Micro method for determination of nonesterified fatty acid in whole blood obtained by fingertip puncture.
Diagnostic tools for early identification of subjects at high risk for type 2 diabetes and other obesity-related disorders are important in prevention of these diseases. Nonesterified fatty acids (NEFAs) have been suggested to serve as a prediagnostic marker of diabetes and obesity-related disorders. In the current study, we developed a sensitive and reproducible micro method for quantification of NEFA in less than 10 microl whole blood. The method involves only two steps: (i) conversion of NEFA to fatty acid acyl-coenzyme A (acyl-CoA) esters using an acyl-CoA synthetase and (ii) quantification of the formed acyl-CoA esters with a fluorescent biosensor based on bovine acyl-CoA binding protein (ACBP). Lys50 of ACBP was mutagenized to a cysteine residue that was covalently modified with 6-bromoacetyl-2-dimethylaminonaphthalene to make a fluorescent acyl-CoA indicator (FACI-50). FACI-50 exhibits high fluorescence emission yield with maximum at 490 nm in the presence of CoA when excited at 387 nm. The addition of palmitoyl-CoA to a CoA-saturated FACI-50 lowered fluorescence emission by eightfold. Ethanol extract from 1 microl whole blood was incubated with ATP, CoA, and FACI-50. Following background fluorescence reading, NEFAs were converted to acyl-CoA by the acyl-CoA synthetase and the NEFA content was calculated from fluorescence emission changes using palmitic acid as external standard. The FACI-50 NEFA method was compared with two commercially available methods for quantification of NEFA.
Hansen
Jesper S
JS
Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense M, Denmark.
Villadsen
Jens K
JK
Gaster
Michael
M
Faergeman
Nils J
NJ
Knudsen
Jens
J
D000214:Acyl Coenzyme A / Q000378:metabolism; D000818:Animals; D001786:Blood Glucose / Q000378:metabolism; D050556:Fatty Acid-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005230:Fatty Acids, Nonesterified / Q000032:analysis / Q000097:blood* / Q000592:standards; D005385:Fingers / Q000098:blood supply*; D006801:Humans; D008239:Lysine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019308:Palmitic Acid / Q000032:analysis; D010171:Palmitoyl Coenzyme A / Q000032:analysis; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011677:Punctures / Q000379:methods*; D012015:Reference Standards; D013050:Spectrometry, Fluorescence
D000214:Acyl Coenzyme A; D001786:Blood Glucose; D050556:Fatty Acid-Binding Proteins; D005230:Fatty Acids, Nonesterified; D050505:Mutant Proteins; D010171:Palmitoyl Coenzyme A; D019308:Palmitic Acid; D008239:Lysine