level of Entropy R 2 was used to assess the strength of association in logistic regression models 19. Odds ratio per standard deviation increase in the skin collagen AGE variables and HbA1c was reported. For univariate AGE effects, Benjamini and Hochberg s false discovery rate (FDR) alpha adjustment for multiple tests was conducted to control the overall FDR at a level of Unless otherwise noted, nominal P values were presented. SAS was used to perform these analyses. RESULTS Clinical Characteristics of Participants As previously reported 7, Table 1 presents the characteristics of the participants in the skin biopsy study. At DCCT baseline, there was a slight but significant difference in age between the intensive and conventional treatment groups in the primary cohort, and there was a 0.6% (6.6 8

9 Page 9 of 47 mmol/mol) HbA1c difference and a 5mmHg difference in systolic BP in the secondary cohort. At DCCT closeout, mean HbA1c, mean blood glucose, extent of retinopathy, AER and neuropathy were all lower in the intensive than in the conventional group in the primary cohort but only neuropathy was less in the secondary cohort. That the beneficial effect of intensive treatment was less evidenced in the secondary cohort may reflect a somewhat smaller sample size, a slightly higher baseline HbA1c in the intensive group, and a lesser HbA1c difference of 1.7% (18.6 mmol/mol) between the intensive and conventional groups in the secondary cohort compared to a difference of 2.4% (26.2 mmol/mol) in the primary cohort at DCCT closeout. Univariate Analysis Table 2 presents univariate risk factors for progression of various complications from DCCT closeout to years of EDIC. Mean DCCT and EDIC HbA1c s were highly statistically significantly associated with greater progression of retinopathy (p<0.0001), nephropathy (<0.0001) and neuropathy (p= ). Of note, EDIC baseline AER was significantly greater in those who later showed progression of all three complications during EDIC (each p<0.008), whereas the degree of retinopathy at EDIC baseline was associated only with progression of nephropathy and neuropathy (each p 0.01). All AGEs except for CEL and G-H1 were associated with progression of retinopathy (each p 0.01) (Fig. 1); all except acid soluble collagen, CEL, GH-1 and MG-H1 were associated with progression of nephropathy (Fig. 2) (each p 0.02); and all except acid soluble collagen, CEL and GH-1 were significantly associated with progression of neuropathy (Fig. 3) (each p 0.02). All of these remained significant after Benjamini and Hochberg s false discovery rate 9

10 Page 10 of 47 (FDR) adjustment (each FDA adjusted p<0.05). As previously reported, furosine, CML, pentosidine, relative fluorescence and pepsin solubility (negatively) were significantly associated with all three complications, but only GSPNE of the four newly measured AGEs was uniformly associated with future progression of all complications (p<0.0001). Multivariable Logistic Regression Analyses Tables 3, 4 and 5 present multivariable logistic regression analyses of covariates associated respectively with progression of retinopathy, nephropathy and neuropathy. Retinopathy: The complete panel comprising the original and the new set of AGEs was significantly associated with progression of retinopathy 16 years after DCCT closeout and remained so after adjustment for mean DCCT HbA1c, mean EDIC HbA1c, and all the other significant factors from univariate analyses in Table 2 (treatment group, AER, and autonomic neuropathy) (Table 3). The same was true for the panel of 6 original AGEs, and backward selected AGEs- furosine and GSPNE, jointly or individually. The four added AGEs strengthened the association between the original AGEs and retinopathy (LRT p=0.02). Interestingly, the mean DCCT HbA1c effect on retinopathy progression during EDIC lost significance when adjusted for the whole panel of AGEs, or furosine alone, or for the combination of furosine and GSPNE, i.e. glycated collagen and its downstream cross-linked product. Nephropathy: The whole panel of 10 AGEs and the panel of 6 original AGEs were both associated with the progression of nephropathy 15 to 16 years after DCCT closeout (Table 4). However, the added AGEs did not strengthen this association (p=0.59). Furosine was selected as 10

