Dietary docosahexaenoic acid (DHA) downregulates liver DHA synthesis by inhibiting eicosapentaenoic acid elongation.

DHA is abundant in the brain where it regulates cell survival, neurogenesis, and neuroinflammation. DHA can be obtained from the diet or synthesized from alpha-linolenic acid (ALA; 18:3n-3) via a series of desaturation and elongation reactions occurring in the liver. Tracer studies suggest that dietary DHA can downregulate its own synthesis, but the mechanism remains undetermined and is the primary objective of this manuscript. First, we show by tracing 13C content (δ13C) of DHA via compound-specific isotope analysis, that following low dietary DHA, the brain receives DHA synthesized from ALA. We then show that dietary DHA increases mouse liver and serum EPA, which is dependant on ALA. Furthermore, by compound-specific isotope analysis we demonstrate that the source of increased EPA is slowed EPA metabolism, not increased DHA retroconversion as previously assumed. DHA feeding alone or with ALA lowered liver elongation of very long chain (ELOVL2, EPA elongation) enzyme activity despite no change in protein content. To further evaluate the role of ELOVL2, a liver-specific Elovl2 KO was generated showing that DHA feeding in the presence or absence of a functional liver ELOVL2 yields similar results. An enzyme competition assay for EPA elongation suggests both uncompetitive and noncompetitive inhibition by DHA depending on DHA levels. To translate our findings, we show that DHA supplementation in men and women increases EPA levels in a manner dependent on a SNP (rs953413) in the ELOVL2 gene. In conclusion, we identify a novel feedback inhibition pathway where dietary DHA downregulates its liver synthesis by inhibiting EPA elongation.

Novel 13C enrichment technique reveals early turnover of DHA in peripheral tissues.

The brain is rich in DHA, which plays important roles in regulating neuronal function. Recently, using compound-specific isotope analysis that takes advantage of natural differences in carbon-13 content (13C/12C ratio or δ13C) of the food supply, we determined the brain DHA half-life. However, because of methodological limitations, we were unable to capture DHA turnover rates in peripheral tissues. In the current study, we applied compound-specific isotope analysis via high-precision GC combustion isotope ratio mass spectrometry to determine half-lives of brain, liver, and plasma DHA in mice following a dietary switch experiment. To model DHA tissue turnover rates in peripheral tissues, we added earlier time points within the diet switch study and took advantage of natural variations in the δ13C-DHA of algal and fish DHA sources to maintain DHA pool sizes and used an enriched (uniformly labeled 13C) DHA treatment. Mice were fed a fish-DHA diet (control) for 3 months, then switched to an algal-DHA treatment diet, the 13C enriched-DHA treatment diet, or they stayed on the control diet for the remainder of the study time course. In mice fed the algal and 13C enriched-DHA diets, the brain DHA half-life was 47 and 46 days, the liver half-life was 5.6 and 7.2 days, and the plasma half-life was 4.7 and 6.4 days, respectively. By using improved methodologies, we calculated DHA turnover rates in the liver and plasma, and our study for the first time, by using an enriched DHA source (very high δ13C), validated its utility in diet switch studies.

Analysis of omega-3 and omega-6 polyunsaturated fatty acid metabolism by compound-specific isotope analysis in humans.

Natural variations in the 13C:12C ratio (carbon-13 isotopic abundance [δ13C]) of the food supply have been used to determine the dietary origin and metabolism of fatty acids, especially in the n-3 PUFA biosynthesis pathway. However, n-6 PUFA metabolism following linoleic acid (LNA) intake remains under investigation. Here, we sought to use natural variations in the δ13C signature of dietary oils and fatty fish to analyze n-3 and n-6 PUFA metabolism following dietary changes in LNA and eicosapentaenoic acid (EPA) + DHA in adult humans. Participants with migraine (aged 38.6 ± 2.3 years, 93% female, body mass index of 27.0 ± 1.1 kg/m2) were randomly assigned to one of three dietary groups for 16 weeks: 1) low omega-3, high omega-6 (H6), 2) high omega-3, high omega-6 (H3H6), or 3) high omega-3, low omega-6 (H3). Blood was collected at baseline, 4, 10, and 16 weeks. Plasma PUFA concentrations and δ13C were determined. The H6 intervention exhibited increases in plasma LNA δ13C signature over time; meanwhile, plasma LNA concentrations were unchanged. No changes in plasma arachidonic acid δ13C or concentration were observed. Participants on the H3H6 and H3 interventions demonstrated increases in plasma EPA and DHA concentration over time. Plasma δ13C-EPA increased in total lipids of the H3 group and phospholipids of the H3H6 group compared with baseline. Compound-specific isotope analysis supports a tracer-free technique that can track metabolism of dietary fatty acids in humans, provided that the isotopic signature of the dietary source is sufficiently different from plasma δ13C.

