Retroconversion is a minor contributor to increases in eicosapentaenoic acid following docosahexaenoic acid feeding as determined by compound specific isotope analysis in rat liver

Abstract

Dietary docosahexaenoic acid (DHA, 22:6n-3) not only increases blood and tissue levels of DHA, but also eicosapentaenoic acid (EPA, 20:5n-3). It is generally believed that this increase is due to DHA retroconversion to EPA, however, a slower conversion of α-linolenic acid (ALA, 18:3n-3) derived EPA to downstream metabolic products (i.e. slower turnover of EPA) is equally plausible. In this study, 21-day old Long Evans rats were weaned onto an ALA only or DHA + ALA diet for 12 weeks. Afterwards, livers were collected and the natural abundance 13C-enrichment was determined by compound specific isotope analysis (CSIA) of liver EPA by isotope ratio mass-spectrometry and compared to dietary ALA and DHA 13C-enrichment. Isotopic signatures (per mil, ‰) for liver EPA were not different (p > 0.05) between the ALA only diet (-25.89 ± 0.39 ‰, mean ± SEM) and the DHA + ALA diet (-26.26 ± 0.40 ‰), suggesting the relative contribution from dietary ALA and DHA to liver EPA did not change. However, with DHA feeding estimates of absolute EPA contribution from ALA increased 4.4-fold (147 ± 22 to 788 ± 153 nmol/g) compared to 3.2-fold from DHA (91 ± 14 to 382 ± 13 nmol/g), respectively. In conclusion, CSIA of liver EPA in rats following 12-weeks of dietary DHA suggests that retroconversion of DHA to EPA is a relatively small contributor to increases in EPA, and that this increase in EPA is largely coming from elongation/desaturation of ALA.

Keywords: Alpha-linolenic acid; Docosahexaenoic acid; Eicosapentaenoic acid; Liver; Metabolism; Retroconversion.

Conflict of interest statement

Ethics approval

All experimental procedures were performed in agreement with the policies set out by the Canadian Council on Animal Care and were approved by the Animal Ethics Committee at the University of Toronto (Protocol # – 20011797).

Consent for publication

Not applicable.

Competing interests

Richard P. Bazinet (RPB) has received research grants from Bunge Ltd., Arctic Nutrition, the Dairy Farmers of Canada and Nestle Inc., as well as travel support from Mead Johnson and mass spectrometry equipment and support from Sciex. In addition, RPB is on the executive of the International Society for the Study of Fatty Acids and Lipids and held a meeting on behalf of Fatty Acids and Cell Signaling, both of which rely on corporate sponsorship. RPB has given expert testimony in relation to supplements and the brain. RPB also provides complimentary fatty acid analysis for farmers, food producers and others involved in the food industry, some of whom provide free food samples. There are no other competing interests.

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Figures

Fig. 1

Liver concentrations of (a) eicosapentaenoic acid and (b) arachidonic acid following 12 weeks ingestion of 2% ALA or 2% DHA + 2% ALA diet in male Long Evans rats. * – represents statistically significant liver concentration between dietary groups as determined by significant Student’s t-test, p < 0.05. N = 6, mean ± SEM. ALA – α-linolenic acid, 18:3n-3; ARA – arachidonic acid, 20:4n-6; DHA – docosahexaenoic acid, 22:6n-3; EPA – eicosapentaenoic acid, 20:5n-3

Fig. 2

Liver carbon isotope signatures for eicosapentaenoic acid and arachidonic acid in male Long Evans rats following 12 weeks ingestion of 2% ALA or 2% DHA + 2% ALA diet in male Long Evans rats. * – represents significantly different liver carbon isotope signatures between dietary groups as determined by significant Student’s t-test, p < 0.05. N = 6, mean ± SEM. ALA – α-linolenic acid, 18:3n-3; ARA – arachidonic acid, 20:4n-6; DHA – docosahexaenoic acid, 22:6n-3; EPA – eicosapentaenoic acid, 20:5n-3; VPDB – Vienna Peedee Belemnite

Fig. 3

Estimates of (a) percent of liver EPA derived from ALA, (b) absolute concentration of liver EPA from ALA and DHA sources, (c) percent of liver ARA derived from LNA and (d) absolute concentration of liver ARA derived from LNA and other sources. * - represents significant differences between diets for proportion of liver EPA from ALA (a) and ARA from LNA (b) as determined by Student’s t-test. * - represents significant differences between diets by Student’s t-test and # represents significant differences between sources of EPA and ARA by paired t-test following significant interaction effect by two-way ANOVA (c and d), p < 0.05. N = 6, mean ± SEM. c Interaction = 0.036, Diet <0.0001, Source = 0.008; d Interaction = 0.001, Diet = 0.07, Source <0.0001. ALA – α-linolenic acid, 18:3n-3; ARA – arachidonic acid, 20:4n-6; DHA – docosahexaenoic acid, 22:6n-3; EPA, eicosapetaenoic acid, 20:5n-3; LNA – linoleic acid, 18:2n-6

Fig. 4

Representation of theoretical carbon flow influencing 13C enrichment of EPA in the ALA + DHA diet. [1] Elongation and desaturation of dietary ALA (−28.22‰), [2] retroconversion of dietary DHA (−22.30‰), [3] 2 carbons via acetyl-CoA provided from glycolysis (−12 to −16‰) for elongation of ALA, [4] slower elongation/desaturation of EPA to DHA, [5] preferential β-oxidation of lighter 12C isotopes from EPA and additional EPA metbaolism. ALA – α-linolenic acid, 18:3n-3; DHA – docosahexaenoic acid, 22:6n-3; EPA – eicosapentaenoic acid, 20:5n-3