Type of Study and Population
We conducted a cross-sectional study on 196 adult patients (over 18 years of age) with type 1 diabetes with more than 10 years of diabetes evolution, recruited in the Adult Endocrine, Diabetes, and Nutrition Department of the Reims University Hospital, in France.
Description of the DIABAGE Study Population and Ethical Considerations
The DIABAGE (DIABetes Advanced Glycation End products) study is one of the tasks of the university research project VIVA (VIeillissement VAsculaire et protéique-« Vascular and protein aging») which aims to study the links between protein molecular aging and vascular complications in different populations of elderly or accelerated-aging patients. A total of 196 patients were identified from the CARéDIAB (Champagne Ardenne Réseau DIABète) database described in a previous study . All patients were included in the study by their endocrinologist and then admitted to the hospital for a day to undergo a complete clinical and biological diabetes examination.
Formal consent was obtained from all patients included in the study and statistical analyses were performed anonymously. The study was conducted in accordance with the Declaration of Helsinki on medical research involving human subjects. CARéDIAB database is approved by the National Committee on data processing and liberties (“CNIL- Comité National Informatique et Libertés”)-Approval number 1434306.
Inclusion and Exclusion Criteria
Patients were consecutively included in the study since they had T1D with a minimum of 10 years of evolution; 518 patients with more than 10 years of diabetes evolution were identified from the CARéDIAB database, which included 1987 T1D patients. Among those patients, 196 were consecutively included in the study on a regular basis during their diabetes check-ups. Patients with acute illness and inflammatory syndrome at the time of investigation were excluded.
Exposure and Outcome Variables
Definitions of T1D and Diabetes Vascular Complications
T1D was defined according to the World Health Organization criteria (WHO) based on the 1997’s American Diabetes Association (ADA) recommendations . In this study, T1D was assessed by the presence of hyperglycemia requiring insulin therapy with one or more positive specific antibodies among the following: anti-Islet cells antibodies (ICA), anti-glutamic acid decarboxylase (GAD), tyrosine kinase, anti-ZnT8, anti-insulin). All patients selected in our study received diabetes typing by determination of specific antibodies. Each complication was assessed by the medical records (history of complications reported in the medical files) and completed by the following exams:
Electrocardiography for coronaropathy complemented, if abnormal, by cardiological exploration performed by a cardiologist.
Ankle Brachial Pressure index for arteriopathy of the lower limbs (less than 0.9) or documented history of arteriopathy with revascularization or not.
Stroke was assessed on the medical history of patients who experienced either transient ischemic attack or constituted stroke.
Pulse wave velocity (PWV) was performed to assess the arterial stiffness using the SphygmoCor device. Carotid-femoral pulse wave velocity (cfPWV), the gold-standard non-invasive tool for evaluating arterial stiffness as recommended by the American Heart Association, was applied in this study . The PWV was determined over the carotid-femoral region by measuring the propagation time of the pressure pulse from the carotid to femoral arteries using an arterial tonometer for recording pressure waveforms. To determine the cfPWV, the traveled path length was measured as the arterial length from the ascending aorta to the measurement point at the femoral artery minus the length from the ascending aorta to the measurement point at the carotid artery. The PWV was expressed in meters per second (m/s).
Direct examination of the retinal fundus by an ophthalmologist.
Urine albumin excretion: normal: < 30 mg/24 h; microalbuminuria: 30–300 mg/24 h; macroalbuminuria > 300 mg/24 h. Nephropathy is defined by a 24-h microalbuminuria ≥ 30 mg/24 h or urine albumin/urine creatinine ratio ≥ 3 mg/mmol.
Polyneuropathy by monofilament test using the standardized technique recommended by the International Working Group on the Diabetic Foot (IWGDF)  or presence of painful neuropathy evaluated by DN4 questionnaire .
