Clinical Chemistry 60:11 Endocrinology and 1409–1418 (2014)

Association of 1,5-Anhydroglucitol with and Microvascular Conditions Elizabeth Selvin,1,2* Andreea M. Rawlings,1 Morgan Grams,1,3 Ronald Klein,4 Michael Steffes,5 and Josef Coresh1,2

BACKGROUND: 1,5-Anhydroglucitol (1,5-AG) is in- CONCLUSIONS: 1,5-AG was associated with long-term versely related to and may be a useful risk of important microvascular outcomes, particularly indicator of short-term (1–2 weeks) hyperglycemia in persons with diagnosed diabetes and even after ad- and glycemic excursions, but its prognostic value is un- justment for Hb A1c. Our results suggest 1,5-AG may clear. We sought to evaluate the associations of 1,5-AG capture risk information associated with hyperglyce- with risk of diabetes and microvascular disease. mic excursions. © 2014 American Association for Clinical Chemistry

METHODS: We measured 1,5-AG in blood samples from over 10000 participants in the ARIC (Atherosclerosis 6 Risk in Communities) Study (baseline, 1990–1992) Hemoglobin A1c (Hb A1c) is the standard clinical mea- and characterized the independent associations with sure used to monitor glycemic control, and it is now prevalent retinopathy and with incident chronic kid- recommended for use in the diagnosis of diabetes (1). ney disease and incident diabetes during approxi- Although Hb A1c has high reliability compared to the mately 20 years of follow-up. more traditional oral (2), there are certain settings in which Hb A1c testing can be problematic (e.g., anemias, hemoglobinopathies, dial- RESULTS: We found that 1,5-AG was associated with ysis, , liver disease). In addition, there is ev- prevalent retinopathy, driven primarily by the strong idence that glycemic excursions (an aspect of diabetes association in persons with diagnosed diabetes: ad- control incompletely captured by Hb A ) may con- justed odds ratio (OR) 11.26 (95% CI, 6.17–20.53) for 1c Ͻ ␮ Ն ␮ tribute to vascular damage independently of mean glu- 6 g/mL compared to 1,5-AG 10 g/mL. This re- cose concentrations (3–5). As such, there is growing sult remained significant after further adjustment for interest in alternative markers of hyperglycemia. hemoglobin A1c (Hb A1c) (OR, 4.85; 95% CI, 2.42– 1,5-Anhydroglucitol (1,5-AG) or 1-deoxyglucose 9.74). In persons with diagnosed diabetes, low 1,5-AG is a monosaccharide, originating mainly from foods Ͻ ␮ Ն ␮ ( 6 g/mL vs 10 g/mL) was also associated with a and closely resembling glucose in structure (6, 7).In Ͼ 2-fold increased risk of incident chronic dis- the normoglycemic setting, 1,5-AG is typically present ease [adjusted hazard ratio (HR), 2.83; 95% CI, 2.15– at high but constant concentrations in the blood. 3.74] and remained significant after adjustment for Hb 1,5-AG is freely filtered by the glomeruli and reab- A1c (HR, 1.43; 95% CI, 1.02–2.00). Nondiabetic par- sorbed in the renal tubule with the small amount ex- ticipants with high 1,5-AG (Ն10 ␮g/mL) had the low- creted corresponding to dietary intake. However, in the est prevalence of retinopathy and lowest risk of kidney setting of hyperglycemia, high amounts of glucose disease. In persons without diagnosed diabetes at base- block tubular reabsorption of 1,5-AG, causing serum line, 1,5-AG Ͻ10 ␮g/mL was also associated with inci- concentrations to fall (i.e., 1,5-AG has an inverse asso- dent diabetes (adjusted HR, 2.29; 95% CI, 2.03–2.58). ciation with serum glucose). Thus, 1,5-AG is thought

1 Department of Epidemiology and the Welch Center for Prevention, Epidemiol- Received June 27, 2014; accepted August 13, 2014. ogy and Clinical Research, Johns Hopkins Bloomberg School of Public Health, Previously published online at DOI: 10.1373/clinchem.2014.229427 Baltimore, MD; 2 Division of General Internal , Department of Medi- © 2014 American Association for Clinical Chemistry 3 6 cine, Johns Hopkins University, Baltimore, MD; Division of Nephrology, De- Nonstandard abbreviations: Hb A1c, hemoglobin A1c; 1,5-AG, 1,5-anhydroglu- partment of Medicine, Johns Hopkins University, Baltimore, MD; 4 Department citol; CKD, chronic kidney disease; ARIC, Atherosclerosis Risk in Communities; of Ophthalmology and Visual Sciences, University of Wisconsin School of eGFR, estimated glomerular filtration rate; CKD-EPI, CKD–Epidemiology Collab- Medicine and Public Health; 5 Department of Laboratory Medicine and Pathol- oration; ETDRS, Early Treatment Study; OR, odds ratio; ogy, University of Minnesota, Minneapolis, MD. AUC, area under the ROC curve; HR, hazard ratio; NRI, net-reclassification * Address correspondence to this author at: Welch Center for Prevention, Epide- improvement; IDI, integrated-discrimination-improvement. miology and Clinical Research and the, Johns Hopkins Bloomberg School of Public Health, 2024 E. Monument St., Suite 2-600, Baltimore, MD 21287. Fax 410-955-0476; e-mail [email protected].

