The radiochemical assay for juvenile hormone biosynthesis in insects: problems and solutions

Measurement of juvenile hormone (JH) production using the radiochemical assay (RCA) for JH biosynthesis and release is usually a reliable and precise technique. However problems with radiolabeled precursors and misunderstanding of the data, the techniques and the calculations have contributed towards uncertainty with respect to published experimental results. Problems with the purity of [methyl−3H]-methionine or determination of its specific radioactivity have had detrimental effects on the reliability of results using the RCA. Proper control procedures and the use of 14C/3H-double-label RCA can be useful in detecting irregularities in the experimental results, and in determining contributing factors to any problems. The use of [methyl−14C]-methionine and an awareness of normally expected RCA values can also assist the researcher in checking the validity of results. The radiolabeled methyl moiety of methionine is incorporated into JH without discrimination relative to unlabeled methyl methionine, by the o-methyl transferase. However unexpected preferential incorporation of the [methyl−14C]- vs. [methyl−3H]-moiety into JH occurs, but is only evident at concentrations of radiolabeled methionine outside the normal range of the RCA. Changes in radioactive precursor formulation have no effect on the RCA.