An initial cohort of 150 asymptomatic T1D consecutively followed at our outpatient clinic and a control group of 50 subjects matched by age, sex and smoke condition were recruited between 2010 and 2012 in Badalona, Barcelona. In this cohort we evaluated the presence of subclinical atherosclerosis as previously reported . Inclusion criteria were an age between 20 and 50 years and an evolution disease of more than 10 years. The exclusion criteria were a previous history of clinical macrovascular or cardiac heart disease (CHD). Current smoking and previously smoking condition for less than 5 years were included in the same category. A group of non-diabetic subjects matched for age, sex, body mass index (BMI) and smoking condition recruited from the relatives and staff of our hospital was also included as control group. All patients were under intensive insulin treatment and 15 % of them using pump devices.
The local ethics committee, in accordance with the Declaration of Helsinki, approved the study; all participants gave their written informed consent prior to inclusion.
Demographic and clinical data including age, sex, history of clinical macrovascular disease and microvascular diabetic complications, family history of early CHD in first degree relatives (defined as CHD occurring before age 55 years in men and before age 65 years in women) and medical treatment (antihypertensive agents, statins and acetylsalicylic acid) were recorded for all patients. BMI was calculated as weight in kilograms divided by height per square meter.
Diabetic nephropathy was evaluated according to urinary albumin excretion. Thus, normal urinary albumin excretion was considered below 30 mg/24 h, microalbuminuria from 30 to 300 mg/24 h and proteinuria above 300 mg/24 h. These results were confirmed on at least two out of three consecutive determinations. Diabetic retinopathy was defined by fundus oculi performed by a specialized ophthalmologist.
Trained personnel collected clinical parameters (age, sex, height, weight, BMI, blood pressure and smoking habit and family history of early CHD).
Blood samples were drawn by venipuncture at between 8.00 and 08.30 h after an overnight fast. Plasma glucose, total cholesterol, high density (HDL) and low density (LDL) lipoprotein cholesterol, triglycerides, calcium (Ca) and phosphate (Ph) were measured by routine clinical chemistry immediately after extraction. HbA1c was measured in blood samples with ethylenediaminetetraacetic acid (EDTA) by high-performance liquid chromatographic (HPLC) using a fully-automated Adams Menarini HI-AUTO A1c 8160 analyzer manufactured by Arkray (Kyoto, Japan) with an inter-assay coefficient of variation of 1.8 and 1.5 % at HbA1c levels of 4.8 and 9.0 % respectively (reference range 4–5.8 %). This method is a cation exchange HPLC method certified by the NGSP (National Glycohemoglobin Standardization Program) of traceability to the Diabetes Control and Complications Trial Reference (DCCT) Method. Mean HbA1c was calculated as an average of three determinations in the previous year before the inclusion in the study.
Serum YKL-40 was analyzed using a commercial ELISA assay (Quidel Speciality products, San Diego, CA USA), Sensitivity of the assay was 20 μg/ml. The average within-run and total CVs were 3.6 and 5.4 %, respectively. The reference interval (central 90 % interval) for chondrex in healthy adults was 25–95 μg/l.
Serum adiponectin concentrations were measured by a commercial radioimmunoassay (Linco Research, Inc., St Louis. MO, USA), using 125I-labeled technique. The intra- and inter-assay coefficients of variation (CVs) at mean adiponectin levels of 3, 6 and 15 μg/ml were 3.6, 6.2 and 1.8 % intra-assay, and 9.2, 6.9 and 9.2 % inter-assay, respectively. Assay sensitivity was 1 μg/ml. All plasma samples were diluted 1:250 yielding an effective range of 0.2–50 μg/ml.
Plasma homocysteine concentrations were measured using an automated enzyme chemiluminescence immunoassay (Immulite 2000® Siemens Healthcare Diagnostics, Llanberis, UK). Assay sensitivity was 1.2 μmol/l. The intra- and inter-assay coefficients of variation (CVs) at mean homocysteine concentrations of 11 and 24.5 μmol/l were 8.1 and 5.6 % intra-assay, and 8.5 and 8.1 % inter-assay, respectively (reference range 5.0–12 μmol/l).
Serum TNF- α, IL-6 and IL-1β concentrations were measured using an enzyme chemiluminescence immunometric assay (Immulite®, Siemens Healthcare Diagnostics, Llanberis, UK). For TNF-α, analytical sensitivity was 1.7 pg/mL and interassay CV <6.8 %. For IL-6 analytical sensitivity was 2 pg/ml and interassay CV <7.3 %. For IL-1β analytical sensitivity was 1.5 pg/ml and interassay CV <9.1 %.
Serum high sensitivity C-reactive protein (hsCRP) were measured using an ultrasensitive CRP test (N High Sensitivity CRP) on a BN-ProSpec nephelometer (Dade Behring, GMBH, Marburg, Germany) with an inter-assay variation coefficient of 3.7 and 3.5 % for CRP concentrations of 2.38 and 52.2 mg/l, respectively. Assay was 0.175 mg/l, performed using a sample dilution of 1:20.
Evaluation of subclinical atherosclerosis
A computed tomography (CT) to quantify coronary artery calcification score (CACS) was performed using a 16-slice high resolution CT ECG-gated, with retrospective reconstruction and with special attention to the coronary arteries (SOMATOM Sensation 16 and Syngo Calcium Scoring software for analysis and calcium calcification). CACS was identified as a dense area in the coronary artery exceeding the threshold of 130 Hounsfield units. A total Agatston score was determined for each patient. The results were expressed according to the classification previously described by Shaw et al.  and results were transformed as a categorical variable considering positive those greater than zero and negative those with negative results equal to zero.
A carotid ultrasound (CU) to measure the CIMT was performed in all participants. Ultrasonographic images were acquired using high resolution B-mode ultrasound (Siemens Acuson Sequoia 512) with an electric linear array 13-5 MHz transducer. The CIMT was the result of the median of the tunica intima and tunica media thickness in per protocol defined carotid areas (left internal carotid, right internal carotid, common carotid and bifurcation). Plaque number and characteristics were recorded.
A single trained radiologist performed evaluation and acquisition of CT images and CU.
Continuous variables were expressed as mean standard deviations (SD) or median (percentile 2.5 and 97.5) and categorical variables as frequency and/or percentage. The Student’s t-test or the non-parametric Mann–Whitney U test, as appropriated, tested differences between groups. A p value less than 0.05 was considered statistically significant. Categorical variables were compared with a χ2 test. Correlation analyses between continuous variables were performed by using univariant Spearman correlation analysis. All statistical analyses were performed using the Statistical Package for Social Science (SPSS, Chicago, IL, USA) for personal computers, version 12.0 (SPSS).