The majority of brain palmitic acid is maintained by lipogenesis from dietary sugars and is augmented in mice fed low palmitic acid levels from birth.

Previously, results from studies investigating if brain palmitic acid (16:0; PAM) was maintained by either dietary uptake or de novo lipogenesis (DNL) varied. Here, we utilize naturally occurring carbon isotope ratios (13 C/12 C; δ13 C) to uncover the origin of brain PAM. Additionally, we explored brain and liver fatty acid concentration, brain metabolomics, and behavior. BALB/c dams were equilibrated onto either a low PAM diet (LP; <2%) or high PAM diet (HP; >95%) prior to producing one generation of offspring. Offspring stayed on the respective diet of the dam until 15-weeks of age, at which time the Open Field test was conducted, prior to euthanasia and tissue lipid extraction. Although liver PAM was lower in mice fed the LP diet, as well as female mice, brain PAM was not affected by diet or sex. Across mice of either sex on both diets, brain 13 C-PAM revealed compared to dietary uptake, DNL from dietary sugars contributed 68.8%-79.5% and 46.6%-58.0% to the total brain PAM pool by both peripheral and local brain DNL, and local brain DNL alone, respectively. DNL was augmented in mice fed the LP diet, and the ability to up-regulate DNL in the liver or the brain depended on sex. Anxiety-like behaviors were decreased in mice fed the LP diet and were correlated with markers of LP diet consumption including increased liver 13 C-PAM, warranting further investigation. Altogether, our results indicate that DNL from dietary sugars is a compensatory mechanism to maintain brain PAM in response to the LP diet.

Tetracosahexaenoylethanolamide, a novel N-acylethanolamide, is elevated in ischemia and increases neuronal output.

N-acylethanolamines (NAEs) are endogenous lipid-signaling molecules derived from fatty acids that regulate numerous biological functions, including in the brain. Interestingly, NAEs are elevated in the absence of fatty acid amide hydrolase (FAAH) and following CO2-induced ischemia/hypercapnia, suggesting a neuroprotective response. Tetracosahexaenoic acid (THA) is a product and precursor to DHA; however, the NAE product, tetracosahexaenoylethanolamide (THEA), has never been reported. Presently, THEA was chemically synthesized as an authentic standard to confirm THEA presence in biological tissues. Whole brains were collected and analyzed for unesterified THA, total THA, and THEA in wild-type and FAAH-KO mice that were euthanized by either head-focused microwave fixation, CO2 + microwave, or CO2 only. PPAR activity by transient transfection assay and ex vivo neuronal output in medium spiny neurons (MSNs) of the nucleus accumbens by patch clamp electrophysiology were determined following THEA exposure. THEA in the wild-type mice was nearly doubled (P < 0.05) following ischemia/hypercapnia (CO2 euthanization) and up to 12 times higher (P < 0.001) in the FAAH-KO compared with wild-type. THEA did not increase (P > 0.05) transcriptional activity of PPARs relative to control, but 100 nM of THEA increased (P < 0.001) neuronal output in MSNs of the nucleus accumbens. Here were identify a novel NAE, THEA, in the brain that is elevated upon ischemia/hypercapnia and by KO of the FAAH enzyme. While THEA did not activate PPAR, it augmented the excitability of MSNs in the nucleus accumbens. Overall, our results suggest that THEA is a novel NAE that is produced in the brain upon ischemia/hypercapnia and regulates neuronal excitation.

