Potential of UCHL1 as biomarker for destruction of pancreatic beta cells.

There is a clinical need for plasma tests for real-time detection of beta cell destruction, as surrogate endpoint in islet transplantation and immunoprevention trials in type 1 diabetes. This study reports on the use of label-free LC-MS/MS proteomics for bottom-up selection of candidate biomarkers. Ubiquitin COOH-terminal hydrolase 1 (UCHL1) was identified as abundant protein in rat and human beta cells, showing promising beta cell-selectivity, and was selected for further validation in standardized toxicity models. In vitro, H2O2-induced necrosis of INS-1 cells and human islets resulted in intracellular UCHL1 depletion and its extracellular discharge. In vivo, streptozotocin progressively depleted UCHL1 from islet cores and in 50% of animals, an associated plasma UCHL1 surge was detected preceding the GAD65 peak. UCHL1 was cleared with a half-life of 20min. Whole-body dynamic planar imaging of (99m)-Technetium-labeled UCHL1 indicated a rapid UCHL1 uptake in the liver and spleen, followed by urinary excretion of mainly proteolytic UCHL1 fragments. We conclude that LC-MS/MS proteomics is a useful tool to prioritize biomarkers for beta cell injury with promising molar abundance. Despite its consistent UCHL1 discharge by damaged beta cells in vitro, its in vivo use might be restrained by its rapid elimination from plasma. BIOLOGICAL SIGNIFICANCE: Our bottom-up LC-MS/MS proteomics represents a pragmatic approach to identify protein-type biomarkers of pancreatic beta cell injury. UCHL1 successfully passed sequential validation steps of beta cell-selectivity, antigenicity and toxic discharge in vitro. Whole-body dynamic planar imaging of radiolabeled recombinant UCHL1 indicated rapid clearance through the liver, spleen and urinary excretion of proteolytic fragments, likely explaining non-consistent detection in vivo. Integration of kinetic biomarker clearance studies in the a priori selection criteria is recommended before engaging in resource-intensive custom development of sensitive immunoassays for clinical translation.

LC-MS/MS identification of doublecortin as abundant beta cell-selective protein discharged by damaged beta cells in vitro.

There is a clinical need for plasma tests that can directly detect injury to pancreatic beta cells in type 1 diabetes. Such tests require biomarkers that are abundantly and selectively released into plasma by damaged beta cells. We combined LC-MS/MS proteomics and tissue-comparative transcriptomics of FACS-purified beta cells for bottom-up identification of candidate markers. Less than 10% of 467 proteins detected in beta cells showed endocrine-enriched expression. One surprising candidate was the neuronal migration marker doublecortin: in situ analysis revealed uniform doublecortin expression in the cytoplasm of all beta cells. Western blotting and real-time PCR confirmed its strong beta cell-selectivity outside the brain and its high molar abundance, indicating promising biomarker properties in comparison to GAD65, a more established marker of beta cell injury. DCX potential was validated in vitro: chemically-induced necrosis of rat and human beta cells led to a discharge of intracellular doublecortin into the extracellular space, proportionate to the amount of injured cells, and similar to GAD65. In vivo, recombinant DCX showed favorable pharmacokinetic properties, with a half-life in plasma of around 3h. Combined, our findings provide first proof-of-principle for doublecortin as biomarker for beta cell injury in vitro, advocating its further validation as biomarker in vivo.

Validation of an enzyme-linked immunosorbent assay for C-peptide analysis in Cameroon.

AIMS: To validate an ELISA method for C-peptide analysis in Cameroon. METHODS: We evaluated the linearity, detection limit, functional sensitivity, precision and accuracy, and further investigated for cross-reactivity by proinsulin, and interferences by lipids, bilirubin and hemoglobin. This method was compared with the Roche electrochemiluminescence immunoassay. C-peptide stability was assessed following a series of freeze-thaw cycles, and after storage at room temperature. The C-peptide reference range was determined by analyzing fifty plasma samples of Cameroonians without diabetes. RESULTS: The ELISA was linear at least up to 7.09 µg/L, and had a detection limit of 0.09 µg/L, and a functional sensitivity of 0.32 µg/L. The inter- and intraassay %CV were 2.9-9.9%, and 5.2-9.4%, respectively. Recoveries were 81-94% in serum, and 93-98% in buffer. Comparison with the ECLIA yielded a good correlation coefficient (R(2)=0.98). There was no cross-reactivity with proinsulin, and no interference with lipids, bilirubin and hemoglobin. C-peptide was stable at room temperature for 24 h and up to 7 freeze-thaw cycles for medium (1-6 µg/L) and high (>6 µg/L) levels (<-15°C and <-70°C). The reference range for C-peptide was 0.38-3.63 µg/L. CONCLUSIONS: This method is suitable for C-peptide analysis in low-income countries like Cameroon.

An important minority of prediabetic first-degree relatives of type 1 diabetic patients derives from seroconversion to persistent autoantibody positivity after 10 years of age.

AIMS/HYPOTHESIS: The appearance of autoantibodies (Abs) before diabetes onset has mainly been studied in young children. However, most patients develop type 1 diabetes after the age of 15 years. In first-degree relatives aged under 40 years, we investigated the frequency of seroconversion to (persistent) Ab positivity, progression to diabetes and baseline characteristics of seroconverters according to age. METHODS: Abs against insulin (IAA), glutamate decarboxylase (GADA), insulinoma-associated protein 2 (IA-2A) and zinc transporter 8 (ZnT8A) were measured during follow-up of 7,170 first-degree relatives. RESULTS: We identified 379 (5.3%) relatives with positivity for IAA, GADA, IA-2A and/or ZnT8A (Ab(+)) at first sampling and 224 (3.1%) at a later time point. Most seroconversions occurred after the age of 10 years (63%). During follow-up, Abs persisted more often in relatives initially Ab(+) (76%) than in seroconverters (53%; p < 0.001). In both groups diabetes developed at a similar pace and almost exclusively with Ab persistence (136 of 139 prediabetic individuals). For both groups, progression was more rapid if Abs appeared before the age of 10 years. Baseline characteristics at seroconversion did not vary significantly according to age. CONCLUSIONS/INTERPRETATION: Seroconversion to (persistent) Ab(+) occurs regardless of age. Although the progression rate to diabetes is higher under age 10 years, later seroconverters (up to age 40 years) have similar characteristics when compared with age-matched initially Ab(+) relatives and generate an important minority of prediabetic relatives, warranting their identification and, eventually, enrolment in prevention trials.