title: Quantitation of in vitro α-1 adrenergic receptor antagonist binding capacity to biologic melanin using tandem mass spectrometry.
abstract: OBJECTIVE
  The purpose of this study was to develop methods to allow evaluation of the binding characteristics for a series of α-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 α-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.
authors.affiliation: Departments of Biochemistry, Rush University Medical Center , Chicago, IL , USA .
authors.forename: Jeffrey S
authors.initials: JS
authors.lastname: Gaynes
authors.forename: Cedomir
authors.initials: C
authors.lastname: Micic
authors.forename: Bruce I
authors.initials: BI
authors.lastname: Gaynes
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Borgia
mesh_terms: D058668:Adrenergic alpha-1 Receptor Antagonists / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D001681:Biological Assay / Q000379:methods; D002417:Cattle; D002738:Chloroquine / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D002829:Choroid / Q000378:metabolism; D017292:Doxazosin / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D066298:In Vitro Techniques; D007498:Iris / Q000378:metabolism; D008543:Melanins / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D055213:Retinal Pigment Epithelium / Q000378:metabolism; D013449:Sulfonamides / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D000077409:Tamsulosin; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D058668:Adrenergic alpha-1 Receptor Antagonists; D008543:Melanins; D013449:Sulfonamides; D002738:Chloroquine; D000077409:Tamsulosin; D017292:Doxazosin