11 Page 11 of 47 the strongest predictor of nephropathy, which remained significantly associated after adjustment for mean DCCT and mean EDIC HbA1c, whereas the mean DCCT HbA1c effect lost significance when adjusted for furosine or the whole panel of AGEs. The nephropathy analysis was repeated by using the outcome of a sustained AER 30mg/24 h (Supplementary Tables 1 and 2). The results were almost identical to what was reported to AER 40mg/24 h, which confirmed the robustness of these findings. Neuropathy: The entire panel of 10 AGEs, the panel of 6 original AGEs, the combination of backward-selected AGEs furosine and MG-H1, and both of these AGEs individually, remained associated with progression of neuropathy in EDIC after adjustment for mean DCCT HbA1c and mean EDIC HbA1c (Table 5). The four new AGEs strengthened the association between the 6 original AGEs and neuropathy (LRT, p=0.04). Moreover, the effect of mean DCCT HbA1c on neuropathy progression lost significance when adjusted for furosine alone or in combination with MG-H1 and when adjusted for the whole panel of AGEs. Furosine vs. fructose-lysine and associations amongst AGEs and with A1c We have reported in this study the yr EDIC data on furosine from the original set instead of fructose-lysine, its native precursor that was automatically included in the analytical procedure. Both markers performed about the same in the Spearman correlation analysis with other AGEs (Supplemental Table 3). As expected, the two markers correlated highly with each other (R 2 = 0.70, p<0.001). The R 2 values with other AGEs were very similar, in general slightly favoring furosine over FL, except for the correlation with glucosepane (0.52 vs 0.56) and MG- H1 (0.13 vs 0.26), whereby only FL was significant (p<0.01). As expected furosine correlated 11

12 Page 12 of 47 better with past (DCCT) than prospective (EDIC) mean glycemia as reflected by A1c values (0.72 vs. 029, both p<0.001). Of interest is that collagen pepsin solubility was highly inversely correlated with most other markers, in particular the matrix crosslink GSPN which is the most abundant AGE in skin. GSPNE was the glycation product most strongly correlated with the other AGEs, followed by furosine and MG-H1. Furosine (r=0.72, p<0.0001) was more strongly associated with mean DCCT HbA1c than FL (r=0.48, p<0.0001). CEL, G-H1 and MG-H1 were not correlated with mean DCCT HbA1c. CML correlated with each component of the original set of AGEs, as previously reported, and also correlated with GSPNE (r=0.50, p<0.0001) and with DCCT mean HbA1c (r = 0.38, p<0.0001) but did not correlate with EDIC mean HbA1c. As for correlation with complications, our previous study on association with progression of complications during DCCT up to the time of the biopsy 7 revealed that the order of individual strength of association (entropy R 2 ) for sustained 3 step progression was FL >furosine (each P <0.05), but for sustained greater than or equal to 3 MA it was furosine>fl (p<0.05), for nephropathy furosine>fl (each P<0.05), and for neuropathy furosine>>>fl(each P<0.05). For the future progression of complications during EDIC we found only trivial differences in the χ 2 and R 2 effects leading to the conclusion that both forms of the Amadori product behave very similarly. DISCUSSION In univariate analysis of 8 AGEs and 2 collagen solubility markers, 4 of which we have measured for the first time in our original DCCT biopsies, i.e. GSPNE, CEL, G-H1 and MG-H1, GSPNE stands out in its significant association with 3 step ETDRS worsening of retinopathy, 12

13 Page 13 of 47 development of microalbuminuria, and development of neuropathy from closeout of DCCT to years later in EDIC. In multivariable analysis using backward selection, GSPNE was associated with the subsequent development of retinopathy even after adjustment for DCCT mean HbA1c and EDIC mean HbA1c, as well as DCCT treatment group and log AER at EDIC baseline (Table 3). In addition, it is noteworthy that the content of GSPNE in skin collagen from people with type I diabetes is 3-4x higher than furosine, CML or MG-H1, 17 x higher than CEL, 42 x higher than GH1, and 100x higher than pentosidine. GSPNE is also quantitatively both the single most prevalent AGE and protein crosslink, and it is exclusively derived from glucose. These facts support GSPNE as perhaps the one AGE that plays the most important role in the pathogenesis of retinopathy from hyperglycemia. This is not to deny a role for other AGEs either individually or in combination. In univariate analysis, furosine, CML and pentosidine were associated with all 3 progressive complications as previously reported 6 and furosine remained significant after adjustment for DCCT mean HbA1c and for EDIC mean HbA1c. MG-H1, derived from methylglyoxal, was also strongly associated with retinopathy (p=0.004) and neuropathy (p=0.006) but not significantly with nephropathy (p=0.1). Not surprisingly, there were numerous correlations between many pairs of AGEs and between AGEs and DCCT mean HbA1c (Supplemental Table 3), which expressed the glycemic exposure prior to measurement of the AGEs. Again the correlations between GSPNE and each of the other AGEs as well as with DCCT mean HbA1c were most often the strongest of any single AGE. Furosine, a measure of glycated collagen, correlated most strongly with DCCT mean HbA1c (r=0.72) while the AGE MG-H1, formed from methylglyoxal originating mainly from the degradation of cellular triosephosphate and influenced by glycolytic activity 21,22 13

