title: Quantification of total content of non-esterified fatty acids bound to human serum albumin.
abstract: Non-esterified fatty acids bound to the human serum albumin (HSA) contribute to several HSAs properties of special concern in pathologies, for instance to the reactivity of the free HSA-Cys34 thiol group (important antioxidative thiol pool in plasma), and to the affinity for binding of molecules and ions (for example cobalt as a prominent biomarker in heart ischemia). Therefore, the method for determination of FAs bound to HSA was developed. FAs were released from HSA (previously isolated from serum by ammonium sulfate precipitation) using acidic copper(II) sulfate in phosphoric acid, extracted by n-heptane-chloroform (4:1, v/v) mixture, spotted on TL silica-gel and then developed with n-heptane-chloroform-acetic acid (5:3:0.3, v/v/v). Common office flatbed scanner and software solution for densitometric image analysis, developed in R, were used. The linearity of calibration curve in concentration range from 0.1 to 5.0mmol/L stearic acid was achieved. The method was proved to be precise (with RSD of 1.4-4.7%) and accurate. Accuracy was examined by standard addition method (recoveries 97.2-102.5%) and by comparison to results of GC. The method is sample saving, technically less demanding, and cheap, and therefore suitable for determination of FAs/HSA ratio when elevated concentrations of free FAs are reliable diagnostic/risk parameter of pathological states.
authors.affiliation: Department of Biochemistry, Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, Belgrade 11158, Serbia.
authors.forename: Ivan D
authors.initials: ID
authors.lastname: Pavićević
authors.affiliation: Department of Biochemistry, Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, Belgrade 11158, Serbia.
authors.forename: Vesna B
authors.initials: VB
authors.lastname: Jovanović
authors.affiliation: Institute for Medical Research, Laboratory for Nutrition and Metabolism, University of Belgrade, Belgrade, Serbia.
authors.forename: Marija M
authors.initials: MM
authors.lastname: Takić
authors.affiliation: Department of Biochemistry, Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, Belgrade 11158, Serbia.
authors.forename: Jelena M
authors.initials: JM
authors.lastname: Aćimović
authors.affiliation: INEP - Institute for the Application of Nuclear Energy in Agriculture, Veterinary Science and Forestry, University of Belgrade, Belgrade, 381, Serbia.
authors.forename: Ana Z
authors.initials: AZ
authors.lastname: Penezić
authors.affiliation: Department of Biochemistry, Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, Belgrade 11158, Serbia. Electronic address: ljmandic@chem.bg.ac.rs.
authors.forename: Ljuba M
authors.initials: LM
authors.lastname: Mandić
mesh_terms: D002626:Chemistry, Pharmaceutical / Q000379:methods*; D002849:Chromatography, Gas / Q000379:methods; D002855:Chromatography, Thin Layer / Q000379:methods; D018360:Crystallography, X-Ray; D005230:Fatty Acids, Nonesterified / Q000032:analysis* / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding / Q000502:physiology; D017433:Protein Structure, Secondary; D012709:Serum Albumin / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*
chemical_list: D005230:Fatty Acids, Nonesterified; D012709:Serum Albumin
keywords: Fatty acid bound to HSA; Fatty acids/HSA ratio; Flatbed scanner and digital image analysis; TLC quantification