Abstract
This study reports methods for the quantitative determination of stable isotope-labeled essential fatty acids (EFAs) as well as an experiment in which deuterium-labeled linoleic acid (18:2n-6) and alpha-linolenic acid (18:3n-3) were compared with those labeled with carbon-13 in rat plasma in vivo. Standard curves were constructed to compensate for concentration and plasma matrix effects. It was observed that endogenous pools of fatty acids had a greater suppressing effect on the measurements of 13C-U-labeled EFAs relative to those labeled with 2H5. Using these methods, the in vivo metabolism of orally administered deuterated-linolenate, 13C-U-labeled linolenate, deuterated-linoleate, and 13C-U-labeled linoleate was compared in adult rats (n = 11). There were no significant differences in the concentrations of the 2H versus 13C isotopomers of 18:2n-6, 18:3n-3, arachidonic acid (20:4n-6), and docosahexaenoic acid (22:6n-3) in rat plasma samples at 24 h after dosing. Thus, there appears to be little isotope effect for 2H5- versus 13C-U-labeled EFAs when the data are calculated using the conventional standard curves and corrected for endogenous fatty acid pool size and matrix effects.
Highlights
This study reports methods for the quantitative determination of stable isotope-labeled essential fatty acids (EFAs) as well as an experiment in which deuterium-labeled linoleic acid (18:2n-6) and ␣-linolenic acid (18:3n-3) were compared with those labeled with carbon-13 in rat plasma in vivo
The effects of the varying concentrations of endogenous fatty acids on 2H5 and 13C ions were quantified by adding a known amount of a pure stable isotope standard (92 pmol of 2H5-18:3n-3, 69 pmol of 13C-U-18:3n-3, 476 pmol of 2H5-18:2n-6, and 476 pmol of 13C-U-18:2n-6) to various volumes of rat plasma (0, 10, 20, 30, 40, and 50 l) with normal saline used to bring the amount up to 50 l
The linear range for each isotope was as follows: 0.01–4.6 pmol for 2H5-18:3n-3, 0.01–6.9 pmol for 13C-U-18:3n-3, 0.005–2.4 pmol for 2H5-18:2n-6, and 0.05–4.8 pmol for 13C-U-18:2n-6. These data clearly demonstrate that the different response factors are dependent upon the concentration of the endogenous fatty acids, which vary in different samples
Summary
This study reports methods for the quantitative determination of stable isotope-labeled essential fatty acids (EFAs) as well as an experiment in which deuterium-labeled linoleic acid (18:2n-6) and ␣-linolenic acid (18:3n-3) were compared with those labeled with carbon-13 in rat plasma in vivo. The exact concentrations of all standard stock solutions, both the labeled and unlabeled fatty acids, were determined by GC analysis. Standard curves for stable isotope-labeled fatty acids.
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