14 Page 14 of 47 correlated not at all with mean DCCT HbA1c. Within the panel of 10 AGEs reported here, two were indirect indices of crosslinking, i.e. acid solubility and pepsin solubility, each representing physicochemical properties that reflect the incorporation of AGEs into tissue proteins. Although GSPNE has been known since , its precise role in the development of diabetic complications remains to be understood. Incorporation of GSPNE may decrease the turnover of collagen and increase its accumulation, perhaps contributing to thickening of basement membranes 24 in the retina, kidney and peripheral nerves in addition to hyperglycemia-mediated increased extracellular matrix deposition 25. Second, the necessity of arginine residues for the formation of GSPNE (Fig. 1), may compete with integrin binding sites to basement membrane 26 (23) and the resultant loss of proper attachment of endothelial cells to basement membrane may promote programmed death of these cells 27. An unexpected finding was that the methylglyoxal AGE MG-H1 remained strongly associated with neuropathy progression in spite of adjustment for all other risk factors. This provides crucial support for the recently reported association between neuropathy and methylglyoxal in 27 participants of the Oslo study 28. These clinical findings together with preclinical data provide a rationale for in-depth studies on the role of methylglyoxal in diabetic neuropathy. Strengths of this study are the availability of tissue specimens from an exceptionally well characterized type 1 diabetes cohort with repeated measurements of retinopathy, nephropathy and neuropathy by the latest and most modern validated methods for up to 17 years after the skin biopsies were obtained and stored. There also are limitations. The data are fundamentally associative; even though the AGE measurements preceded the subsequent development of 14

15 Page 15 of 47 complications and have a predictive quality, this by itself does not prove a causal role. It is conceivable that an antecedent pathogenic factor induces independently both the complications and AGEs in parallel fashion. It is also not certain that qualitative or quantitative findings in skin collagen AGEs reflect those of retinal, renal or neural tissue. In conclusion, skin collagen AGEs are strongly associated with the future development of diabetic retinopathy, nephropathy and neuropathy up to 17 years later. Glucosepane is the single most prevalent measured AGE in human skin collagen and is systematically most associated with progression of these complications, along with its immediate glucose-derived precursor, fructose lysine, judged herein by the surrogate marker, furosine. ACKNOWLEDGMENTS A complete list of participants in the DCCT/EDIC research group can be found in New England Journal of Medicine, 2011; 365: Industry contributors have had no role in the DCCT/EDIC study but have provided free or discounted supplies or equipment to support participants adherence to the study. A complete list can be found in N Engl J Med 2011; 365: Funding/Support: This study was supported by NIDDK grant DK to DRS and JDRF to VMM. The DCCT/EDIC has been supported by multiple grants from the National Institutes of Health including U01 Cooperative Agreement grants ( , ), and contracts ( ) with the Division of Endocrinology and Metabolic Diseases of the National Institute of and Digestive and Kidney Disease (current grant numbers U01 DK and U01 DK094157), and through support by the National Eye Institute, the National Institute of Neurologic Disorders and Stroke, the Genetic Clinical 15

16 Page 16 of 47 Research Centers Program ( ), and Clinical Translational Science Center Program (2006-present), Bethesda, Maryland, USA. Trial Registration: clinicaltrials.gov NCT and NCT The authors disclose no conflicts of interest. S.G. drafted the manuscript and interpreted the results. WS and XG performed the statistical analyses and co-wrote the manuscript. DRS. performed the laboratory analyses and reviewed the manuscript. PC and JLM reviewed the manuscript. VMM interpreted the results and co-wrote the manuscript. The authors acknowledge Mary Hawkins of the Biostatistics Center, the George Washington University for technical assistance and for editorial compliance during manuscript submission. The DCCT/EDIC research group collected the data and reviewed the manuscript. Our gratitude goes to the DCCT participants who volunteered for the skin biopsies that made this study possible. Patricia Cleary is the guarantor of this work and, as such, had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. 16

17 Page 17 of 47 Legends References: 1. Genuth SM. The case for blood glucose control. Adv Intern Med 1995;40: The DCCT Research Group. The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. The Control and Complications Trial Research Group. N Engl J Med 1993;329: The DCCT Research Group. The relationship of glycemic exposure (HbA1c) to the risk of development and progression in the Control and Complications Trial. 1995;44: Monnier VM, Sell DR, Genuth S. Glycation products as markers and predictors of the progression of diabetic complications. Ann N Y Acad Sci 2005;1043: Monnier VM, Bautista O, Kenny D, et al. Skin collagen glycation, glycoxidation, and crosslinking are lower in subjects with long-term intensive versus conventional therapy of type 1 diabetes: relevance of glycated collagen products versus HbA1c as markers of diabetic complications. DCCT Skin Collagen Ancillary Study Group. Control and Complications Trial. 1999;48: Genuth S, Sun W, Cleary P, et al. Glycation and carboxymethyllysine levels in skin collagen predict the risk of future 10-year progression of diabetic retinopathy and nephropathy in the diabetes control and complications trial and epidemiology of diabetes interventions and complications participants with type 1 diabetes. 2005;54: Monnier VM, Sell DR, Strauch C, et al. The association between skin collagen glucosepane and past progression of microvascular and neuropathic complications in type 1 diabetes. J Complications 2013;27:

