Radiotracer binding to brain microsomes determined by thin-layer chromatography.
A thin-layer chromatography (TLC) assay was developed to monitor the interaction of radiotracers with brain microsomes. Murine brain microsomes were coated onto a zone of a TLC strip, the unreacted sites blocked with gelatin, and the radiotracers chromatographed over the microsomes. Radiotracers bound to the microsomes and were separated from the unreacted materials which migrated at or near the solvent front. Up to 80% of the applied radioactivity bound to the brain microsomes when using 99mTc-(d,l) hexamethyl-propyleneamine oxime (HMPAO) and 123I-(S)-2-hydroxy-3-iodo-6-methoxy-N-[(1-ethyl-2-pyrrolidinyl) methyl]-benzamide (123I-IBZM) as tracers. On the other hand, the presumptive negative control materials p-I-15-phenyl-pentadecanoic acid-123I (123I-IPPA) and 99mTc-mercapto-acetyl triglycine (MAG3) bound poorly (7% and 4%, respectively). 99mTc-ethyl cysteinate dimer (ECD) interacted poorly (9.9%), a result thought to be consistent with its known inability to be metabolized by nonprimate brain tissue. Radiolabeled octreotide analogues (radiolabeled with 111In, I-131 or 99mTc) also bound, and the binding could be reduced by excess unlabeled octreotide. Also, chemical modification by acylation of Lys5 in 111In-labeled octreotide led to decreased binding (approximately 70%) compared to the original radiotracer. Chromatography of the various radiotracers over TLC strips coated only with gelatin was used to monitor nonspecific binding and was low and frequently below 5%. This technique does not require wash steps or centrifugation, and assays are rapidly completed. The assay could be useful in monitoring the interaction of radiotracers with brain microsomes and in evaluating and developing new radiotracers.