1409 to be an indicator of postprandial glucose excursions ASSESSMENT OF RETINOPATHY and short-term (1–2 weeks) hyperglycemia (8, 9). Retinal photographs were taken at visit 3 (1993–1995) Although the assay for 1,5-AG is commercially following a standardized protocol that has been previ- available, there are few data linking concentrations to ously described (13, 14). Briefly, after 5 minutes of long-term outcomes. It is unclear if 1,5-AG is associ- dark adaptation, a nonmydriatic 45-degree retinal ated with microvascular complications of diabetes and photograph centered on the optic disc and macula was if 1,5-AG adds prognostic value to Hb A1c.Wecon- taken of 1 randomly selected eye. Trained graders ducted this study to characterize the association of masked to participant information evaluated each of 1,5-AG with prevalent retinopathy, incident chronic the photographs. We defined any retinopathy as a se- kidney disease (CKD), and incident diabetes. verity score Ն20 according to a modification of the Airlie House classification system, as used in the mod- Methods ified Early Treatment Diabetic Retinopathy Study (ETDRS) (13). Level 20 is commonly considered the STUDY POPULATION earliest stage of diabetic retinopathy (15). The Atherosclerosis Risk in Communities (ARIC) Study is a community-based prospective cohort of over ASSESSMENT OF INCIDENT CHRONIC KIDNEY DISEASE 15000 participants sampled from 4 US communities. We used the 2009 CKD-EPI creatinine equation to de- The first clinic examinations (visit 1) took place from termine the eGFR. Among persons with normal kidney 1987 to 1989, with 3 follow-up visits approximately function at baseline (visit 2), we defined incident CKD Ϫ Ϫ every 3 years (10). A fifth visit was completed in 2011– as either eGFR Ͻ60 mL ⅐ min 1 ⅐ (1.73 m2) 1 esti- 2013. The second clinic examination (visit 2) took mated from serum creatinine measured at visit 4 place from 1990 to 1992 and is the baseline for the (1996–1998), accompanied by at least a 25% decrease present study. There were 14348 participants who at- in eGFR from baseline (visit 2), or a kidney disease tended visit 2. Institutional review boards at each clin- hospitalization or death identified during continuous ical site reviewed the study and informed consent was active surveillance (16). obtained from all participants. In the present study, we excluded all persons ASSESSMENT OF INCIDENT DIABETES whose race/ethnicity was recorded as other than white We identified incident (new) cases of diabetes on the or black (n ϭ 42), who were fasting Ͻ8h(nϭ 446), or basis of a self-reported diabetes diagnosis or use of di- who were missing variables of interest (n ϭ 1552). Ret- abetes medications during the ARIC visits and subse- inal photographs were not taken at baseline. Thus, for quent annual telephone calls with follow-up through our analyses of retinopathy, we included only those April 2011. In sensitivity analyses, we evaluated 2 addi- participants who met our inclusion criteria and who tional definitions of incident diabetes incorporating also attended visit 3 (1993–1995), at which time retinal information on undiagnosed cases identified using glu- photographs were taken (final analytic sample, n ϭ cose measurements at 2 subsequent ARIC visits (17). 9447). For analyses of incident CKD, we excluded per- sons with reduced kidney function, defined as an esti- OTHER VARIABLES mated glomerular filtration rate (eGFR) [calculated Serum glucose was measured using the hexokinase from serum creatinine using the 2009 CKD–Epidemi- method. Hb A1c was measured in stored whole blood ology Collaboration (CKD-EPI) equation] of Ͻ60 samples using HPLC with instruments standardized to mL ⅐ minϪ1 ⅐ (1.73 m2)Ϫ1 at baseline (final analytic the Diabetes Control and Complications Trial assay sample, n ϭ 12083). For analyses of incident diabetes, (Tosoh A1c 2.2 Plus Glycohemoglobin Analyzer and we excluded persons with diagnosed diabetes at base- Tosoh G7) (18). Plasma lipid concentrations (19–22), line (final analytic sample, n ϭ 10948). body mass index (23), and blood pressure (24) were also measured. Hypertension was defined as the mean MEASUREMENT OF 1,5-AG of the second and third readings at the visit (with cutoff 1,5-AG (GlycoMark) was measured in 2012–2013 in for systolic blood pressure of 140 mmHg or higher stored serum samples from visit 2 (1990–1992) using a and/or a cutoff for diastolic blood pressure of 90 Roche Modular P800 system. The interassay CV was mmHg or higher) or the use of hypertension medica- 5%. The reliability coefficient for n ϭ 610 masked du- tion. Participants reported their education level, alco- plicate sample pairs was 0.99. Previous studies have hol use, and smoking status. The level of physical activ- shown this 1,5-AG assay to be reliable in long-term ity was assessed with Baecke’s questionnaire at visit 1 stored samples (11, 12). (25).

