We recruited 74 participants who had had type 1 diabetes for longer than 5 years. Participants had either been diagnosed at less than 30 years of age (n = 68) or when older than 30 years and with islet autoantibodies present (n = 6). All patients had been on insulin since diagnosis. Of the 74 participants, 38 (51%) were male. Age at diagnosis was 16 (9–23) years, median (interquartile range [IQR]), and duration of diabetes was 30 (19–41) years, with BMI of 25 (23–28) kg/m2, HbA1c 7.9 (7.2–9.0)% (63 [55–75] mmol/mol), insulin dose 0.55 (0.44–0.69) units per kg of body weight per day and an estimated GFR of 89 (82–102) ml min−1 1.73 m−2.
Informed consent was obtained from all participants and the study was approved by the National Research Ethics Service Committee South West (09/H0206/25).
Mixed-meal tolerance test
Participants attended the Exeter National Institute for Health Research (NIHR) Clinical Research Facility for a standard mixed-meal tolerance test [3, 12, 13], having fasted from midnight, not taken their usual morning insulin and fully emptied their bladder upon waking. Fasting blood was taken for measurement of C-peptide, creatinine, glucose, HbA1c, and GAD and islet antigen 2 autoantibodies, and a second void urine sample  was collected for UCPCR determination. Participants were given a standard mixed meal (Ensure Plus HP; Abbott Nutrition, Columbus, OH, USA) consisting of 6 ml/kg (maximum 360 ml) water and containing per 100 ml: 15.9 g carbohydrate, 7.9 g protein, 3.3 g fat and 125 kJ energy. Blood was taken for C-peptide and glucose analysis at 90 min post-completion of the mixed meal, with urine being collected for UCPCR determination at 120 min. All participants were asked to provide a home urine sample in a boric acid container [11, 12], taken 2 h after an evening meal (and having voided the bladder before the meal). Serum and urine samples were stored at −80°C for subsequent analysis.
We analysed serum C-peptide with a direct electrochemiluminescence immunoassay using mouse monoclonal anti-C-peptide antibody (Roche Diagnostics, Mannheim, Germany) on an E170 analyser (Roche). The reported limit of detection is 3.3 pmol/l with a CV of 0.6% at 33 pmol/l.
We compared our Roche assay with another assay, Ultrasensitive C-peptide ELISA (Mercodia, Sylveniusgatanm, Sweden), which is a solid-phase two-site enzyme immunoassay that uses a peroxidase-TMB (3,3′,5,5′-tetramethybenzidine) label on an automated ELISA system (Dynex DSX; Launch Diagnostics, Longfield, UK). The comparison involved dual analysis of 67 samples selected for having low levels of serum C-peptide. The reported limit of detection is 1.5 pmol/l with a CV of 5.5% at 37 pmol/l.
Urinary C-peptide was analysed on the E170 analyser (Roche) as previously described .
As C-peptide results were not normally distributed, we used non-parametric statistical tests. A Wilcoxon’s signed rank test was used to compare fasted and stimulated C-peptide levels. McNemar’s test was used to compare proportions detectable by different limits. Bland–Altman plots were used to assess the comparability of results from the Roche and Mercodia assays. Statistical analysis was performed using IBM SPSS version 20 (IBM, Armonk, NY, USA).