Brain eicosapentaenoic acid metabolism as a lead for novel therapeutics in major depression.

The results of several meta-analyses suggest that eicosapentaenoic acid (EPA) supplementation is therapeutic in managing the symptoms of major depression. It was previously assumed that because EPA is extremely low in the brain it did not cross the blood-brain barrier and any therapeutic effects it exerted would be via the periphery. However, more recent studies have established that EPA does enter the brain, but is rapidly metabolised following entry. While EPA does not accumulate within the brain, it is present in microglia and homeostatic mechanisms may regulate its esterification to phospholipids that serve important roles in cell signaling. Furthermore, a variety of signaling molecules from EPA have been described in the periphery and they have the potential to exert effects within the brain. If EPA is confirmed to be therapeutic in major depression as a result of adequately powered randomized clinical trials, future research on brain EPA metabolism could lead to the discovery of novel targets for treating or preventing major depression.

Docosahexaenoic acid is both a product of and a precursor to tetracosahexaenoic acid in the rat.

Tetracosahexaeoic acid (THA; 24:6n-3) is thought to be the immediate precursor of DHA in rodents; however, the relationship between THA and DHA metabolism has not been assessed in vivo. Here, we infused unesterified 2H5-THA and 13C22-DHA, at a steady state, into two groups of male Long-Evans rats and determined the synthesis-secretion kinetics, including daily synthesis-secretion rates of all 20-24 carbon n-3 PUFAs. We determined that the synthesis-secretion coefficient (a measure of the capacity to synthesize a given fatty acid) for the synthesis of DHA from plasma unesterified THA to be 134-fold higher than for THA from DHA. However, when considering the significantly higher endogenous plasma unesterified DHA pool, the daily synthesis-secretion rates were only 7-fold higher for DHA synthesis from THA (96.3 ± 31.3 nmol/d) compared with that for THA synthesis from DHA (11.4 ± 4.1 nmol/d). Furthermore, plasma unesterified THA was converted to DHA and secreted into the plasma at a 2.5-fold faster rate than remaining as THA itself (26.2 ± 6.3 nmol/d), supporting THA's primary role as a precursor to DHA. In conclusion, using a 3 h infusion model in rats, we demonstrate for the first time in vivo that DHA is both a product and a precursor to THA.

Brain oxylipin concentrations following hypercapnia/ischemia: effects of brain dissection and dissection time.

PUFAs are precursors to bioactive oxylipin metabolites that increase in the brain following CO2-induced hypercapnia/ischemia. It is not known whether the brain-dissection process and its duration also alter these metabolites. We applied CO2 with or without head-focused microwave fixation for 2 min to evaluate the effects of CO2-induced asphyxiation, dissection, and dissection time on brain oxylipin concentrations. Compared with head-focused microwave fixation (control), CO2 followed by microwave fixation prior to dissection increased oxylipins derived from lipoxygenase (LOX), 15-hydroxyprostaglandin dehydrogenase (PGDH), cytochrome P450 (CYP), and soluble epoxide hydrolase (sEH) enzymatic pathways. This effect was enhanced when the duration of postmortem ischemia was prolonged by 6.4 min prior to microwave fixation. Brains dissected from rats subjected to CO2 without microwave fixation showed greater increases in LOX, PGDH, CYP and sEH metabolites compared with all other groups, as well as increased cyclooxygenase metabolites. In nonmicrowave-irradiated brains, sEH metabolites and one CYP metabolite correlated positively and negatively with dissection time, respectively. This study presents new evidence that the dissection process and its duration increase brain oxylipin concentrations, and that this is preventable by microwave fixation. When microwave fixation is not available, lipidomic studies should account for dissection time to reduce these artifacts.

DHA Cycling Halves the DHA Supplementation Needed to Maintain Blood and Tissue Concentrations via Higher Synthesis from ALA in Long-Evans Rats.

