Micro method for determination of nonesterified fatty acid in whole blood obtained by fingertip puncture

Jesper S. Hansen, Jens Kristian Munk, Michael Gaster, Nils J. Faergeman, Jens Knudsen

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

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 mu l 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-dimethylaminoiiaphthalene 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 mu l 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. (c) 2006 Elsevier Inc. All rights reserved.
Original languageEnglish
JournalAnalytical Biochemistry
Volume355
Issue number1
Pages (from-to)29-38
ISSN0003-2697
DOIs
Publication statusPublished - 2006
Externally publishedYes

Keywords

  • 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
  • 1763-10-6 Palmitoyl Coenzyme A
  • 2V16EO95H1 Palmitic Acid
  • K3Z4F929H6 Lysine
  • acyl coenzyme A
  • adenosine triphosphate
  • alcohol
  • binding protein
  • coenzyme A
  • fatty acid
  • long chain fatty acid coenzyme A ligase
  • palmitoyl coenzyme A
  • article
  • blood analysis
  • controlled study
  • covalent bond
  • diagnostic accuracy
  • fatty acid analysis
  • fatty acid blood level
  • fluorescence analysis
  • human
  • incubation time
  • optical biosensor
  • priority journal
  • puncture
  • reproducibility
  • Bos taurus
  • Acyl-CoA binding protein
  • Diagnostics
  • Nonesterified fatty acids
  • Type 2 diabetes
  • BIOCHEMICAL
  • BIOCHEMISTRY
  • CHEMISTRY,
  • PROLIFERATOR-ACTIVATED RECEPTORS
  • CORONARY-HEART-DISEASE
  • BINDING-PROTEIN
  • SKELETAL-MUSCLE
  • ACYL-COENZYME
  • ENZYMATIC DETERMINATION
  • COLORIMETRIC METHOD
  • LIGAND-BINDING
  • RISK FACTOR
  • SERUM
  • diagnostics
  • type 2 diabetes
  • nonesterified fatty acids
  • acyl-CoA binding protein
  • obesity Obesity (MeSH) nutritional disease diagnosis
  • type 2 diabetes Diabetes Mellitus, Non-Insulin-Dependent (MeSH) endocrine disease/pancreas, metabolic disease diagnosis
  • Artiodactyla Mammalia Vertebrata Chordata Animalia (Animals, Artiodactyls, Chordates, Mammals, Nonhuman Vertebrates, Nonhuman Mammals, Vertebrates) - Bovidae [85715] bovine common
  • Primates Mammalia Vertebrata Chordata Animalia (Animals, Chordates, Humans, Mammals, Primates, Vertebrates) - Hominidae [86215] human common
  • 6-bromo-acetyl-dimethylaminonaphthalene
  • acyl-coenzyme A synthetase 9013-18-7
  • ATP 111839-44-2
  • cysteine 3374-22-9
  • FAC1-50
  • lysine 50
  • nonesterified fatty acid biomarker
  • palmitic acid 57-10-3
  • palmitoyl-coenzyme A 1763-10-6
  • 10006, Clinical biochemistry - General methods and applications
  • 10062, Biochemistry studies - Nucleic acids, purines and pyrimidines
  • 10064, Biochemistry studies - Proteins, peptides and amino acids
  • 10066, Biochemistry studies - Lipids
  • 12504, Pathology - Diagnostic
  • 13002, Metabolism - General metabolism and metabolic pathways
  • 13020, Metabolism - Metabolic disorders
  • 13203, Nutrition - Malnutrition and obesity
  • 15002, Blood - Blood and lymph studies
  • 15004, Blood - Blood cell studies
  • 17002, Endocrine - General
  • 17008, Endocrine - Pancreas
  • Allied Medical Sciences
  • Human Medicine, Medical Sciences
  • serum blood and lymphatics
  • whole blood blood and lymphatics
  • micro method laboratory techniques
  • Clinical Chemistry
  • Clinical Endocrinology
  • Metabolism
  • Methods and Techniques

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