Abstract
BackgroundDetermination of C-peptide is important in the investigation of unexplained hyperinsulinemic hypoglycemia because a high C-peptide concentration usually indicates endogenous insulin hypersecretion. Insulin autoimmune syndrome (IAS) denotes hyperinsulinemic hypoglycemia due to insulin-binding antibodies that prolong insulin half-life. C-peptide clearance is considered to be unaffected, and although a marked C-peptide immunoreactivity in hypoglycemic samples has been reported, it has been suspected to be artifactual. High-resolution mass spectrometry enables examination of the basis of C-peptide-immunoreactivity in IAS.MethodsPrecipitation of plasma with polyethylene glycol was followed by C-peptide immunoassay. Plasma peptides extracted by solvent precipitation were characterized by nano-LC–MS/MS and analyzed using an untargeted data-dependent method. Peptides related to proinsulin, in amino acid sequence, were identified using proprietary bioinformatics software and confirmed by repeat LC–MS/MS analysis. Gel filtration chromatography coupled to LC–MS/MS was used to identify proinsulin-related peptides present in IAS immunocomplexes. Results were compared with those from C-peptide immunoassay.ResultsPolyethylene glycol precipitation of IAS plasma, but not control plasma, depleted C-peptide immunoreactivity consistent with immunoglobulin-bound C-peptide immunoreactivity. LC–MS/MS detected proinsulin and des 31,32 proinsulin at higher abundance in IAS plasma compared with control plasma. Analysis by gel filtration chromatography coupled to LC–MS/MS demonstrated proinsulin and des 31,32 proinsulin, but no C-peptide, in plasma immunocomplexes.ConclusionsAntibody binding can enrich proinsulin and des 31,32 proinsulin in IAS immunocomplexes. Proinsulin cross-reactivity in some C-peptide immunoassays can lead to artifactually increased C-peptide results.
Highlights
Insulin autoimmune syndrome (IAS, or Hirata disease) denotes spontaneous hyperinsulinemic hypoglycemia due to insulin-binding autoantibodies in individuals not receiving insulin therapy [1, 2]
This has the potential to confuse managing clinicians since normal endogenous C-peptide production by the pancreas is inhibited in response to exogenous insulin-induced hypoglycemia aUniversity of Cambridge Metabolic Research Laboratories, Wellcome Trust–MRC Institute of Metabolic Science, Cambridge, UK; bCore Biochemical Assay Laboratory, Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK; cDepartment of Diabetes & Endocrinology, Royal Free London NHS Foundation Trust, London, UK; dDepartment of Clinical Biochemistry and Immunology, Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK; eNational Institute for Health Research Cambridge Biomedical Research Centre, Cambridge, UK; fUniversity of Edinburgh Centre for Cardiovascular
We analyzed IAS plasma using LC–MS/MS with an untargeted peptidomic approach with the aim of identifying whether C-peptide immunoreactivity was attributable to cross-reacting proinsulin/proinsulinderived molecules
Summary
Determination of C-peptide is important in the investigation of unexplained hyperinsulinemic hypoglycemia because a high C-peptide concentration usually indicates endogenous insulin hypersecretion. Insulin autoimmune syndrome (IAS) denotes hyperinsulinemic hypoglycemia due to insulin-binding antibodies that prolong insulin half-life. C-peptide clearance is considered to be unaffected, and a marked C-peptide immunoreactivity in hypoglycemic samples has been reported, it has been suspected to be artifactual. High-resolution mass spectrometry enables examination of the basis of C-peptide-immunoreactivity in IAS
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