1410 Clinical Chemistry 60:11 (2014) 1,5-AG and Microvascular Disease

STATISTICAL ANALYSES the prevalence estimates using the Z-test and the 20- Baseline characteristics of the study population were year probabilities using the log-rank test. calculated overall and by categories of 1,5-AG at base- All analyses were conducted using Stata/SE ver- line. We categorized 1,5-AG according to cutpoints sion 13.0 (StataCorp). recommended by the manufacturer. In persons with a diagnosis of diabetes, we divided the population into 3 Results groups on the basis of concentrations of 1,5-AG at baseline (Ͻ6,6toϽ10, and Ն10 ␮g/mL). In persons Baseline characteristics of the study population accord- without a diagnosis of diabetes, we divided the popu- ing to categories of 1,5-AG in persons with and without lation into 2 groups on the basis of concentrations of diagnosed diabetes are shown in Table 1. In general, Ͻ10 and Ն10 ␮g/mL. baseline risk factor associations for 1,5-AG were in- Adjusted odds ratios (ORs) and their corresponding verse to but highly consistent with known risk factors 95% CIs for retinopathy were estimated using multivari- of diabetes and hyperglycemia. Older age, black race, able logistic regression models. Model accuracy was as- higher body mass index, hypertension, lower educa- sessed using the area under the ROC curve (AUC). For tion, higher proportion family history of diabetes, analyses of incident CKD and diabetes, adjusted hazard lower HDL cholesterol, and higher triglycerides were ratios (HRs) and their corresponding 95% CIs were esti- all associated with lower baseline concentrations of mated using Cox proportional hazards models. We veri- 1,5-AG. 1,5-AG was also strongly inversely associated fied that the proportional hazards assumption was met with both Hb A1c and fasting glucose, although the as- using log–log plots and by testing for risk factor-by-time sociations were nonlinear, with a flat association be- interactions. To characterize the shape and assess the con- tween 1,5-AG, Hb A1c and fasting glucose at 1,5-AG tinuous associations of 1,5-AG with each of the clinical concentrations Ͼ10 ␮g/mL (see Fig. 1 in the Data Sup- outcomes, we fit restricted cubic splines (26). Model dis- plement that accompanies the online version of this crimination was assessed using Harrell’s c-statistic (27). article at http://www.clinchem.org/content/vol60/ To evaluate the overall improvement in risk classification issue11). In persons with diagnosed diabetes, the Spear- Ϫ for the addition of 1,5-AG to adjusted models (including man correlations with 1,5-AG were 0.84 for Hb A1c and Ϫ models with Hb A1c or fasting glucose), we calculated the 0.77 for fasting glucose. Among persons without diag- continuous net-reclassification improvement (NRI) sta- nosed diabetes the correlations of Hb A1c and fasting glu- tistic and the integrated-discrimination-improvement cose with 1,5-AG were both low (Spearman correlations Ϫ Ϫ (IDI) statistic (28). of 0.08 and 0.02 for Hb A1c and fasting glucose, re- We constructed 4 models for each of the outcomes. spectively) (see online Supplemental Fig. 1). In persons Model 1 was adjusted for age, sex, race-center (5 cate- with diagnosed diabetes, the 1,5-AG cut-point values of gories: 3 categories being white participants in Minnesota, 10 ␮g/mL and 6 ␮g/mL represented the 64th and 50th Maryland, and North Carolina; and 2 categories being percentiles, respectively, corresponding to fasting glucose black participants in North Carolina and Mississippi). values of approximately 165 mg/dL and 194 mg/dL (9.16 Model 2 was adjusted for all variables in model 1 plus LDL and 10.77 mmol/L) and Hb A1c values of 7.3% and 8.2% cholesterol, HDL cholesterol, triglycerides, body mass in- (see online Supplemental Table 1). Spearman correla- dex, waist-to-hip ratio, systolic blood pressure, blood tions between 1,5-AG and the other continuous variables pressure–lowering medication use, family history of dia- were generally low regardless of diabetes status (see online betes, education level, alcohol use, smoking status, and Supplemental Fig. 2). physical activity level. Model 3 was adjusted for all vari- In the 9447 persons with retinal photographs ables in model 2 plus Hb A1c. Model 4 was adjusted for all available, there were 332 cases of retinopathy. In per- variables in model 2 plus fasting glucose. In analyses com- sons with diagnosed diabetes, low concentrations of paring categories of 1,5-AG at baseline, persons with di- 1,5-AG were strongly associated with retinopathy (Ta- agnosed diabetes and 1,5-AG concentrations Ն10 ␮g/mL ble 2). The lowest prevalence of retinopathy was in per- served as the reference group. We tested for interactions sons without diagnosed diabetes and 1,5-AG of Ն10 by sex and race. ␮g/mL, but this association was attenuated after ad- To evaluate whether 1,5-AG added prognostic in- justment for major risk factors and especially after fur- formation within clinically relevant categories of Hb ther adjustment for Hb A1c or fasting glucose. In per- Ͻ A1c among persons with diagnosed diabetes, we calcu- sons with diagnosed diabetes, those with 1,5-AG 6 lated the crude prevalence of retinopathy and 20-year ␮g/mL were 11 times more likely to have retinopathy cumulative probability (Kaplan–Meier method) of at visit 3 compared to persons with diagnosed diabe- CKD by categories of 1,5-AG (Ͻ10 and Ն10 ␮g/mL) tes and 1,5-AG of Ն10 ␮g/mL even after adjustment within categories of glycemic control at baseline [Hb (model 2, OR, 11.26; 95% CI, 6.17–20.53) (Table 2). Ͻ Ն A1c, 7% and 7% (53 mmol/mol)]. We compared These results were attenuated but remained signifi-

