Compound-specific isotope analysis resolves the dietary origin of docosahexaenoic acid in the mouse brain

Abstract

DHA (22:6n-3) may be derived from two dietary sources, preformed dietary DHA or through synthesis from α-linolenic acid (ALA; 18:3n-3). However, conventional methods cannot distinguish between DHA derived from either source without the use of costly labeled tracers. In the present study, we demonstrate the proof-of-concept that compound-specific isotope analysis (CSIA) by GC-isotope ratio mass spectrometry (IRMS) can differentiate between sources of brain DHA based on differences in natural ¹³C enrichment. Mice were fed diets containing either purified ALA or DHA as the sole n-3 PUFA. Extracted lipids were analyzed by CSIA for natural abundance ¹³C enrichment. Brain DHA from DHA-fed mice was significantly more enriched (-23.32‰ to -21.92‰) compared with mice on the ALA diet (-28.25‰ to -27.49‰). The measured ¹³C enrichment of brain DHA closely resembled the dietary n-3 PUFA source, -21.86‰ and -28.22‰ for DHA and ALA, respectively. The dietary effect on DHA ¹³C enrichment was similar in liver and blood fractions. Our results demonstrate the effectiveness of CSIA, at natural ¹³C enrichment, to differentiate between the incorporation of preformed or synthesized DHA into the brain and other tissues without the need for tracers.

Keywords: fatty acid; omega-3 fatty acids; stable isotope analysis.

Fig. 1.

GC-IRMS trace of FAMEs derived from a mouse brain total lipid extract. The upper plot shows the signal ratio for m/z 45/44. The lower plot shows the signal for m/z 45 for the duration of the IRMS run. The exploded peak (A) highlights the complete baseline resolution of DHA from surrounding peaks. “STD” indicates signal peaks from the admitted calibrating CO₂ reference gas; >10 pulses of calibrant were admitted prior to each run.

Fig. 2.

Multipoint normalization curve for the reporting of true δ¹³C values for GC-IRMS data. FAME (20-carbon) certified reference materials, USGS70, USGS71, and USGS72, were injected periodically during each programmed sequence, totaling at least three injections per run.

Fig. 3.

DHA tissue concentrations of mice maintained on purified n-3 PUFA-containing diets over multiple generations. DHA concentrations were consistently higher in brain (A), liver (B), serum (C), and RBCs (D) of mice maintained on the DHA diet. All data are mean ± SD (n = 5–6 per group). Data were compared by two-way ANOVA for the interaction of diet and generation; results are presented within each figure. The dashed line represents the mean DHA concentrations of baseline mice; the gray band indicates SD.

Fig. 4.

ARA tissue concentrations of mice maintained on purified n-3 PUFA-containing diets over multiple generations. ARA concentrations were consistently lower in brain (A), liver (B), serum (C), and RBCs (D) of mice maintained on the DHA diet. All data are mean ± SD (n = 5–6 per group). Data were compared by two-way ANOVA for the interaction of diet and generation; results are presented within each figure. The dashed line represents the mean ARA concentrations of baseline mice; the gray band indicates SD.

Fig. 5.

DHA carbon isotope signatures of mice maintained on purified n-3 PUFA-containing diets over multiple generations. Brain (A), liver (B), serum (C), and RBC (D) δ¹³C_DHAs were more isotopically enriched in the DHA-fed animals when compared with the ALA-fed group across all tissues. Each data point represents one animal; solid and dashed lines indicate the mean values for DHA and ALA diets, respectively (n = 5–6 per group). Data were compared by two-way ANOVA for the interaction of diet and generation; results are presented within each figure. A Bonferroni multiple-comparisons test was conducted following a significant (P < 0.05) interaction. ^aA significant difference between diet groups within a generation. ^#A significant difference between generation on the same diet. The upper and lower dotted lines indicate the δ¹³C signature of dietary DHA and ALA, respectively. The dashed line represents the δ¹³C of baseline animals; the gray band indicates ±SD.

Fig. 6.

ARA carbon isotope signatures of mice maintained on purified n-3 PUFA-containing diets over multiple generations. Brain (A), liver (B), serum (C), and RBC (D) δ¹³C_ARAs were found to be similar between dietary groups. Serum δ¹³C_ARA was more enriched in mice fed the DHA diet. Each data point represents one animal; solid and dashed lines indicate the mean values for DHA and ALA diets, respectively (n = 5–6 per group). Data were compared by two-way ANOVA for the interaction of diet and generation; results are presented within each figure. A Bonferonni multiple-comparisons test was conducted following a significant (P < 0.05) interaction. ^aA significant difference between diet groups within a generation. The dotted line indicates the δ¹³C signature of dietary LNA. The dashed line represents the δ¹³C of baseline animals; the gray band indicates ±SD.

Fig. 7.

Carbon isotopic discrimination factors calculated for brain DHA and ARA. Data show the enrichment of ARA and DHA derived from 18-carbon precursors over dietary signatures. Each data point represents one animal; solid and dashed lines indicate the mean values for DHA and ALA diets, respectively (n = 5–6 per group). Data were compared by two-way ANOVA for the interaction of diet and generation; results are presented within each figure. A Bonferroni multiple-comparisons test was conducted following a significant (P < 0.05) interaction. ^aA significant difference between diet groups within a generation. ^#A significant difference between generation on the same diet.