Abstract
We describe a method for assessing tissue-specific plasma free fatty acid (FFA) utilization in vivo using a non-β-oxidizable FFA analog, [9,10-3H]-(R)-2-bromopalmitate (3H-R-BrP). Ideally 3H-R-BrP would be transported in plasma, taken up by tissues and activated by the enzyme acyl-CoA synthetase (ACS) like native FFA, but then 3H-labeled metabolites would be trapped. In vitro we found that 2-bromopalmitate and palmitate compete equivalently for the same ligand binding sites on albumin and intestinal fatty acid binding protein, and activation by ACS was stereoselective for the R-isomer. In vivo, oxidative and non-oxidative FFA metabolism was assessed in anesthetized Wistar rats by infusing, over 4 min, a mixture of 3H-R-BrP and [U-14C] palmitate (14C-palmitate). Indices of total FFA utilization (R*f) and incorporation into storage products (Rfs′) were defined, based on tissue concentrations of 3H and 14C, respectively, 16 min after the start of tracer infusion. R*f, but not Rfs′, was substantially increased in contracting (sciatic nerve stimulated) hindlimb muscles compared with contralateral non-contracting muscles. The contraction-induced increases in R*f were completely prevented by blockade of β-oxidation with etomoxir. These results verify that 3H-R-BrP traces local total FFA utilization, including oxidative and non-oxidative metabolism. Separate estimates of the rates of loss of 3H activity indicated effective 3H metabolite retention in most tissues over a 16-min period, but appeared less effective in liver and heart. In conclusion, simultaneous use of 3H-R-BrP and [14C]palmitate tracers provides a new useful tool for in vivo studies of tissue-specific FFA transport, utilization and metabolic fate, especially in skeletal muscle and adipose tissue.—Oakes, N. D., A. Kjellstedt, G-B. Forsberg, T. Clementz, G. Camejo, S. M. Furler, E. W. Kraegen, M. Ölwegård-Halvarsson, A. B. Jenkins, and B. Ljung. Development and initial evaluation of a novel method for assessing tissue-specific plasma free fatty acid utilization in vivo using (R)-2-bromopalmitate tracer. J. Lipid Res. 1999. 40: 1155–1169.
Highlights
We describe a method for assessing tissue-specific plasma free fatty acid (FFA) utilization in vivo using a non-oxidizable FFA analog, [9,10-3H]-(R)-2-bromopalmitate (3H-R-BrP)
One principle for assessing tissue-specific FFA utilization is based on the use of a labeled fatty acid analog with a structural modification that does not interfere with cellular uptake and metabolic sequestration, but which alters subsequent metabolism to result in local trapping of the label
It has been previously established that intestinal fatty acid binding protein (iFABP) has a single FFA binding site and that dansylamino undecanoic acid (DAUDA) binds to this site [28]
Summary
We describe a method for assessing tissue-specific plasma free fatty acid (FFA) utilization in vivo using a non-oxidizable FFA analog, [9,10-3H]-(R)-2-bromopalmitate (3H-R-BrP). Development and initial evaluation of a novel method for assessing tissue-specific plasma free fatty acid utilization in vivo using (R)-2-bromopalmitate tracer. One principle for assessing tissue-specific FFA utilization is based on the use of a labeled fatty acid analog with a structural modification that does not interfere with cellular uptake and metabolic sequestration, but which alters subsequent metabolism to result in local trapping of the label. Several fatty acid analogs have been designed for the clinical assessment of myocardial metabolism [10,11,12,13,14] These radiolabeled compounds are not readily available, utilize radioisotopes for external imaging, require specialist chemical facilities for synthesis, and have not been extensively evaluated for use in tissues other than myocardium. High specific activity 3H- or 14C-labeled 2bromopalmitate can be produced using commercially available 3H- or 14C-labeled palmitate as starting material
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