Document Detail


Micro method for determination of nonesterified fatty acid in whole blood obtained by fingertip puncture.
MedLine Citation:
PMID:  16814738     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Jesper S Hansen; Jens K Villadsen; Michael Gaster; Nils J Faergeman; Jens Knudsen
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2006-05-19
Journal Detail:
Title:  Analytical biochemistry     Volume:  355     ISSN:  0003-2697     ISO Abbreviation:  Anal. Biochem.     Publication Date:  2006 Aug 
Date Detail:
Created Date:  2006-07-18     Completed Date:  2006-09-18     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  0370535     Medline TA:  Anal Biochem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  29-38     Citation Subset:  IM    
Affiliation:
Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense M, Denmark.
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MeSH Terms
Descriptor/Qualifier:
Acyl Coenzyme A / metabolism
Animals
Blood Glucose / metabolism
Fatty Acid-Binding Proteins / chemistry,  genetics,  metabolism
Fatty Acids, Nonesterified / analysis,  blood*,  standards
Fingers / blood supply*
Humans
Lysine / chemistry,  genetics,  metabolism
Mutagenesis, Site-Directed
Mutant Proteins / chemistry,  genetics,  metabolism
Palmitic Acid / analysis
Palmitoyl Coenzyme A / analysis
Protein Binding / genetics
Protein Conformation
Protein Structure, Tertiary
Punctures / methods*
Reference Standards
Spectrometry, Fluorescence
Chemical
Reg. No./Substance:
0/Acyl Coenzyme A; 0/Blood Glucose; 0/Fatty Acid-Binding Proteins; 0/Fatty Acids, Nonesterified; 0/Mutant Proteins; 1763-10-6/Palmitoyl Coenzyme A; 56-87-1/Lysine; 57-10-3/Palmitic Acid

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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