1.Type of study:
This descriptive study was conducted between January 2018 and November 2019 in the pediatric endocrinology unit of Fundación Jiménez Díaz Hospital, located in Madrid, Spain.
2. Sample size calculation:
To obtain hsCRP values inpatients with type 1 diabetes that were at least twice as high as those of an age- and sex-matched control group with an estimated hsCRP value of 0.7 mg/l (standard deviation of 1.2)11, a minimum of 33 subjects in each group is needed to achieve a statistical power (b) of 90% and level of significance a of 0.05.
3.Subjects:
- Group with type 1 diabetes: children between 6 and 18 years of age diagnosed with type 1 diabetes according to the criteria published by the American Diabetes Association17, with confirmed positive pancreatic autoimmunity (ICAS, anti-GAD, and/or anti IA-2). We ruled out patients who presented another chronic disease or complications of diabetes, including renal impairment (no evidence of microalbuminuria at baseline), retinopathy, neuropathy, and cardiac disfunctions.
- Control group: healthy children with a body mass index (BMI) of between –1.5 and +1.5 SDS relative to the mean according to reference charts18 paired with an age- and sex-matched group with type 1 diabetes.
- Group with obesity: children with a BMI of more than 2 SDS over the mean according to reference charts18 and no other chronic disease, matched with the group with DM1 by age and sex.
In these three groups, patients with levels of hsCRP >10 mg/L were removed from analysis since higher levels of hsCRP are considered a marker of an infection or inflammatory process in accordance with American Heart Association (AHA) Guidelines19 .
4. Measures:
Clinical and demographic variables: age, sex, BMI (kg/m2 and Z-score)18, waist circumference (cm and Z-score)20,and blood pressure (mmHg and Z-score)21.
Specific clinical and demographic variables per group
Group with type 1 diabetes: age at diabetes diagnosis, presence of ketoacidosis at onset, duration of diabetes, insulin regimen [multiple doses or continuous subcutaneous insulin infusion (CSII)],total daily insulin (TDI) requirement, urine albumin to creatinine ratio (ACR), and change in HbA1C over the previous year.
Biochemical data
Blood samples were obtained by venipuncture in the morning on hospital premises after a 12-h fasting period. Samples were kept on ice and sent to the laboratory for analysis. Once centrifuged, fractions were separated and frozen at -70 º C for future analyses.
Capillary whole blood via finger-prick was collected to measure HbA1c using monoclonal antibody agglutination reaction, DCA Vantage®.
ACR was determined in second morning urine sample.
Serum hsCRP was measured with commercial enzyme-linked immunosorbent assays (ELISA) from Aviscera Bioscience (CRP High Sensitivity SK00080-02) according to the manufacturer’s instructions. The coefficient of variation intra-assay and inter-assay was 4.2% and 9.0%, respectively.
5.Statistical Analyses
Statistical analyses was performed using SPSS version 21.0 (SPSS Chicago, Illinois). Data are expressed as mean and 95% confidence intervals (95%CI).The Kolmogorov-Smirnov test was used to determine whether the variables under study were normally distributed. When possible, variables that were not normally distributed were log-transformed before analysis.
Following this, and in order to evaluate the relationship between hsCRP and the different variables included in the study, a correlation analysis was carried out. Subsequently, a multiple linear regression analysis was performed including hsCRP as the dependent variable.
ANOVA was used to compare mean hsCRP values among the three groups and to compare means of biochemical and anthropometric variables by hsCRP risk group according to the classification of the American Cardiovascular Association19.
p values <0.05 were considered statistically significant.