18 Page 18 of Ahmed MU, Brinkmann Frye E, Degenhardt TP, Thorpe SR, Baynes JW. N-epsilon- (carboxyethyl)lysine, a product of the chemical modification of proteins by methylglyoxal, increases with age in human lens proteins. Biochem J 1997;324: Ahmed N, Argirov OK, Minhas HS, Cordeiro CA, Thornalley PJ. Assay of advanced glycation endproducts (AGEs): surveying AGEs by chromatographic assay with derivatization by 6-aminoquinolyl-N-hydroxysuccinimidyl-carbamate and application to Nepsiloncarboxymethyl-lysine- and Nepsilon-(1-carboxyethyl)lysine-modified albumin. Biochem J 2002;364: Ahmed N, Thornalley PJ. Quantitative screening of protein biomarkers of early glycation, advanced glycation, oxidation and nitrosation in cellular and extracellular proteins by tandem mass spectrometry multiple reaction monitoring. Biochem Soc Trans 2003;31: Writing Team for the DDCT-EDIC. Sustained effect of intensive treatment of type 1 diabetes mellitus on development and progression of diabetic nephropathy: the Epidemiology of Interventions and Complications (EDIC) study. JAMA 2003;290: White NH, Sun W, Cleary PA, et al. Effect of prior intensive therapy in type 1 diabetes on 10-year progression of retinopathy in the DCCT/EDIC: comparison of adults and adolescents. 2010;59: EDTRS. Early Treatment Diabetic Retinopathy Study design and baseline patient characteristics. ETDRS report number 7. Ophthalmology 1991;98: The Control and Complications Research Group. The effect of intensive diabetes treatment on the progression of diabetic retinopathy in insulin-dependent diabetes mellitus. Arch Ophthalmol 1995;113:

19 Page 19 of Molitch ME, Steffes MW, Cleary PA, Nathan DM. Baseline analysis of renal function in the Control and Complications Trial. The Control and Complications Trial Research Group [corrected]. Kidney Int 1993;43: Perkins BA, Ficociello LH, Silva KH, Finkelstein DM, Warram JH, Krolewski AS. Regression of microalbuminuria in type 1 diabetes. N Engl J Med 2003;348: Martin CL, Albers J, Herman WH, et al. Neuropathy among the diabetes control and complications trial cohort 8 years after trial completion. Care 2006;29: Pop-Busui R, Lu J, Lopes N, Jones TL. Prevalence of diabetic peripheral neuropathy and relation to glycemic control therapies at baseline in the BARI 2D cohort. Journal of the peripheral nervous system : JPNS 2009;14: Lachin J. Biostatistical Methods: The Assessment of Relative Risks. New York: John Wiley & Sons; Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. Journal of the Royal Statistical Society, Series B 1995;54: Thornalley PJ. Modification of the glyoxalase system in human red blood cells by glucose in vitro. Biochem J 1988;254: Phillips SA, Thornalley PJ. The formation of methylglyoxal from triose phosphates. Investigation using a specific assay for methylglyoxal. Eur J Biochem 1993;212: Lederer MO, Klaiber RG. Cross-linking of proteins by Maillard processes: characterization and detection of lysine-arginine cross-links derived from glyoxal and methylglyoxal. Bioorg Med Chem 1999;7: Kilo C, Vogler N, Williamson JR. Muscle capillary basement membrane changes related to aging and to diabetes mellitus. 1972;21:

