Effect of X-Rays on Trace-Labeled I¹³¹-Insulin and Its Relevance to Biologic Studies with I¹³¹-Labeled Proteins

In vivo metabolic studies in man and animals have revealed that the major fraction of insulin-I131 is degraded with a half-time of the order of forty to sixty minutes or less following its intravenous administration (1). However, a smaller fraction, the magnitude of which varies with different lots of radioiodinated insulin, is removed from the blood stream at a much slower rate, with a half-time of six to twenty-four hours. It has also been shown by paper electrophoresis and paper chromatography that the slowly degraded moiety is bound to the serum proteins in contrast to the rapidly degraded fraction which is free of such association. Appraisal of the factors influencing the relative amounts of rapidly and slowly degraded fractions led to the conclusion that the serum protein-bound components represent alterations of the native insulin and to the suspicion that these alterations are manifestations of radiation damage to the hormone. The present study shows that similar changes can be produced by external irradiation with x-rays, peak energy 1 Mev, at dosage levels comparable to those received by the I131 trace-labeled proteins of high specific activity which are currently being employed in many biologic investigations. Trace amounts of unaltered insulin-I131 were adsorbed to Whatman No. 3MM filter paper at the site of application. The extent of alteration was then determined from the fraction of radioactivity which migrated during electrophoretic or chromatographic separation. The results show that, in addition to the production of I131-containing products which bind to serum proteins, I131 is also released as iodide ion following irradiation of insulin-I131. The radiation dose required for the alteration of a fixed fraction of insulin-I131 is an inverse function not only of the concentration of stable insulin but also of the concentration of other substances such as purified human serum albumin or cysteine. At insulin concentrations of 30 mg. per liter, irradiation at 600 r/min. for fifteen minutes produces approximately 18 per cent alteration and at 1,800 r/min. for thirty minutes produces 75 per cent alteration. The pH of the solution is another important variable, since irradiation-induced changes are barely detectable at pH7 with dosage levels sufficient to alter almost completely the same lots of insulin-I131 by irradiation at pH3. Hydrogen peroxide, in concentrations far exceeding those produced by irradiation, had no significant effect on the insulin-I131.