High-performance liquid chromatographic preparation of single-site carrier-free pancreatic polypeptide hormone radiotracers.
The preferred radiotracer for use in radioimmuno- and receptor assays is one which is as similar as possible to the native hormone in both physicochemical and biological properties. Until recently, rapid detailed evaluation of iodination methodology as well as separation of site-specific tracers and their evaluation relative to the native protein has not been possible. Using reversed-phase high-performance liquid chromatographic methodology (C8 or C18 columns) we have evaluated a variety of iodination techniques and conditions. Procedures were found that allow the isolation of site-specific radiolabelled protein hormones in a rapid, reproducible and quantitative manner. The radiotracers are of the highest possible specific activity, have very low levels of damage and are more stable than tracers prepared by conventional techniques. This methodology has been applied to preparing tracers of beef, pork and human insulins, proinsulins and C-peptides as well as analogues of these proteins. In addition, the methodology has been applied to somatostatin, glucagon, pancreatic polypeptide, vasoactive intestinal peptide and luteinizing hormone-releasing hormone. The use of these tracers yielded increased sensitivity and reproducibility in a number of radioimmunoassays (e.g. C-peptide and glucagon), more reproducible results in radioreceptor assays (e.g. insulin receptors) and more defined studies on drug absorption and degradation. Finally, scale-up of our isolation procedures has yielded the 127I-analogue for homologous binding and displacement studies.
D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D007328:Insulin / Q000302:isolation & purification; D007457:Iodine Radioisotopes; D008722:Methods; D010191:Pancreatic Polypeptide / Q000302:isolation & purification*; D011384:Proinsulin / Q000302:isolation & purification
D007328:Insulin; D007457:Iodine Radioisotopes; D010191:Pancreatic Polypeptide; D011384:Proinsulin