Clinical Chemistry 60:11 (2014) 1411 Table 1 Characteristics of the study population by categories of 1,5-AG at baseline in persons with and a.(12308 ؍ without diagnosed diabetes (n

No diagnosed diabetes (958 ؍ Diagnosed diabetes (n (11,350 ؍ n)

1,5-AG, >10 1,5-AG, <10 1,5-AG, >10 1,5-AG, 6 to 1,5-AG, <6 Characteristics Total ␮g/mL ␮g/mL ␮g/mL <10 ␮g/mL ␮g/mL n 12308 10619 731 349 132 477 1,5-AG, maximum–minimum, ␮g/mL 0.5–49.4 10.0–49.4 0.6–9.9 10.0–33.9 6.0–9.8 0.5–5.9 1,5-AG, ␮g/mL 17.8 (6.55) 19.4 (5.04) 6.82 (2.49) 17.0 (4.96) 8.08 (1.08) 2.32 (1.39)

Hb A1c,% 5.7 (1.1) 5.5 (0.5) 6.2 (1.5) 6.2 (0.8) 7.3 (1.0) 9.7 (1.9)

Hb A1c, mmol/mol 39 (12) 37 (5.5) 44 (16.4) 44 (8.7) 56 (10.9) 83 (20.8) Fasting glucose, mg/dL 111 (36.6) 103 (12.5) 125 (52.4) 131 (34.1) 164 (41.9) 242 (75.8) Age, years 56.9 (5.71) 56.7 (5.68) 57.4 (5.99) 58.3 (5.79) 59.5 (5.22) 57.9 (5.7) Female, % 56.7 56.3 62.1 55.9 54.6 58.3 Black, % 23.1 21.1 30.1 35.2 33.3 45.7 Body mass index, kg/m2 27.9 (5.39) 27.7 (5.24) 28.3 (5.64) 30.2 (6.08) 31.0 (5.73) 31.2 (5.95) Body mass index Ն30, % 28.6 26.3 32.4 44.1 50.8 56.0 Hypertension, % 34.9 32.5 37.9 56.5 60.6 59.6 Family history of diabetes, % 24.2 22.3 28.6 39.5 43.9 43.6 Education, % Less than high school 20.7 19.5 20.3 37.0 37.9 31.7 High school or equivalent 42.0 42.3 40.1 36.1 36.4 43.0 College or above 37.3 38.2 39.7 26.9 25.8 25.4 Current alcohol use, % 57.1 59.2 55.1 41.3 39.4 30.0 Current smoking status, % 21.7 22.1 18.3 25.5 17.4 16.4 Physical activity index 2.45 2.47 2.42 2.29 2.23 2.25 LDL-cholesterol, mg/dL 134 (36.7) 133 (36.2) 133 (39.6) 133 (36.4) 134 (39.3) 141 (42.4) HDL-cholesterol, mg/dL 50.0 (16.6) 50.6 (16.7) 49.3 (16.5) 45.5 (14.5) 41.0 (12.5) 43.6 (13.6) Triglycerides, mg/dL 113 [81, 159] 110 [80, 155] 117 [83, 163] 127 [91, 183] 163 [116, 211] 151 [107, 212] eGFR, mL ⅐ minϪ1 ⅐ (1.73 m2)Ϫ1 96.4 (15.3) 96.4 (14.5) 95.7 (18.3) 95.3 (18.9) 97.0 (16.0) 97.3 (23.0) eGFR, Ͻ60 mL ⅐ minϪ1 ⅐ (1.73 m2)Ϫ1,% 1.80 1.35 3.97 5.73 2.27 5.45 Retinopathy,b % 3.51 1.92 3.45 6.03 12.1 42.4

a Continuous variables are mean (SD) or median [25th percentile, 75th percentile]. Categorical variables are reported as a percentage. b Retinopathy was evaluated at visit 3 (n ϭ 9447) and defined as an ETDRS severity score of Ն20. To convert from milligrams per deciliter to millimoles per liter, multiply by 0.0555 for glucose, 0.0259 for cholesterol, and 0.0113 for triglycerides.

