Quantitation of in vitro a-1 adrenergic receptor antagonist binding capacity to biologic melanin using tandem mass spectrometry. Journal Article

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Authors: Gaynes, JS; Micic, C; Gaynes, BI; Borgia, JA
Article Title: Quantitation of in vitro a-1 adrenergic receptor antagonist binding capacity to biologic melanin using tandem mass spectrometry.
Abstract: PURPOSE: The purpose of this study was to develop methods to allow evaluation of the binding characteristics for a series of a-1 antagonists to biologically-derived melanin. METHODS: Fresh bovine globes were used to obtain iridal and choroid/retinal pigment epithelial (CRPE) derived melanin. Binding characteristics of chloroquine, tamsulosin and doxazosin were then evaluated in vitro using tandem mass spectroscopy. RESULTS: Tandem mass spectrometry-based assays were developed for three a-1 antagonists that provided linear assay ranges which spanned (minimally) 0.01-10 µg/mL, while exhibiting excellent inter-assay precision and accuracy. When applied to the evaluation of binding characteristics for iridal melanin, mean chloroquine and tamsulosin fractions were found to be 41.9 ± 14.2 pmoles mg(-1) and 25.34 ± 6.186?pmoles mg(-1), respectively. Mean iridal doxazosin binding was found to be 6.36?±?2.19 pmoles mg(-1). Interestingly, mean levels of tamsulosin, but not doxazosin found bound to choroid/CRPE derived melanin approached that of chloroquine (27.91 µg/mL, 25.68 µg/mL and 5.94 µg/mL for chloroquine, tamsulosin and doxazosin, respectively). One way ANOVA for binding affinity for chloroquine, tamsulosin and doxazosin was statistically significant for both iridal and CRPE-derived melanin (p = 0.0012 and 0.0023), respectively. A Bonferroni post-hoc analysis demonstrated a statistically significant difference in the amount of binding between tamsulosin, doxazosin and chloroquine to iridal but not CRPE derived melanin (p 0.05). CONCLUSIONS: Tamsulosin appears to demonstrate melanin binding affinity which approaches chloroquine and exceeds doxazosin for both iridal and CRPE-derived bovine melanin.
Journal Title: Current eye research
Publisher: Unknown  
Date Published: 2013