[A study of the absorption of NPH insulin administered into subcutaneous tissue: in vitro study of the mechanism of insulin release from NPH insulin crystal].

NPH (Neutral Protamine Hagedorn) insulin has been widely used for the patients with diabetes mellitus. NPH insulin crystal (NPH-C) is an insoluble ionically bonded complex which insulin and protamine formed at neutral pH. It has been thought that NPH-C dissolve in the injected site by splitting of crystals into protamine and insulin. But the mechanism of the dissolution of NPH-C injected into subcutaneous tissue has not been fully clarified. To investigate the mechanism of the release of insulin from NPH-C, we measured the concentrations of immunoreactive insulin (IRI) and arginine, major component of protamine, recovered from NPH-C incubated with human serum. The concentrations of IRI and arginine, recovered from NPH-C incubated with human serum at 37 degrees C, time-dependently increased, but remained unchanged at 4 degrees C. And the concentrations of IRI and arginine recovered from NPH-C incubated with Krebs-Ringer phosphate buffer at 37 degrees C or 4 degrees C did not change. The release of IRI and arginine from NPH-C with human serum was enhanced by the addition of CoCl2 and was inhibited by the addition of CdCl2 or EDTA. Human serum was fractionated using starch block electrophoresis and the NPH-C dissolving activities of each fractions were investigated. NPH-C dissolved in carboxypeptidase N rich-fraction, but not in plasmin-rich fraction. Carboxypeptidase N was purified from human serum and the activity of NPH-C dissolution was investigated. The relative activity of NPH-C dissolution in the purified Carboxypeptidase N was about 250-fold higher than that in the unpurified human serum.(ABSTRACT TRUNCATED AT 250 WORDS)