20 Page 20 of Muona P, Jaakkola S, Zhang RZ, et al. Hyperglycemic glucose concentrations upregulate the expression of type VI collagen in vitro. Relevance to alterations of peripheral nerves in diabetes mellitus. Am J Pathol 1993;142: Dobler D, Ahmed N, Song L, Eboigbodin KE, Thornalley PJ. Increased dicarbonyl metabolism in endothelial cells in hyperglycemia induces anoikis and impairs angiogenesis by RGD and GFOGER motif modification. 2006;55: Bhatwadekar AD, Glenn JV, Li G, Curtis TM, Gardiner TA, Stitt AW. Advanced glycation of fibronectin impairs vascular repair by endothelial progenitor cells: implications for vasodegeneration in diabetic retinopathy. Invest Ophthalmol Vis Sci 2008;49: Sveen KA, Karime B, Jorum E, et al. Small- and large-fiber neuropathy after 40 years of type 1 diabetes: associations with glycemic control and advanced protein glycation: the Oslo Study. Care 2013;36: Figure 1 AGE levels in those with (yes) or without (no) 3 step progression of retinopathy on the ETDRS scale from DCCT closeout up to EDIC year 16 Figure 2 AGE levels in those with (yes) or without (no) development of AER>40mg/24 hours from DCCT closeout to EDIC year Figure 3 20

21 Page 21 of 47 AGE levels in those with (yes) or without (no) development of confirmed clinical neuropathy or abnormal autonomic nervous system function from DCCT closeout to EDIC year

22 Page 22 of 47 Table 1 Clinical Characteristics of DCCT Participants at DCCT Baseline and Closeout Primary Cohort Secondary Cohort Intensive Convention al P Value Intensive Convention al P Value N DCCT Baseline Age(years) 28 ± 6 26 ± ± 7 30 ± 6 Duration(months) 31 ± ± ± ± 43 A 1c (%) (mmol/mol) 8.8 ± ± ± ± ± ± ± ± Mean blood glucose (mg/dl) 221 ± ± ± ± 72 Triglycerides (mg/dl) 79 ± ± ± ± 56 Cholesterol (mg/dl) 182 ± ± ± ± 37 Retinopathy (%) 10/10: None / 20: Microaneurysms Only / 30: Mild NPDR / 45: Moderate NPDR AER>40mg/24 h (%) Confirmed Clinical Neuropathy (%) SBP (mmhg) 116 ± ± ± ± DBP (mmhg) 73 ± 9 72 ± 9 73 ± 8 76 ± 10 DCCT Closeout Age (years) 34 ± 6 31 ± ± 7 36 ± 6 22

23 Page 23 of 47 Duration (months) 101 ± ± ± ± 46 DCCT Mean HbA 1c (%) (mmol/mol) 7.1 ± ± ± ± 15.3 < ± ± ± ± 16.4 <0.001 Mean blood glucose (mg/dl) 149 ± ± 76 < ± ± 83 <0.001 Triglycerides (mg/dl) 72 ± ± ± ± 36 Cholesterol (mg/dl) 175 ± ± ± ± 33 Retinopathy (%) /10: None / 20: Microaneurysms Only / 30: Mild NPDR / 45: Moderate NPDR AER>40 mg/24 hr Confirmed Clinical Neuropathy (%) sbp (mmhg) 115 ± ± ± ± 14 dbp (mmhg) 75 ± 9 74 ± 8 77 ± 8 77 ± 11 23

24 Page 24 of 47 Table 2. Univariate analysis of risk factors versus further worsening retinopathy, new onset of nephropathy or new onset of neuropathy during EDIC * Characteristics Three or more-step ETDRS progression from DCCT closeout through EDIC study year Development of microalbuminuria or worse (AER 40mg/24hr) from DCCT closeout through EDIC study year Development of confirmed clinical neuropathy or abnormal autonomic function from DCCT closeout to EDIC year or Event Nonevent P value Event Nonevent P value Event Nonevent P value N Demographic Age (yrs) at EDIC study baseline 33.6± ± ± ± ± ± Women (%) duration at EDIC baseline (years) 10.5± ± ± ± ± ± DCCT treatment group Intensives (%) Glycemia control DCCT mean A1c, (%) 8.7± ±1.3 < ± ±1.4 < ± ± (mmol/mol) (72±18.6) (58±14.2) (73±17.5) (61±15.3) (67±18.6) (61±14.2) EDIC mean A1c up to EDIC 8.6± ±0.9 < ± ±1.0 < ± ±

25 Page 25 of 47 study year 16. % (mmol/mol) (70±13.1) (60±9.8) (74±13.1) (61±10.9) (67±14.2) (61±9.8) Medical at EDIC baseline Mean blood pressure (mmhg) 90.2± ± ± ± ± ± Hypertension (%) Triglycerides (mg/dl) 81.3± ± ± ± ± ± HDL cholesterol (mg/dl) 52.9± ± ± ± ± ± LDL cholesterol (mg/dl) 113±28 108± ±27 108± ±31 106± Overweight (%) Smoker (%) Retinopathy at EDIC baseline (%) No retinopathy (10/10) Microaneurismsonly20/(<) Mild NPDR 35/(<) Moderate NPDR or worse 43/(<) Renal at EDIC baseline AER (mg/24 hr), l median (inter-quartiles) (6, 20) (4, 12) (7, 17) (4, 10) (6, 20) (6, 12) 25

