Development of an isotope dilution assay for precise determination of insulin, C-peptide, and proinsulin levels in non-diabetic and type II diabetic individuals with comparison to immunoassay.
We describe the application of a stable isotope dilution assay (IDA) to determine precise insulin, C-peptide, and proinsulin levels in blood by extraction from serum and quantitation by mass spectrometry using analogues of each target protein labeled with stable isotopes. Insulin and C-peptide levels were also determined by immunoassay, which gave consistently higher results than by IDA, the relative difference being larger at low concentrations. Insulin, C-peptide, and proinsulin levels were all shown by IDA to be higher in type II diabetics than in non-diabetics, with mean values rising from 22 (+/- 2) to 92 (+/- 8), 335 (+/- 11) to 821 (+/- 24), and 6 (+/- 1) to 37 (+/- 3) pM, respectively. Interestingly, the ratio between IDA and immunoassay values for insulin levels increased from 1.3 in non-diabetics to 1.7 in type II diabetics. The ratio between proinsulin and insulin levels by IDA increased from 0.24 in non-diabetics to 0.36 in type II diabetics, whereas the ratio between C-peptide and insulin levels by IDA decreased from 17.6 to 10.7. This disproportionate change in protein levels between different types of individuals has implications for the metabolism of insulin in the diabetics studied (type II) and suggests that C-peptide levels are not always a reliable guide as to pancreatic insulin secretion. In addition, levels of the 33-residue C-peptide (partially trimmed form) were shown to be less than 10% that of the fully trimmed 31-residue C-peptide levels, and we tested IDA in a clinical context by two post-pancreatic graft studies. IDA was shown to give direct, positive identification of the target protein with unrivaled accuracy, avoiding many of the problems associated with present methodology for protein determination.
Kippen
A D
AD
Department of Medical Biochemistry, University Medical Centre, Switzerland.
Cerini
F
F
Vadas
L
L
Stöcklin
R
R
Vu
L
L
Offord
R E
RE
Rose
K
K
D002096:C-Peptide / Q000097:blood*; D002851:Chromatography, High Pressure Liquid; D003922:Diabetes Mellitus, Type 1 / Q000097:blood*; D003924:Diabetes Mellitus, Type 2 / Q000097:blood*; D005260:Female; D006801:Humans; D007118:Immunoassay; D007328:Insulin / Q000097:blood*; D008297:Male; D008875:Middle Aged; D016035:Pancreas Transplantation; D011384:Proinsulin / Q000097:blood*
D002096:C-Peptide; D007328:Insulin; D011384:Proinsulin