Necessity of ascorbic acid in the radioligand binding assay for [3H]5-hydroxytryptamine.
Evidence is presented to suggest that ascorbic acid is required in the radioligand binding assay for [3H]5-hydroxytryptamine ([3H]5-HT, [3H]serotonin). In the absence of ascorbic acid, oxidation of [3H]5-HT occurred if the radioligand solution was left on ice for 1 or 3 hr. The oxidative products were detectable by thin-layer chromatography. They increased the binding significantly, although there was only slight oxidation (less than 1%) of the [3H]5-HT. When ascorbic acid was not used in the radioligand binding assay for [3H]5-HT, even though [3H]5-HT was prepared immediately before incubating with the membranes from the cortex of the rat, the binding also increased. The increased binding of [3H]5-HT had a low affinity (Kd = 14 nM) and high B max (1180 fmol/mg protein), compared to that in the presence of ascorbic acid (Kd = 5 nM; B max = 210 fmol/mg protein). However, the increased binding was not receptor-related because the additional radioactivity was not displaceable by excess of unlabelled 5-HT (10 microM) or d-LSD (d-lysergic acid iethylamide) and anomalous "specific binding" occurred in boiled membranes from cortex, in which the binding sites for 5-HT were destroyed. These results suggest that oxidation of [3H]5-HT may occur during the incubation with membranes and that ascorbic acid is therefore required as an antioxidant.
D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology*; D002540:Cerebral Cortex / Q000032:analysis* / Q000378:metabolism; D008297:Male; D010084:Oxidation-Reduction; D011869:Radioligand Assay; D051381:Rats; D011919:Rats, Inbred Strains; D011985:Receptors, Serotonin / Q000032:analysis* / Q000378:metabolism; D012701:Serotonin / Q000378:metabolism*
D011985:Receptors, Serotonin; D012701:Serotonin; D001205:Ascorbic Acid