title: Quantification of cholesterol solubilized in bile salt micellar aqueous solutions using (13)C nuclear magnetic resonance.
abstract: In this work, we develop a methodology to quantitatively follow the solubilization of cholesterol on glycodeoxycholic acid (GDCA) micelles using (13)C nuclear magnetic resonance (NMR). The amount of solubilized cholesterol enriched in (13)C at position 4, [4-(13)C]cholesterol, was quantified from the area of its resonance, at 44.5 ppm, using the CH(2) groups from GDCA as an internal reference. The loading of the micelles with cholesterol leads to a quantitative upper field shift of most carbons in the nonpolar surface of GDCA, and this was used to follow the solubilization of unlabeled cholesterol. The solubilization followed a pseudo first-order kinetics with a characteristic time constant of 3.6 h, and the maximum solubility of cholesterol in 50 mM total lipid (GDCA + cholesterol) is 3.0 ± 0.1mM, corresponding to a mean occupation number per micelle ≥1. The solubilization profile indicates that the affinity of cholesterol for the GDCA micelles is unaffected by the presence of the solute, leading essentially to full solubilization up to the saturation limit. The relaxation times of GDCA carbons at 50mM give information regarding its aggregation and indicate that GDCA is associated in small micelles (hydrodynamic [Rh] = 1.1 nm) without any evidence for formation of larger secondary micelles. This was confirmed by dynamic light scattering results.
authors.affiliation: Department of Chemistry, University of Coimbra, 3004-535 Coimbra, Portugal.
authors.forename: Filipe M
authors.initials: FM
authors.lastname: Coreta-Gomes
authors.forename: Winchil L C
authors.initials: WL
authors.lastname: Vaz
authors.forename: Emeric
authors.initials: E
authors.lastname: Wasielewski
authors.forename: Carlos F G C
authors.initials: CF
authors.lastname: Geraldes
authors.forename: Maria João
authors.initials: MJ
authors.lastname: Moreno
mesh_terms: D015057:1-Naphthylamine / Q000031:analogs & derivatives / Q000737:chemistry; D001647:Bile Acids and Salts / Q000032:analysis* / Q000737:chemistry; D002244:Carbon / Q000737:chemistry; D002250:Carbon Radioisotopes / Q000737:chemistry; D002784:Cholesterol / Q000032:analysis* / Q000737:chemistry; D006002:Glycodeoxycholic Acid / Q000737:chemistry*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008823:Micelles; D012995:Solubility; D012996:Solutions / Q000737:chemistry*; D014867:Water / Q000737:chemistry
chemical_list: D001647:Bile Acids and Salts; D002250:Carbon Radioisotopes; D008823:Micelles; D012996:Solutions; D014867:Water; D006002:Glycodeoxycholic Acid; D002244:Carbon; C005444:N-phenyl-1-naphthylamine; D015057:1-Naphthylamine; D002784:Cholesterol