Summary
A routine radioimmunoassay for human C-peptide in serum is described. Antibodies against human C-peptide were raised by immunizing guinea pigs with human b-component. Nine out of 12 animals produced useful antibodies within 6 months. Insulin antibodies coupled to Sepharose were used to bind human proinsulin and insulin in the serum and after centrifugation C-peptide was determined in the supernatant. The detection limit of the assay (calculated as 2 SD from zero) was about 0.003 pmole of C-peptide (in 100 μl). The main sources of error were: (1) Normal and diabetic sera devoid of C-peptide gave a displacement of125I-Tyr-C-peptide varying from 0 to 0.16 nM (6 different antisera). Only one antiserum (M 1181) showed no displacement, and the values of C-peptide determined with this antiserum in normal and diabetic sera were lower than the values determined with another antiserum, which gave a value of 0.07 nM in the sera free of C-peptide. It is suggested that displacement found with most antisera is due to substances in serum that are not related to C-peptide or proinsulin. (2) Serial dilutions of pancreatic extracts and sera may yield dilution curves slightly different to those of the synthetic standard. Possible explanations are discussed. These sources of error can be eliminated or reduced by the proper selection of antisera. Fasting sera from 15 normals, 8 maturity-onset diabetics and 10 insulin-requiring diabetics showed the following concentrations of C-peptide: (M 1181) 0.35±0.09, 0.74+0.51 and 0.21±0.14 (nM, mean±SD). One hour after 1.75 g/kg oral glucose the values increased to 2.24±0.71, 2.34±1.18 and 0.42 ± 0.24 nM.
Article PDF
Similar content being viewed by others
References
Block, M.B., Mako, M., Steiner, D.F., Rubenstein, A.H.: Elevated circulating proinsulin levels in insulin-requiring diabetic patients. J. Lab. clin. Med.78, 811–812 (1971)
Block, M.B., Mako, M.E., Steiner, D.F., Rubenstein, A.H.: Circulating C-peptide immunoreactivity. Studies in normals and diabetic patients. Diabetes21, 1013–1026 (1972)
Fink, G., Cresto, J.C., Gutman, R.A., Lavine, R.L., Rubenstein, A.H., Recant, L.: Plasma proinsulin-like material in insulin treated diabetics. Horm. Metab. Res.6, 439–443 (1974)
Heding, L.G.: Determination of total serum insulin (IRI) in insulin treated diabetic patients. Diabetologia8, 260–266 (1972)
Heding, L.G.: Radioimmunological determination of pancreatic and gut glucagon in plasma. Diabetologia7, 10–19 (1972)
Heding, L.G., Larsen, U.D., Markussen, J., Jorgensen, K.H. Hallund, O.: Radioimmunoassays for human, pork and ox C-peptides and related substances. Horm. Metab. Res. Suppl. Vol.5, 40–44 (1974)
Heding, L.G., Turner, R.C., Harris, E.: C-peptide, proinsulin and insulin responses to fish-insulin induced hypoglycaemia in the diagnosis of insulinomas. Diabetes24 (Suppl. 2), 412 (1975)
Kaneko, T., Oka, H., Munemura, M., Oda, T., Yamashita, K., Suzuki, S., Yanaihara, N., Hashimoto, T., Yanaihara, C.: Radioimmunoassay of human proinsulin C-peptide using synthetic human connecting peptide. Endocr. jap.21, 141–145 (1974)
Markussen, J., Sundby, F., Smyth, D.G., Ko, A.: Preparation of human C-peptide. Horm. Metab. Res.3, 229–232 (1971)
Markussen, J., Heding, L.G., Jørgensen, K.H., Sundby, F.: Proinsulin, insulin and C-peptide. Horm. Metab. Res. Suppl. Series No.3, 33–35 (1971)
Naithani, V.K., Dechesne, M., Markussen, J., Heding, L.G., Larsen, U.D.: Studies on Polypeptides, VI1, synthesis, circular dichroism and immunological studies of tyrosyl human C-peptide. Hoppe-Seylers Z. physiol. Chem.356, 1305–1312 (1975)
Naithani, V.K., Dechesne, M., Markussen, J., Heding L.G.: Studies on polypeptides, V1. Improved synthesis of human proinsulin C-peptide and its benzyloxy-carbonyl derivative. Circular dichroism and immunological studies of human C-peptide. Hoppe-Seylers Z. physiol. Chem.356, 997–1010 (1975)
Östman, J., Arner, P., Groth, C.-G., Heding, L., Jorfeldt, L., Lundgren, G.: Pancreastransplantation vid diabetes mellitus: Studier av pankreas endokrina funktion och intermedicinska synspunkter. (paper in preparation)
Rubenstein, A.H., Block, M.B., Starr, J., Melani, F., Steiner, D.F.: Proinsulin and C-peptide in blood. Diabetes21 (Suppl. 2), 661–672 (1972)
Turner, R.C., Harris, E.: Diagnosis of insulinomas by suppression tests. Lancet1974 II, 188–190
Schlichtkrull, J.: Proinsuline et substances apparantées. Path. et Biol.19, 885–892 (1971)
Yanaihara, N., Hashimoto, T., Yanaihara, C., Sakagami, M., Steiner, D.F., Rubenstein, A.H.: Synthesis of human connecting peptide derivatives and their immunological properties. Biochem. biophys. Res. Commun.59, 1124–1130 (1974)
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Heding, L.G. Radioimmunological determination of human C-peptide in serum. Diabetologia 11, 541–548 (1975). https://doi.org/10.1007/BF01222104
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF01222104