In 1982, the maternity hospital in Pelotas, a southern Brazilian city, were visited daily and all births were identified. The neonates whose families lived in the urban area were examined and the mothers interviewed soon after birth (n 5914). These individuals have been prospectively followed at different moments of their life cycles.
In 2004, an attempt was made to locate all participants of the cohort, using multiple strategies. The subjects were interviewed at home and invited to visit the research laboratory to donate a blood sample . At 30 ages, a new attempt was made to locate all cohort members, who were invited to visit the research clinic, where they were interviewed, examined, and blood samples were collected .
Anthropometric variables of weight, was measured at 23 years, using Seca (Uniscale®, Germany) portable electronic scales with 100 g precision. Aluminum anthropometers with 1 mm precision were used to measure height according to Lohman et al. . At 30 years, weight was measured with a scale connected to Bod Pod (Cosmed®, USA) device and height with an aluminum vertical stadiometer with 1 mm precision. Waist circumference was measured while the subjects were standing upright with feet together and arms relaxed along the body. The measuring tape was extended on the horizontal plane at waist height (narrowest portion of the trunk) between the last rib and the iliac crest at the end of a regular exhaling and without compressing the skin, and the value was recorded with 0.1 cm precision. Body mass index (BMI) was calculated by dividing the weight in kilograms (kg) by the square of the height in meters (m).
Cardiometabolic variables were measured at 23 years: capillary glycaemia (mg/dL), total cholesterol (mg/dL), HDL cholesterol (mg/dL), triglycerides (mg/dL), C-reactive protein (mg/dL). At age 30, serum glycaemia (mg/dL), total cholesterol (mg/dL); HDL cholesterol (mg/dL); LDL cholesterol (mg/dL), triglycerides (mg/dL); C-reactive protein (mg/dL); glycated hemoglobin (%) were measured. At 23 years, glycaemia was measured using an Accu-Chek Advantage (Roche®) portable glucometer, total cholesterol, HDL, and triglycerides were measured using a Selectra 2 (Merck®, Darmstadt, Germany. C-Peptide was measured in the blood samples collected at 23 years, using the chemiluminescence technique (Immulite – Siemens) , because the analyses were performed on samples collected at random, the estimates were adjusted for fasting time.
At 30 years, serum glucose was measured using the colorimetric enzyme assay with K082 -Glicose Monoreagente kits (Bioclin®, Brazil), LDL cholesterol was measured with K088 (Bioclin®, Brazil) kits and glycated hemoglobin (HbA1c), with K09 -HbA1c Bi-reagent (Bio-Rad®, Berkeley, California, USA) kits. High-sensitivity C-reactive protein (Hs-CRP) was measured using the automated turbidimetry technique with a BS 380 (Shenzhen-Mindray Bio-Medical Electronics Co.; Ltd®, China) chemical analyzer. Those measures with values below the lower limit of detection of 0.1 mg/L were converted to 0.05 mg/L. Expressing acute inflammatory conditions, we excluded values above 10 mg / L . We also excluded those subjects whose serum glucose level was > 200 mg / dL at 23 and 30 years due to diabetes mellitus .
Blood pressure was measured at 23 years, using an Omron® wrist monitor at the beginning and at the end of the interview, about 60 min apart, with the respondent sited with the arm on a support. At 30 years, blood pressure was measured twice with an HEM705 CPINT (Omron®) automated device coupled to an arm cuff, which was replaced by a proper model for obese subjects. The mean of two measurements was used in the analyses.
The following variables were considered as confounding factors: physical activity was measured at 23 and 30 years of age using the International Physical Activity Questionnaire (IPAQ)  maternal skin color (assessed by the interviewer in the perinatal study), monthly family income in minimum wage, tobacco use (those subjects who reported smoking at least one cigarette a day at 23 years were considered as smokers), alcohol consumption (daily intake in mg/L), family history of hypertension (father and/or mother), birthweight (grams), and rapid weight gain between 2 and 4 years (change in Z score above 0.67 standard deviation)  . Paternal and maternal history of dyslipidemia, hypertension, myocardial infarction, and/or stroke was also considered.
The means of the variables according to the quartiles of Pep-C were evaluated according to the trend tests of the ordered groups. Due to the asymmetric distribution, C-reactive protein and triglycerides were transformed into logarithm. Multiple linear regression was used to evaluate the association between C-Peptide and cardiovascular risk factors, estimates were adjusted for confounding variables like: fasting time (estimated as the time between the last meal and blood sample collection), physical activity practice per week (minutes), family income, mother’s skin color, birthweight, rapid growth between 2 and 4 years old, smoking at 23 years, alcohol consumption, family history of dyslipidemia, arterial hypertension and stroke, myocardial infarction .
The analyses were carried out using the software STATA version 13.1. The study was approved by the Research Ethics Committee of the Medical School of the Federal University of Pelotas under protocol OF. 16/12. All interviews and blood collections were performed after written consent was obtained from the participants.