Description of the Variables Studied
Measurement of Circulating AGEs
Three protein-bound AGEs (carboxymethyllysine (CML), pentosidine, and methylglyoxal-hydroimidazolone (MGH1)) were measured by liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) as previously described [20, 21]. Serum CML and pentosidine were quantified by LC–MS/MS (API4000 system ABSciex, Les Ulis, France) in dialyzed (24 h at 4 °C against 150 mM NaCl) serum samples. For MG-H1 measurement, 35 μl of dialyzed serum spiked with 20 μM d3-MG-H1, used as internal standard, were hydrolyzed (6 M HCl, 110 °C, 18 h, final volume: 1 ml). Hydrolysates were then evaporated to dryness under nitrogen stream and dried hydrolyzates were resuspended in 100 μl of 125 mM ammonium formate, before being filtered using Uptidisc PTFE filters (4 mm, 0.45 μm, Interchim, France) prior to LC–MS/MS analysis. The chromatographic separation was performed using a Kinetex PFP column (100 × 4.6 mm, 2.6 μm – Phenomenex) with a gradient program composed of 5 mM ammonium formate (pH 2.9) as mobile phase A and 100% acetonitrile as mobile phase B. The flow rate was constant at 0.7 ml/min during all separation steps. The injection volume was 4 μl and oven temperature was set at 30 °C. Detection was performed using an API4000 system in positive-ion mode with an electrospray ionization (ESI) source. Multiple reaction monitoring (MRM) transitions were as follows: 229.2 > 166.1 and 229.2 > 212.2 for the quantification and confirmation transitions, whereas 232.2 > 169.1 was used for the internal standard.
Measurement of AGEs by Skin Auto Fluorescence (SAF)
Tissue AGEs were evaluated non-invasively by measuring SAF using the AGE-Reader® device (Diagnostics Technologies B.V., Groningen, The Netherlands) . The autofluorescence reader illuminates a skin area of about 1 cm2, protected from surrounding light, with an excitation light source between 300 and 420 nm. The emission light and the reflected excitation light of the skin are measured with a spectrometer in the range of 300–600 nm, using 200 μm of fiberglass. The measurements were performed at ambient temperature at the flying side of the arm about 10 cm under the bend, while the patients were seated. Since skin pigmentation can influence autofluorescence by light absorption, autofluorescence was calculated by dividing the average light intensity emitted by nanomole in the range of 420 to 600 nm by the average excited light intensity per nanometer in the 300–420 nm range. The autofluorescence was expressed in arbitrary units (AU) and multiplied by 100. The reflection of the skin was calculated in the range of 300–420 nm by dividing the average intensity reflected by the skin by the average intensity reflected by a white Teflon block (assuming a 100% reflection). Intra-series and between-series coefficients of variation for SAF were 4.4 and 6.4%, respectively, as previously reported in our center [20, 21]. The value used for statistical analysis was the average of three successive measurements.
Other Variables Collected
Several variables were selected as covariates: age at examination, gender, duration of diabetes, body mass index, smoking, systolic blood pressure, diastolic blood pressure, HbA1c, total cholesterol, LDL cholesterol, HDL cholesterol, triglycerides, and glomerular filtration rate (GFR) by CKD-EPI method. Smokers were classified into two groups: current smokers (active smokers or smoking cessation less than 3 years) and former smokers (smoking cessation for 3 years or more). We included the current smokers as a covariate in the analyses.
We selected six vascular complications as dependent variables: retinopathy, nephropathy, neuropathy, coronaropathy, arteriopathy of lower limbs, and stroke. These six outcomes were subdivided into two groups: microangiopathy defined by the presence of at least one microvascular complication (i.e., retinopathy, nephropathy, and neuropathy) and macroangiopathy defined by the presence of at least one macrovascular complication (i.e., coronaropathy, arteriopathy of lower limbs, and stroke).
For the descriptive analysis, continuous variables were expressed as mean and standard deviation if the distribution was normal, otherwise as median and interquartile range (Q1–Q3). Patients with any of the complications were compared to patients without complications for each variable using Student’s t test or Mann–Whitney test, accordingly. Categorical variables were presented as numbers and percentages and comparisons were performed by Chi-square or Fisher’s exact test, accordingly. Associations between different types of AGEs (i.e., SAF and circulating pentosidine, CML, and MGH1) and complications defined as dependent variables were analyzed in a binary logistic regression model and corresponding odd ratios (OR) identified in univariate and multivariate analysis adjusting for potential confounders (age, gender, duration of diabetes, glycated hemoglobin, body mass index, hypertension, dyslipidemia, GFR: glomerular filtration rate by CKD-EPI; smoking status). A linear regression was performed to analyze the correlation between the PWV and different AGEs listed above. Analyses were performed with the software SPSS 17.0. All tests were considered significant at p values less than 0.05.