26 Page 26 of 47 AER>=40 (%) ~ 0~ n/a Neuropathy at EDIC baseline Confirmed clinical neuropathy (%) Abnormal autonomic function (%) n/a n/a Skin Collagens at EDIC baseline Furosine (pmol/mg) 878± ±179 < ± ±194 < ± ± CML (pmol/mg) 580± ±117 < ± ± ± ±130 <.0001 Pentosidine (pmol/mg) 27.7± ± ± ± ± ± Fluorescence (pmol/mg) 193±43 183± ±42 181± ±46 175± Acid-soluble collagen (%) 0.54± ± ± ± ± ± Pepsin-soluble collagen (%) 6.0± ± ± ± ± ± GSPNE (nmoles/mg) 2.8± ±0.5 < ± ± ± ±0.6 <.0001 CEL (pmol/mg) 164± ± ±83 144± ± ± G-H1 (pmol/mg) 66±42 65± ±47 64± ±36 62± MG-H1(nmol/mg) 0.91± ± ± ± ± ±

27 Page 27 of 47 *Analyses only included subjects who were at risk of an outcome during EDIC, those with a pre-existing outcome during the DCCT being excluded. The retinopathy analysis excluded those who underwent scatter photocoagulation during DCCT (n=2). The nephropathy analyses excluded those who had AER>40mg/24 hr at DCCT closeout (n=20). The neuropathy analysis excluded those who had confirmed clinical neuropathy or abnormal autonomic neuropathy (n=30) at DCCT closeout. confirmed clinical neuropathy at EDIC years or abnormal autonomic function at EDIC years or Hypertension was defined as systolic blood pressure 140 mmhg or diastolic blood pressure 90 mmhg. Overweight was defined as body mass index 25 kg/m 2. 27

28 Page 28 of 47 Table 3. Summary of multivariable logistic regressions of AGE associations with the prevalence of retinopathy without and with adjustment for HbA1c, and vice versa. Covariate effects evaluated Df X 2 P value Entropy All AGEs: Furosine, CML, pentosidine, Fluorescence, acid soluble collagen, pepsin soluble collagen,cel, G-H1, MG-H1, GSPNE Unadjusted < DCCT mean A1c EDIC mean A1c < Original AGEs published in 2005 Furosine, CML, pentosidine, Fluorescence, acid soluble collagen, pepsin soluble collagen Unadjusted < DCCT mean A1c EDIC mean A1c < Backward selected all AGEs: Furosine, GSPNE Unadjusted < DCCT mean A1c EDIC mean A1c < CML, pentosidine, Fluorescence, acid < soluble collagen, pepsin soluble collagen, CEL, G-H1, MG-H1 Selected Factors R 2 OR* (95% CI) 28

29 Page 29 of 47 Furosine Unadjusted < (1.8, 3.6) DCCT mean A1c (1.3, 3.2) EDIC mean A1c < (1.6, 3.2) Selected Factors (1.3, 4.0) GSPNE Unadjusted < (1.7, 3.4) DCCT mean A1c (1.4, 2.9) EDIC mean A1c < (1.6, 3.2) Selected Factors (1.3, 3.5) DCCT mean A1c effect Unadjusted < (1.7, 3.3) Furosine, CML, pentosidine, Fluorescence, acid soluble collagen, pepsin soluble collagen, (0.7, 1.9) CEL, G-H1, MG-H1, GSPNE Backward Selected AGEs: Furosine and (0.8, 2.0) GSPNE Furosine alone (0.9, 2.2) GSPNE alone (1.3, 2.6) EDIC mean A1c effect Unadjusted < (1.7, 3.3) 29

30 Page 30 of 47 Furosine, CML, pentosidine, Fluorescence, acid soluble collagen, pepsin soluble collagen, CEL, GH1, MG < (1.6, 3.6) H1, GSPNE Backward Selected AGEs: Furosine and GSPNE < (1.5, 3.0) * Odds ratio is based on 1 standard deviation increase in the continuous variables (DCCT mean A1c: 1.6% (17.5 mmol/mol);; EDIC mean A1c: 1.1% (12.0 mmol/mol); furosine: 231; GSPNE: 0.66; MG-H1: 0.44). Within each block multiple model effects are presented using the set of variables named within each block. For the first block the joint effect of all 10 AGEs is assessed. The Unadjusted model shows the effect of the named variables without adjustment for other factors, e.g. the effect of the 10 AGEs in combination. This is followed by the effect of the covariates after adjustment for one or more specified factors, each from a separate model, e.g. the effect of the 10 AGEs in a model adjusted for the DCCT mean HbA1c and in a model adjusted for the EDIC mean HbA1c. A model adjusted for the following factors that were nominally statistically significantly associated with the outcome in univariate analyses in Table 2: DCCT mean A1c, EDIC mean A1c up to year 16, DCCT treatment group, and both the log(aer) and abnormal autonomic function at EDIC baseline 30

