Abstract
The recoveries of human C-peptide and insulin from human serum were determined utilizing the method for extraction of insulin. Serum samples were obtained from healthy subjects before and after glucose loads. The elution profiles from Bio-Gel P-30 of acid-ethanol extracts of these sera revealed that the C-peptide immunoreactivity (CPR) from healthy subjects was eluted at the fractions corresponding to Cpeptide.
First, precipitation of extracted CPR and immunoreactive insulin (IRI) was performed by the addition of 1.5 vol of absolute ethanol and 2.5 vol of ethyl ether to each volume of the extract. The CPR and IRI recovered by this procedure were 32.8 ± 2.8 and 66.0 ± 2.9%(mean ±S. E.), respectively. When gel filtration was employed after the acid-ethanol extraction and the ethanol-ether precipitation, recovery of CPR was calculated as 29.7 ± 1.8%(mean ± S. E.). Differences in recoveries of CPR by these two methods were not significant.
About one third of CPR remained in the supernatant of alcohol-ether precipitation. When the higher proportion of ether to ethanol was adopted to the step of ethanol-ether precipitation, a considerable improvement of recovery of CPR was achieved. The minimum volumes of ethanol and ether to obtain the highest recoveries of CPR and IRI were 1.5 vol of ethanol and 5.0 vol of ether to each volume of the extract. Reextraction of the residue, obtained from the first step of acidethanol extraction, resulted in a considerable improvement of recoveries of these two peptides. The improved recoveries of CPR and IRI by the modified extraction method were calculated as 60.7 ± 1.5 and 83.3 ± 3.4%(mean ± S. E.), respectively.
