Effect of dilution on immunoreactive insulin activity of human plasma

1. Plasma from normal fasting subjects showed significant augmentation of immunoreactive insulin activity on serial dilution. This augmentation effect was not observed in samples obtained after glucose loading or in samples mixed with high concentrations of human standard insulin. 2. Low concentrations of human standard insulin (13 and 25 μU/ml) added to undiluted plasma were fully recovered. Serial dilution of such plasma-insulin mixtures showed augmentation of immunoreactive insulin activity. 3. High concentration of human standard insulin (65.5 μU/ml) added to undiluted plasma was fully recoverable. No augmentation of immunoreactive insulin activity was observed on dilution of such plasma-insulin mixtures. 4. Altering the pH of plasma from pH 7.4 to pH 3.0 did not reveal augmentation. 5. Human standard insulin curve was not significantly altered by the presence of porcine proinsulin. Immunoreactive insulin activity of human plasma mixed with porcine proinsulin did not change, while the same activity was augmented on serial dilution. 6. The results presented here suggest that: a. the augmentation effect is due to the presence of a component of inhibitory nature, b. the 'component' present in undiluted plasma specifically influences the first step, i.e. antigen-antibody complex formation, c. the influence of the 'component' is dependent on the level of immunoreactive insulin and d. this pH dependent 'component' of inhibitory nature does not appear to be proinsulin-like material.