Study Population
We conducted an observational cross-sectional study including 60 asymptomatic T1DM-patients, from a larger cohort (clinicaltrials.gov Identifier: NCT02910271) designed to comprehensively address their subclinical atherosclerosis profile [7]. T1DM required age at onset of diabetes <30 yr-old, previous episode of ketoacidosis or diabetic autoimmunity, and mandatory use of insulin for survival, as defined by ADA criteria. Exclusion criteria were: i) symptomatic intermittent claudication according to Edinburgh Claudication Questionnaire [8]; ii) previous diagnosis of peripheral artery disease, diabetic foot or leg amputation; iii) previous diagnosis of cerebrovascular disease iv) end-stage renal disease; and v) ongoing pregnancy. A detailed description of this trial has been reported elsewhere [7]. The study protocol conformed to the ethical guidelines of the Declaration of Helsinki, and was approved by Ramón y Cajal ethics committee (Date of approval: January 22, 2016; Reference number: 464/15). Written consent has been obtained from each subject after full explanation of the purpose and nature of all procedures used.
Clinical, biochemical variables and copeptin assay
We review the medical history and recorded clinical parameters related to T1DM and cardiovascular risk factors of all patients at recruitment. Then, participants were submitted to a complete anthropometric evaluation that included weight, height, waist circumference, and hip circumference measurements. A fasting blood sample and urine collection were collected to assess renal function and to measure the urinary albumin-to-creatinine ratio, serum lipids, and HbA1c. Technical characteristics of assays used for biochemical measurements have been described elsewhere in detail [7].
Copeptin concentration was measured in fasting serum samples. Blood samples were left to clot for one hour and then centrifuged at 1500 x g for 10 min. Then, serum was kept frozen at -80°C until assayed. After thawing, serum copeptin concentrations were measured in duplicate using a commercial ELISA kit from a single manufacturer and assay lot (High Sensitive ELISA kit for Copeptin, CPP HEA365Hu Cloud-Clone, USA) The lower limit of detection of the assay was 2.63 pmol/L and its intra- and inter-assay coefficients of variation were below 10% and 12%, respectively. The technician in charge of these assays was blinded to patient’s features.
Assessment of carotid ultrasound examination
All study participants underwent a carotid ultrasound examination. Patients rested in supine position for at least 10 min before measurements were taken. Vascular tests were conducted under standardized conditions after an overnight fasting to avoid the possible interference of a postprandial surge in glucose levels. cIMT was calculated for both common carotid arteries of each patient, and the means of such measurements was used for analysis. A Toshiba Nemio model SSA-550A Basic Diagnostic Ultrasound System (Toshiba Medical System S.A., Alcobendas, Madrid, Spain) with a 7.5-MHz probe was used in these assessments. Common carotid, internal carotid, external carotid, and vertebral arteries were also scanned for the presence of carotid plaques (CP), defined as IMT ≥ 1.5 mm protruding into the lumen [7].
Statistical analysis
Data are shown as means ± SD and counts (percentages). Normality of continuous variables was assured as needed by applying logarithmic transformation and checked using the Kolmogorov-Smirnov test. Because copeptin concentrations were not normally distributed, their results were expressed as medians [interquartile range]. We used χ2 or Fisher’s exact tests for categorical variables, and Student’s t or Mann–Whitney U tests for continuous variables as appropriate. We used Spearman’s correlations to evaluate the association between clinical and biochemical variables, and copeptin concentrations. We used Pearson’s correlations between cIMT and continuous variables of interest.
Finally, a multiple stepwise linear regression model was used to explore the main determinants of cIMT levels among those variables with a P value < 0.10 in univariate analyses. Statistical significance was set at a P value < 0.05.
Participants were classified as a function of their normal (≤95th percentile) or abnormal (>95th percentile) carotid IMT values – using normative values from the healthy Spanish population stratified by age and sex [9] – and into high copeptin (HighCp) and low copeptin (LowCp) subgroups – using 13 pmol/L concentration as cut-off value, which is the upper limit of normality of copeptin levels in healthy adults under normoosmotic conditions [2].