Due to our shared interest in this topic, we were eager to read the recent article by Sims et al. (1). We agree with the authors that proinsulin (PI) secretion can occur in individuals with long-standing type 1 diabetes (T1D), even in the absence of measurable serum C-peptide (2). However, our work is discordant with the observations of the authors, and we think it is important to understand the differences.
We have previously reported (2) a 16% prevalence of PI secretion in the same T1D Exchange cohort, which is significantly less than the 89.9% reported by Sims et al. (1). In addition, we have similarly completed mixed-meal tolerance tests on a cohort of 13 young healthy individuals as well as 12 adult individuals with long-standing T1D (3), and our observations are again in stark contrast to those presented by the authors. In particular, PI secretion is not ubiquitous in long-standing T1D, and the measured PI concentrations in all of our cohorts are multiple-fold lower. Why the discrepancy?
In our preparatory work for our recent publications (2,3), we found a marked difference in measured PI concentrations depending on the assay used. In order to ensure we were most precisely and accurately measuring PI, we performed extensive assay validation on three commercially available PI assays: the one used in the current study, Millipore’s total PI radioimmunoassay (catalog no. HPI-15K; Millipore), as well as ALPCO’s ultrasensitive STELLUX Chemi Human Total Proinsulin ELISA with corrected standards (catalog no. 80-PINHUT-CH01) and Mercodia’s total PI ELISA (catalog no. 10-1118-01). We purchased total PI from the National Institute for Biological Standards and Control (Potters Bar, Hertfordshire, England), which is the repository for biological standards of the World Health Organization, and performed spike and recovery and dilutional linearity assays to quantify PI and settle ourselves on the most accurate assay. In our hands, the Millipore radioimmunoassay consistently overrecovered total PI such that the measured PI was significantly more than expected (often by 160%). In contrast, the Mercodia and ALPCO ELISAs performed best, with the ALPCO assay working better in the very low range. We independently confirmed that the ALPCO assay fully measures total as well as Des (31,32) and Des (64,65) PI and has <0.6% cross-reactivity to human insulin, 0% to human C-peptide, and 0% to human glucagon.
Because of the differences in total PI measured in our studies versus the current one, we believe it is scientifically prudent for these samples to be reassayed, but with the use of total PI kits that have been independently validated for accuracy by performing spike and recovery as well as dilutional linearity assays. This could even be done by a neutral party. In summary, we agree that PI secretion occurs in individuals with T1D and applaud the authors’ effort to qualitatively confirm PI through mass spectrometry. However, the assay chosen for PI measurement is important to avoid potentially overstating the prevalence and quantified PI concentrations in both healthy and long-standing T1D cohorts.
Article Information
Duality of Interest. No potential conflicts of interest relevant to this article were reported.