BACKGROUND: Eicosapentaenoic acid (EPA) plus docosahexaenoic acid (DHA) recommendations are frequently stated at 500 mg/d; however, adherence to these recommendations would result in a large global commercial EPA/DHA production deficit. Previously, our laboratory demonstrated that acute DHA intake in rats can increase the capacity for synthesis-secretion of n-3 (ω-3) polyunsaturated fatty acids (PUFAs). OBJECTIVE: We aimed to investigate the utility of a dietary DHA cycling strategy that employs 2 wk of repeated DHA feeding for a total of 3 cycles over 12 wk. METHODS: Male Long-Evans rats were fed a 10% fat diet by weight comprised of either 1) a 2-wk, 2% α-linolenic acid (ALA, DHA-ALA group 18:3n-3) diet followed by a 2-wk, 2% DHA + 2% ALA diet over 3 consecutive 4-wk periods ("DHA cycling," DHA-ALA group); 2) a 2% DHA + 2% ALA diet (DHA group) for 12 wk; or 3) a 2% ALA-only diet (ALA group) for 12 wk. At 15 wk old, blood and tissue fatty acid concentrations and liver mRNA expression and 13C-DHA natural abundances were determined. RESULTS: DHA concentrations in plasma, erythrocytes, and whole blood between the DHA-ALA group and the DHA groups were not different (P ≥ 0.05), but were 72-110% higher (P < 0.05) than in the ALA group. Similarly, DHA concentrations in liver, heart, adipose, and brain were not different (P ≥ 0.05) between the DHA-fed groups, but were at least 62%, 72%, 320%, and 68% higher (P < 0.05) than in the ALA group in liver, heart, adipose, and skeletal muscle, respectively. Compound-specific isotope analysis indicated that 310% more liver DHA in the DHA-ALA group compared with the DHA group is derived from dietary ALA, and this was accompanied by a 123% and 93% higher expression of elongation of very long-chain (Elovl)2 and Elovl5, respectively, in the DHA-ALA group compared with the ALA group. CONCLUSIONS: DHA cycling requires half the dietary DHA while achieving equal blood and tissue DHA concentrations in rats. Implementation of such dietary strategies in humans could reduce the gap between global dietary n-3 PUFA recommendations and commercial production.

Complete assessment of whole-body n-3 and n-6 PUFA synthesis-secretion kinetics and DHA turnover in a rodent model.

Previous assessments of the PUFA biosynthesis pathway have focused on DHA and arachidonic acid synthesis. Here, we determined whole-body synthesis-secretion kinetics for all downstream products of PUFA metabolism, including direct measurements of DHA and n-6 docosapentaenoic acid (DPAn-6, 22:5n-6) turnover, and compared n-6 and n-3 homolog kinetics. We infused labeled α-linolenic acid (ALA, 18:3n-3), linoleic acid (LNA, 18:2n-6), DHA, and DPAn-6 as 2H5-ALA, 13C18-LNA, 13C22-DHA, and 13C22-DPAn-6. Eight 11-week-old Long Evans rats fed a 10% fat diet were infused with the labeled PUFAs over 3 h, and plasma enrichment of labeled products was measured every 30 min. The DHA synthesis-secretion rate (94 ± 34 nmol/day) did not differ from other PUFA products (range, 21.8 ± 4.3 nmol/day to 408 ± 116 nmol/day). Synthesis-secretion rates of n-6 and n-3 PUFA homologs were similar, except 22:4n-6 and DPAn-6 had lower synthesis rates. However, daily turnover from newly synthesized DHA (0.067 ± 0.023%) was 56-fold to 556-fold slower than all other PUFA turnover and was 130-fold slower than that determined directly from the total plasma unesterified DHA pool. In conclusion, n-6 and n-3 PUFA synthesis-secretion kinetics suggest that differences in turnover, not in synthesis-secretion rates, primarily determine PUFA plasma levels.

Whole-body DHA synthesis-secretion kinetics from plasma eicosapentaenoic acid and alpha-linolenic acid in the free-living rat.