cant after adjustment for Hb A1c or fasting glucose HR, 2.83; 95% CI, 2.15–3.74). After adjustment for Hb (Table 2). A1c or fasting glucose, the association between lower In the 12083 persons with normal kidney function 1,5-AG concentrations and incident CKD in persons at baseline, there were 1534 incident cases of CKD dur- with diabetes was attenuated but remained significant ing a median of 19 years of follow-up. 1,5-AG was (Table 2, Models 3 and 4). strongly associated with incident CKD in a largely In the 10 948 persons without a diagnosis of di- graded fashion. Persons without diagnosed diabetes abetes at baseline, there were 2882 incident cases of and with high 1,5-AG concentrations (Ն10 ␮g/mL) diabetes during a median of 18 years of follow-up. had the lowest risk of incident CKD, whereas, persons Over this time period, persons with 1,5-AG concen- with diagnosed diabetes and 1,5-AG concentrations trations of Ͻ10 ␮g/mL at baseline were more than Ͻ6 ␮g/mL had almost a 3-fold increased risk of CKD twice as likely to develop diabetes compared to per- even after multivariable adjustment (Table 2, Model 2: sons with 1,5-AG Ն10 ␮g/mL. The association per-

1412 Clinical Chemistry 60:11 (2014) 1,5-AG and Microvascular Disease

Table 2 Adjusted ORs (95% CIs) or HRs (95% CIs) of baseline categories of 1,5-AG with prevalent retinopathy, CKD, or incident diabetes in the overall study population.

Outcome Model 1a OR (95% CI) Model 2b OR (95% CI) Model 3c OR (95% CI) Model 4d OR (95% CI) Prevalent retinopathye No diagnosis of diabetes Ն10 ␮g/mL 0.36 (0.20–0.63) 0.43 (0.24–0.76) 0.51 (0.28–0.91) 0.52 (0.29–0.93) Ͻ10 ␮g/mL 0.62 (0.31–1.27) 0.69 (0.34–1.42) 0.62 (0.30–1.29) 0.68 (0.33–1.39) Diagnosed diabetes Ն10 ␮g/mL 1 (Reference) 1 (Reference) 1 (Reference) 1 (Reference) 6toϽ10 ␮g/mL 2.19 (0.95–5.06) 2.21 (0.95–5.13) 1.72 (0.73–4.03) 1.84 (0.78–4.31) Ͻ6 ␮g/mL 11.62 (6.43–21.01) 11.26 (6.17–20.53) 4.85 (2.42–9.74) 5.72 (2.92–11.17) P for trendf Ͻ0.001 Ͻ0.001 Ͻ0.001 Ͻ0.001 Model 1 HR (95% CI) Model 2 HR (95% CI) Model 3 HR (95% CI) Model 4 HR (95% CI)

Incident chronic kidney diseaseg No diagnosis of diabetes Ն10 ␮g/mL 0.53 (0.41–0.67) 0.66 (0.52–0.85) 0.77 (0.60–0.98) 0.72 (0.56–0.92) Ͻ10 ␮g/mL 0.69 (0.51–0.93) 0.87 (0.64–1.17) 0.82 (0.61–1.12) 0.87 (0.64–1.18) Diagnosed diabetes Ն10 ␮g/mL 1 (Reference) 1 (Reference) 1 (Reference) 1 (Reference) 6toϽ10 ␮g/mL 1.66 (1.15–2.41) 1.72 (1.19–2.50) 1.42 (0.98–2.07) 1.59 (1.09–2.30) Ͻ6 ␮g/mL 2.63 (2.00–3.46) 2.83 (2.15–3.74) 1.43 (1.02–2.00) 2.11 (1.52–2.94) P for trend Ͻ0.001 Ͻ0.001 Ͻ0.001 Ͻ0.001 Incident diagnosed diabetesh No diagnosis of diabetes Ն10 ␮g/mL 1 (Reference) 1 (Reference) 1 (Reference) 1 (Reference) Ͻ10 ␮g/mL 2.27 (2.01, 2.55) 2.29 (2.03, 2.58) 1.42 (1.24, 1.63) 1.22 (1.05, 1.43)

