RADIOIMMUNOASSAY OF BOUND AND FREE MELATONIN IN PLASMA

We describe a nonextraction procedure, and two extraction procedures, for RIA of melatonin inhuman plasma. All procedures shouted a diurnal rhythm of melatonin int human subjects, will interindividual differenc es greater than interprocedure differences. However, further investiga tions demonstrated considerable variability of recovery in the nonextr action procedure, suggesting a variability of binding proteins between samples, Combining recovery and dialysis experiments in the extractio n procedures, we demonstrated that chloroform was unable to extract al bumin-bound melatonin from a human serum albumin solution but, paradox ically, was able to extract bound and free melatonin from a plasma sam ple. The methanol extraction procedure extracted free and bound melato nin from all sources. These results indicate that albumin binding can substantially affect the RIA procedures. We conclude that assays shoul d be validated against free and bound melatonin and that the two forms should be independently investigated when assessing bioactivity.