Measurement of circulating human proinsulin concentrations using a proinsulin-specific antiserum.
Antibodies have been raised against biosynthetic human proinsulin that show less than 1% cross-reactivity with human insulin and C-peptide. A sensitive (IC50 0.16 pmol/ml; minimum detectable concentration 0.004 pmol/ml) radioimmunoassay has been developed using this antiserum and 125I-proinsulin that will measure proinsulin-like immunoreactivity in human serum without the need for prior separation of insulin or C-peptide. In healthy, fasted subjects (N = 23), the serum proinsulin concentration was 0.015 +/- 0.001 pmol/ml (mean +/- SEM). In six healthy subjects, serum proinsulin rose to 250% of basal after 120 min in response to 100 g oral carbohydrate, but to only 130% after 60 min following 25 g oral carbohydrate. The proinsulin/total immunoreactive insulin ratio and the proinsulin/C-peptide ratio fell sharply after both high and low carbohydrate loads. Endogenous human serum proinsulin-like immunoreactivity released into the circulation after 100 g carbohydrate was eluted from a Mono Q high-performance, ion-exchange column with the same retention time as biosynthetic human proinsulin. Treatment of biosynthetic proinsulin with trypsin under mild conditions led to a decrease in proinsulin-like immunoreactivity concomitant with an increase in C-peptide and insulin-like immunoreactivity, indicating that the proinsulin-specific antiserum did not preferentially recognize intermediates of proinsulin cleavage.
D000328:Adult; D000918:Antibody Specificity; D002096:C-Peptide / Q000097:blood; D002241:Carbohydrates; D002852:Chromatography, Ion Exchange / Q000379:methods; D005260:Female; D006801:Humans; D007328:Insulin / Q000097:blood; D008297:Male; D011384:Proinsulin / Q000097:blood* / Q000276:immunology; D011863:Radioimmunoassay; D014357:Trypsin
D002096:C-Peptide; D002241:Carbohydrates; D007328:Insulin; D011384:Proinsulin; D014357:Trypsin