a Model 1: age (years), race-center, sex (male, female). b Model 2: variables in model 1 ϩ LDL cholesterol (mg/dL), HDL -cholesterol (mg/dL), triglycerides (mg/dL), body mass index (kg/m2), waist-to-hip ratio, mean systolic blood pressure (mmHg), blood pressure–lowering medication use (yes, no), family history of diabetes (yes, no), education (less than high school, high school or equivalent, more than high school), drinking status (current, former, never), smoking status (current, former, never), physical activity index. c ϩ Model 3: variables in model 2 Hb A1c (per %-point). d Model 4: variables in model 2 ϩ fasting glucose (per 1 mg/dL). e Baseline study population limited to persons with valid retinal photographs (n ϭ 9447). f Baseline study population was limited to persons with normal kidney function [estimated glomerular filtration rate Ն60 mL ⅐ minϪ1 ⅐ (1.73 m2)Ϫ1] and to persons with follow-up data (n ϭ 12083). g The overall P for trend was obtained by modeling the category median (for all 5 categories) as a continuous variable. h Baseline study population was limited to persons without a history of diabetes and to persons with follow-up data (n ϭ 10948). sisted after comprehensive adjustment for tradi- 1,5-AG with retinopathy or CKD. We did, however, ob- tional diabetes risk factors (Table 2, Model 2: HR, serve a somewhat stronger association of 1,5-AG Ն10 2.29 (95% CI, 2.03–2.58) and was attenuated after ␮g/mL (vs Ͻ10 ␮g/mL) with incident diabetes in men further adjustment for Hb A1c (Model 3: 1.42; 95% (Model 2: HR, 3.05; 95% CI, 2.56–3.63) compared to CI, 1.24–1.63) or fasting glucose (Model 4: 1.22; women (Model 2: HR, 1.83; 95% CI, 1.56–2.15). 95% CI, 1.05–1.43). Sensitivity analyses demon- 1,5-AG significantly improved prediction of ret- strated that these results were robust to different def- inopathy, CKD, and diabetes when added to a model initions of incident diabetes (see online Supplemen- with basic risk factors (Table 3). When 1,5-AG was tal Table 2). added to models already containing Hb A1c, the im- We did not observe statistically significant interac- provement in the c-statistic was statistically signifi- tions by race for 1,5-AG with any of the outcomes. There cant for retinopathy but not incident CKD or inci- were also no statistically significant interactions by sex for dent diabetes. The continuous NRI and IDI statistics

Clinical Chemistry 60:11 (2014) 1413 Table 3 Prediction statistics and differences between models.

Difference in AUC from P value for Difference in AUC from P value for AUC reference model difference model with Hb A1c difference Prevalent retinopathy Model 1a 0.6629 (Reference) — — — ϩ Ͻ Model 1 Hb A1c 0.7845 0.1216 0.0001 (Reference) — Model 1 ϩ 1,5-AG 0.7861 0.1232 Ͻ0.0001 — — ϩ ϩ Ͻ Model 1 Hb A1c 1,5-AG 0.7943 0.1314 0.0001 0.0098 0.0352 Model 2b 0.7411 (Reference) — — — ϩ Ͻ Model 2 Hb A1c 0.8044 0.0633 0.0001 (Reference) — Model 2 ϩ 1,5-AG 0.8092 0.0681 Ͻ0.0001 — — ϩ ϩ Ͻ Model 2 Hb A1c 1,5-AG 0.8130 0.0719 0.0001 0.0086 0.0275 Incident chronic kidney c-Statistic Difference in c-statistic P value for Difference in c-statistic P value for disease from reference model difference from model with difference

Hb A1c Model 1 0.6749 (Reference) — — — ϩ Ͻ Model 1 Hb A1c 0.7208 0.0459 0.0001 (Reference) — Model 1 ϩ 1,5-AG 0.7017 0.0268 Ͻ0.0001 — — ϩ ϩ Ͻ Model 1 Hb A1c 1,5-AG 0.7226 0.0477 0.0001 0.0017 0.0647 Model 2 0.7385 (Reference) — — — ϩ Ͻ Model 2 Hb A1c 0.7563 0.0178 0.0001 (Reference) — Model 2 ϩ 1,5-AG 0.7494 0.0110 Ͻ0.0001 — — ϩ ϩ Ͻ Model 2 Hb A1c 1,5-AG 0.7565 0.0180 0.0001 0.0002 0.5616 Incident diabetes Model 1 0.5761 (Reference) — — — ϩ Ͻ Model 1 Hb A1c 0.7342 0.1581 0.0001 (Reference) — Model 1 ϩ 1,5-AG 0.6016 0.0256 Ͻ0.0001 — — ϩ ϩ Ͻ Model 1 Hb A1c 1,5-AG 0.7344 0.1583 0.0001 0.0002 0.7922 Model 2 0.7134 (Reference) — — — ϩ Ͻ Model 2 Hb A1c 0.7760 0.0626 0.0001 (Reference) — Model 2 ϩ 1,5-AG 0.7134 0.0094 Ͻ0.0001 — — ϩ ϩ Ͻ Model 2 Hb A1c 1,5-AG 0.7764 0.0630 0.0001 0.0004 0.4876

a Model 1: age (years), race-center, sex (male, female). b Model 2: variables in model 1 ϩ LDL cholesterol (mg/dL), HDL cholesterol (mg/dL), triglycerides (mg/dL), body mass index (kg/m2), waist-to-hip ratio, mean systolic blood pressure (mmHg), blood pressure–lowering medication use (yes, no), family history of diabetes (yes, no), education (less than high school, high school or

equivalent, more than high school), drinking status (current, former, never), smoking status (current, former, never), physical activity index. HbA1c and 1,5-AG were modeled using restricted cubic splines with knots at the 5th, 35th, 65th, and 95th percentiles.