31 Page 31 of 47 Table 4. Summary of multivariable logistic regressions of AGE associations with the prevalence of nephropathy without and with adjustment for HbA1c, and vice versa OR * Covariate effects evaluated Df X 2 P value Entropy R 2 (95% CI) All AGEs: Furosine,CML,pentosidine, Fluorescence, acid soluble collagen, pepsin soluble collagen, CEL, G-H1, MG-H1, GSPNE Unadjusted DCCT mean A1c EDIC mean A1c Original AGEs published in 2005: Furosine, CML, pentosidine, Fluorescence, acid soluble collagen, pepsin soluble collagen Unadjusted DCCT mean A1c EDIC mean A1c Backward selected AGEs: Furosine Unadjusted < (1.6, 3.3) DCCT mean A1c (1.0, 3.2) EDIC mean A1c (1.3, 2.5) CML,pentosidine, Fluorescence, acid soluble collagen, pepsin soluble collagen, CEL, G-H1, MG (1.0, 3.2) H1, GSPNE Selected Factors 31

32 Page 32 of (1.3, 5.0) DCCT mean A1c effect Unadjusted < (1.5, 2.1) Furosine, CML, pentosidine, Fluorescence, acid soluble collagen, pepsin soluble collagen, (0.7, 2.2) CEL, G-H1, MG-H1, GSPNE Backward Selected AGEs: (0.8, 2.3) Furosine EDIC mean A1c effect Unadjusted < (2.2, 5.1) Furosine, CML, pentosidine, Fluorescence, acid soluble collagen, pepsin soluble collagen, < (2.0, 5.0) CEL, G-H1, MG-H1, GSPNE Backward Selected AGEs: Furosine < (2.0, 4.8) * Odds ratio is based on 1 standard deviation increase in the continuous variables (DCCT mean A1c: 1.6% (17.5 mmol/mol); EDIC mean A1c: 1.1% (12.0 mmol/mol); furosine: 231; GSPNE: 0.66; MG-H1: 0.44). Within each block multiple model effects are presented using the set of variables named within each block. For the first block the joint effect of all 10 AGEs is assessed. The Unadjusted model shows the effect of the named variables without adjustment for other factors, e.g. the effect of the 10 AGEs in combination. This is followed by the effect of the covariates after adjustment for one or more specified factors, each from a separate model, e.g. the effect of the 10 AGEs in a model adjusted for the DCCT mean HbA1c and in a model adjusted for the EDIC mean HbA1c. A model adjusted for the following factors that were nominally statistically significantly associated with the outcome in univariate analyses in Table 2: DCCT mean A1c, EDIC mean A1c up to year 16, an both retinopathy and log(aer) at EDIC baseline 32

33 Page 33 of 47 Table 5. Summary of multivariable logistic regressions of AGE associations with the prevalence of neuropathy without and with adjustment for HbA1c, and vice versa. Covariate effects evaluated Df X 2 P value Entropy All AGEs: Furosine, CML, pentosidine, Fluorescence, acid soluble collagen, pepsin soluble collagen, CEL, G- H1, MG-H1, GSPNE Unadjusted DCCT mean A1c EDIC mean A1c Original AGEs published in 2005: Furosine, CML, pentosidine, Fluorescence, acid soluble collagen, pepsin soluble collagen Unadjusted DCCT mean A1c DIC mean A1c Backward selected AGEs: MG-H1, Furosine Unadjusted < DCCT mean A1c < EDIC mean A1c < CML, pentosidine, Fluorescence, acid soluble collagen, pepsin soluble collagen, CEL, G-H1, MG-H1 Selected Factors < MG-H1 R 2 OR* (95% CI) 33

34 Page 34 of 47 Unadjusted (1.3, 3.1) DCCT mean A1c (1.3, 3.0) EDIC mean A1c < (1.5, 3.5) Selected Factors < (1.4, 3.7) Furosine Unadjusted (1.3, 2.6) DCCT mean A1c (1.3, 3.2) EDIC mean A1c (1.3, 2.5) Selected Factors (0.8, 3.2) DCCT mean A1c effect Unadjusted (1.1, 2.1) Furosine, CML, pentosidine, (0.4, 1.3) Fluorescence, acid soluble collagen, pepsin soluble collagen, CEL, G-H1, MG-H1, GSPNE Backward Selected AGEs: (0.5, 1.5) Furosine, MG-H1 Furosine (0.5, 1.5) MG-H (1.1, 2.1) EDIC mean A1c effect Unadjusted (1.2, 2.3) Furosine, CML, pentosidine, Fluorescence, acid soluble collagen, pepsin soluble collagen, CEL, G-H1, MG-H1, GSPNE (1.1, 2.4) Backward Selected AGEs: (1.1, 2.5) 34

