Plasma Proinsulin: Comparison of Insulin Specific Protease and Gel Filtration Assays

Abstract

Our laboratory has had a good experience using the gel filtration assay for proinsulin. Since the proteolytic enzymatic assay for proinsulin has the reported advantage of being simpler and requiring less plasma per assay, a comparison was made of the enzyme and gel filtration assays for plasma proinsulin. The proteolytic enzyme was prepared from rat muscle and a 30 to 60 per cent ammonium sulfate fraction was used. The kinetics of the enzyme were characterized with regard to substrate concentration (proinsulin and insulin), enzyme concentration, time of incubation and effect of plasma. The studies of enzyme using labeled substrates revealed identical behavior as with cold substrates. The Km for insulin was .476 × 10−6M and for proinsulin was .956 × 10−6M. Lineweaver-Burk plot showed competitive inhibition between insulin and proinsulin. A noncompetitive inhibitor of the enzyme was identified in a plasma sample from a “normal” human subject. The plasma proinsulin assay was performed as described by Kitabchi et al. with the modification that each plasma had an internal control incubated with labeled insulin or proinsulin. It was seen that the activity of the enzyme was somewhat variable in different plasmas. In noninsulinoma samples of plasma, the per cent proinsulin varied between 0 and 72 per cent. In the insulinoma plasmas, the values were 30 to 143 per cent. Twenty-one samples were compared with the gel filtration assay for proinsulin. This latter procedure utilizes G-50 Sephadex columns with size separation of proinsulin and insulin. Enzymatic assay values appeared consistently higher but still had a degree of correlation with those obtained by column. However, the enzyme assay has significant limitations both in screening of normal subjects as well as in diagnosing insulinomas, since false high proinsulin values may be found. Gel filtration appears at this point in time to be the most reliable method for assay of plasma proinsulin-like material.