Journal article
Micro method for determination of nonesterified fatty acid in whole blood obtained by fingertip puncture
Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense M, Denmark.1
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.
Language: | English |
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Year: | 2006 |
Pages: | 29-38 |
ISSN: | 10960309 and 00032697 |
Types: | Journal article |
DOI: | 10.1016/j.ab.2006.04.054 |
Acyl Coenzyme A Animals Blood Glucose Fatty Acid-Binding Proteins Fatty Acids, Nonesterified Fingers Humans Lysine Mutagenesis, Site-Directed Mutant Proteins Palmitic Acid Palmitoyl Coenzyme A Protein Binding Protein Conformation Protein Structure, Tertiary Punctures Reference Standards Spectrometry, Fluorescence