for retinopathy, incident CKD, and incident diabe- ations at higher values of 1,5-AG. The substantially tes showed largely similar results (see online Supple- different distributions of 1,5-AG in persons with and mental Table 3). without diabetes are evident from the frequency histo- In the overall population (persons with and without grams included in Fig. 1. Consistent with the results pre- diabetes combined), the relative association of 1,5-AG sented in Table 2, analyses of prevalent retinopathy and was much stronger for retinopathy compared to incident incident CKD stratified by diabetes status at baseline re- CKD, although the shapes of the associations of 1,5-AG veal that much of the association is being driven by per- were similar (Fig. 1). The associations of 1,5-AG with sons with diabetes and/or low concentrations of 1,5-AG at prevalent retinopathy, incident CKD, and incident diabe- baseline (see online Supplemental Fig. 3). tes appeared strong and linear at values below approxi- Among persons with a diagnosis of diabetes, the mately 15 ␮g/mL, but there was little evidence for associ- prevalence of retinopathy and absolute risk of CKD

1414 Clinical Chemistry 60:11 (2014) 1,5-AG and Microvascular Disease

A Retinopathy B CKD 32 800 32 800

16 16

600 600 8 8 Frequency 4 Frequency

400 4 400 2 HR (95% CI) OR (95% CI)

1 2 200 200

1

0 0 0 10 20 30 0 10 20 30 1,5-AG (µg/mL) 1,5-AG (µg/mL)

C Diabetes 32 800

16

600 8 Frequency

4 400 HR (95% CI) 2

200 1

0 0 10 20 30 1,5-AG (µg/mL)

Fig. 1. Adjusted associations for baseline 1,5-anhydroglucitol with prevalent retinopathy (ORs) and incident CKD and incident diabetes (HRs) in the overall population. Frequency histograms for 1,5-AG are shown separately for persons with diagnosed diabetes (dark grey bars) and without diagnosed diabetes (light grey bars). Adjusted ORs (for prevalent retinopathy) are from logistic regression models and adjusted HRs (for incident CKD and incident diabetes) are from Cox proportional hazards models. Baseline 1,5-AG was modeled using restricted cubic splines (solid lines) with knots at the 5th, 35th, 65th, and 95th percentiles. Models were centered at the 10th percentile of 1,5-AG. Display of the data was truncated at the 1st and 99th percentiles. The shaded areas are the 95% CIs for restricted cubic spline models. Models were adjusted for age (years), race-center, sex (male, female), LDL cholesterol (mg/dL), HDL cholesterol (mg/dL), triglycerides (mg/dL), body mass index (kg/m2), waist-to-hip ratio, mean systolic blood pressure (mmHg), blood pressure–lowering medication use (yes, no), family history of diabetes (yes, no), education (less than high school, high school or equivalent, more than high school), drinking status (current, former, never), smoking status (current, former, never), and physical activity index (score).

Clinical Chemistry 60:11 (2014) 1415 Fig. 2. Prevalence of retinopathy (A) and 20-year cumulative incidence of CKD (B) by categories of 1,5-AG (<10 ␮ > ␮ > g/mL, 10 g/dL) within categories of glycemic control (Hb A1c <7%, Hb A1c 7%) among persons with diagnosed diabetes at baseline. Vertical bars are 95% CIs.

associated with categories of 1,5-AG (Ͻ10 and Ն10 persons with 1,5-AG Ͻ10 ␮g/mL had substantially more ␮ Ͻ Ն Ն g/mL) by categories of Hb A1c [ 7 and 7%) retinopathy than those with 1,5-AG concentrations 10 (Ͻ53 and Ն53 mmol/mol)] are shown in Fig. 2. ␮g/mL (P Ͻ 0.001) (Fig. 2A). Among persons with Hb Ն Ն Ͻ Ͻ Among persons with Hb A1c 7% ( 53 mmol/mol), A1c 7% ( 53 mmol/mol), 1,5-AG categories did not