35 Page 35 of 47 Furosine, MG-GH1 * Odds ratio is based on 1 standard deviation increase in the continuous variables (DCCT mean A1c: 1.6% (17.5 mmol/mol); EDIC mean A1c: 1.1% (12.0 mmol/mol); furosine: 231; GSPNE: 0.66; MG-H1: 0.44). Within each block multiple model effects are presented using the set of variables named within each block. For the first block the joint effect of all 10 AGEs is assessed. The Unadjusted model shows the effect of the named variables without adjustment for other factors, e.g. the effect of the 10 AGEs in combination. This is followed by the effect of the covariates after adjustment for one or more specified factors, each from a separate model, e.g. the effect of the 10 AGEs in a model adjusted for the DCCT mean HbA1c and in a model adjusted for the EDIC mean HbA1c. A model adjusted for the following factors that were nominally statistically significantly associated with the outcome in univariate analyses in Table 2: DCCT mean A1c, EDIC mean A1c up to year 16, and also Age, LDL, and log(aer) at EDIC baseline 35

36 Page 36 of A Furosine pmol/mg ** CML pmol/mg ** Fluorescence units * CEL pmol/mg G-H1 pmol/mg Yes No 0 N= P-Value= <.0001 <

37 Page 37 of B Pepsin Soluble % ** Pentosidine ** Acid Soluble % * MG-H1 nmol/mg ** Glucosepane nmoles/mg ** Yes No 0 N= P-Value= <

38 Page 38 of A ** Furosine pmol/mg 800 CML pmol/mg ** 600 Yes Fluorescence units * CEL pmol/mg G-H1 pmol/mg No 0 N= P-Value= <

39 Page 39 of 47 B 12 Pepsin Soluble % 40 Pentosidine 10 * * 8 6 Glucosepane nmol/mg Yes No 4 2 Acid Soluble % MG-H1 nmol/mg ** N= P-Value=

40 Page 40 of 47 A Furosine pmol/mg ** CML pmol/mg ** Fluorescence units ** CEL pmol/mg G-H1 pmol/mg Yes No 0 N= P-Value= <

41 Page 41 of 47 B Acid Soluble % Pepsin Soluble % ** MG-H1 nmol/mg ** Glucosepane nmol/mg ** Pentosidine ** Yes No 0 N= P-Value= <

42 Page 42 of 47 SupplementaryTable 1. Univariate analysis of risk factors for Sustained Microalbuminuria (AER>30) Characteristics Development of sustained microalbuminuria (AER 30mg/24hr) from DCCT closeout through EDIC study year Event Nonevent P value N Demographic Age at EDIC study baseline 32.8± ± Women (%) duration at EDIC baseline (years) DCCT treatment group 9.6± ± Intensives (%) Glycemia control DCCT mean A1c (%) 8.9± ±1.5 <.0001 EDIC mean A1c up to EDIC study year 16 (%) 9.0± ±1.0 <.0001 Retinopathy at EDIC baseline (%) No retinopathy (10/10) Microaneurismsonly20/(<) Mild NPDR 35/(<) Moderate NPDR or worse 43/(<) Renal at EDIC baseline AER (mg/24 hr), <.0001

43 Page 43 of 47 median (inter-quartiles) (7.2, 20.2) (4.3, 10.1) AER>=40 (%) <.0001 Neuropathy at EDIC baseline Confirmed clinical neuropathy (%) Abnormal autonomic function (%) Skin Collagens at EDIC baseline Furosine (pmol/mg) 886± ±214 <.0001 CML (pmol/mg) 590± ± Pentosidine (pmol/mg) 29.1± ± Fluorescence (pmol/mg) 197±45 182± Acid-soluble collagen (%) 0.48± ± Pepsin-soluble collagen (%) 5.7± ± GSPNE (nmol/mg) 2.8± ±0.6 <.0001 CEL (pmol/mg) 158±98 144± G-H1 (pmol/mg) 74±51 64± MG-H1(nmol/mg) 0.95± ± ~ The nephropathy analyses excluded those who had sustained AER 30mg/24 hr at DCCT closeout (n=18).