1416 Clinical Chemistry 60:11 (2014) 1,5-AG and Microvascular Disease

provide significant additional risk stratification informa- Nonetheless, certain limitations of our study should tion (P value ϭ 0.10). A similar pattern was observed for be considered in the interpretation of these data. These the cumulative incidence of CKD (Fig. 2B). include the reliance on a single measurement of 1,5-AG, the limited number of fasting glucose and serum creati- Conclusions nine measurements during follow-up, and the fact that the retinal photographs were obtained only at visit 3 (in This study demonstrated strong associations between 1993–1994), whereas measurements of 1,5-AG were ob- 1,5-AG and diabetic microvascular outcomes in a tained at baseline (in 1990–1992). We were not able to community-based setting and suggests that 1,5-AG has validate the incident self-reported cases of diabetes iden- prognostic value in persons with diabetes and particu- tified after visit 4. Nonetheless, in sensitivity analyses of Ն Ն larly those with Hb A1c 7% ( 53 mmol/mol). Low incident diabetes defined on the basis of a combination of concentrations of 1,5-AG were strongly associated with glucose measurement, medication use, and self-reported prevalent retinopathy and incident CKD, particularly information available for the first 6 years of follow-up, our among those persons with diagnosed diabetes. In addi- results were similar. Strengths of this study include the tion, 1,5-AG added significant information beyond Hb large, community-based sample, the rigorous measure- A1c for discrimination of prevalent retinopathy. These ment of diabetes risk factors, and the long-term prospec- Ͼ results suggest that 1,5-AG may complement Hb A1c tive follow-up with high retention ( 90% contact rate for monitoring recent postprandial glycemic excur- during follow-up in ARIC). sions in persons with diabetes, but additional studies In summary, this study demonstrated robust asso- are needed to establish its clinical utility. ciations between low concentrations of 1,5-AG and mi- In persons without diabetes, the association of crovascular complications in the setting of diabetes and 1,5-AG with the development of diabetes and complica- supports a possible role for 1,5-AG as a useful bio- tions of diabetes was moderate to weak. Indeed, among marker of hyperglycemia. Additional studies are persons without diabetes, the correlations of 1,5-AG with needed to fully evaluate the clinical utility of 1,5-AG in fasting glucose and Hb A1c were very low. These findings the setting of . are in contrast to those for other markers of hyperglyce- mia, such as glycated albumin and , which are more strongly associated with Hb A1c and fasting glu- cose (29), and predict long-term outcomes even in per- Author Contributions: All authors confirmed they have contributed to the sons without a diagnosis of diabetes (30). There was no intellectual content of this paper and have met the following 3 requirements: (a) evidence of associations of 1,5-AG with long-term out- significant contributions to the conception and design, acquisition of data, or ␮ analysis and interpretation of data; (b) drafting or revising the article for intel- comes at values of approximately 15 g/mL or higher. lectual content; and (c) final approval of the published article. This suggests that while low concentrations of 1,5-AG are relatively insensitive for the identification of early hyper- Authors’ Disclosures or Potential Conflicts of Interest: Upon man- uscript submission, all authors completed the author disclosure form. glycemic states and future diabetes, they may be useful in Disclosures and/or potential conflicts of interest: the setting of overt diabetes. Our findings are consistent with the of 1,5-AG, in which plasma concen- Employment or Leadership: None declared. Consultant or Advisory Role: None declared. trations of 1,5-AG are thought to show decreases only at Stock Ownership: None declared. the highest concentrations of blood glucose and reflect Honoraria: None declared. glucose excursions (31–33). Research Funding: The Atherosclerosis Risk in Communities 1,5-AG has been shown in other studies to corre- Study is carried out as a collaborative study supported by National late with complications of diabetes. In a community- Heart, Lung, and Blood Institute contracts (HHSN268201100005C, HHSN268201100006C, HHSN268201100007C, HHSN268201100008C, based cross-sectional study of 517 persons with type 2 HHSN268201100009C, HHSN268201100010C, HHSN268201100011C, diabetes in Korea, low 1,5-AG concentrations were as- and HHSN268201100012C) and reagents for the 1,5-AG assays were do- sociated with both retinopathy and albuminuria (34). nated by the GlycoMarkTM Corporation; E. Selvin, NIH/NIDDK grant R01 We have previously shown cross-sectional associations DK089174; A. Rawlings, NIH/NHLBI grant T32 HL007024. Expert Testimony: None declared. of 1,5-AG with retinopathy and albuminuria in 1600 Patents: None declared. older adults in ARIC (mean age, 70 years) (35). In one previous prospective study of approximately 2000 per- Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation sons in Japan, 1,5-AG at baseline was significantly as- of data, or preparation or approval of manuscript. sociated with incident cardiovascular events during 11 years of follow-up (36). Our results extend the findings Acknowledgments: The authors thank the staff and participants of the ARIC study for their important contributions. Dr. E. Selvin is the guarantor reported in this and suggest that 1,5-AG may of this work and, as such, had full access to all the data in the study and takes be a useful biomarker of prognosis for microvascular responsibility for the integrity of the data and the accuracy of the data outcomes in the setting of diabetes. analysis.

Clinical Chemistry 60:11 (2014) 1417 References

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