Whole body docosahexaenoic acid (DHA, 22:6n-3) synthesis from α-linolenic acid (ALA, 18:3n-3) is considered to be very low, however, the daily synthesis-secretion of DHA may be sufficient to supply the adult brain. The current study aims to assess whether whole body DHA synthesis-secretion kinetics are different when comparing plasma ALA versus eicosapentaenoic acid (EPA, 20:5n-3) as the precursor. Male Long Evans rats (n=6) were fed a 2% ALA in total fat diet for eight weeks, followed by surgery to implant a catheter into each of the jugular vein and carotid artery and 3h of steady-state infusion with a known amount of (2)H-ALA and (13)C-eicosapentaenoic acid (EPA, 20:5n3). Blood samples were collected at thirty-minute intervals and plasma enrichment of (2)H- and (13)C EPA, n-3 docosapentaenoic acid (DPAn-3, 22:5n-3) and DHA were determined for assessment of synthesis-secretion kinetic parameters. Results indicate a 13-fold higher synthesis-secretion coefficient for DHA from EPA as compared to ALA. However, after correcting for the 6.6 fold higher endogenous plasma ALA concentration, no significant differences in daily synthesis-secretion (nmol/day) of DHA (97.6±28.2 and 172±62), DPAn-3 (853±279 and 1139±484) or EPA (1587±592 and 1628±366) were observed from plasma unesterified ALA and EPA sources, respectively. These results suggest that typical diets which are significantly higher in ALA compared to EPA yield similar daily DHA synthesis-secretion despite a significantly higher synthesis-secretion coefficient from EPA.

Fatty acid amide hydrolase (FAAH) regulates hypercapnia/ischemia-induced increases in n-acylethanolamines in mouse brain.

N-acylethanolamines (NAEs) are endogenous lipid ligands for several receptors including cannabinoid receptors and peroxisome proliferator-activated receptor-alpha (PPAR-α), which regulate numerous physiological functions. Fatty acid amide hydrolase (FAAH) is largely responsible for the degradation of NAEs. However, at high concentrations of ethanolamines and unesterified fatty acids, FAAH can also catalyze the reverse reaction, producing NAEs. Several brain insults such as ischemia and hypoxia increase brain unesterified fatty acids. Because FAAH can catalyze the synthesis of NAE, we aimed to test whether FAAH was necessary for CO2 -induced hypercapnia/ischemia increases in NAE. To test this, we examined levels of NAEs, 1- and 2-arachidonoylglycerols as well as their corresponding fatty acid precursors in wild-type and mice lacking FAAH (FAAH-KO) with three Kill methods: (i) head-focused, high-energy microwave irradiation (microwave), (ii) 5 min CO2 followed by microwave irradiation (CO2 + microwave), and (iii) 5 min CO2 only (CO2 ). Both CO2 -induced groups increased, to a similar extent, brain levels of unesterified oleic, arachidonic, and docosahexaenoic acid and 1- and 2-arachidonoylglycerols compared to the microwave group in both wild-type and FAAH-KO mice. Oleoylethanolamide (OEA), arachidonoylethanolamide (AEA), and docosahexaenoylethanolamide (DHEA) levels were about 8-, 7-, and 2.5-fold higher, respectively, in the FAAH-KO mice compared with the wild-type mice. Interestingly, the concentrations of OEA, AEA, and DHEA increased 2.5- to 4-fold in response to both CO2 -induced groups in wild-type mice, but DHEA increased only in the CO2 group in FAAH-KO mice. Our study demonstrates that FAAH is necessary for CO2 - induced increases in OEA and AEA but not DHEA. Targeting brain FAAH could impair the production of NAEs in response to brain injuries.

Phospholipid class-specific brain enrichment in response to lysophosphatidylcholine docosahexaenoic acid infusion.

Recent studies suggest that at least two pools of plasma docosahexaenoic acid (DHA) can supply the brain: non-esterified DHA (NE-DHA) and lysophosphatidylcholine (lysoPtdCho)-DHA. In contrast to NE-DHA, brain uptake of lysoPtdCho-DHA appears to be mediated by a specific transporter, but whether both forms of DHA supply undergo the same metabolic fate, particularly with regards to enrichment of specific phospholipid (PL) subclasses, remains to be determined. This study aimed to evaluate brain uptake of NE-DHA and lysoPtdCho-DHA into brain PL classes. Fifteen-week-old rats were infused intravenously with radiolabelled NE-14C-DHA or lysoPtdCho-14C-DHA (n=4/group) over five mins to achieve a steady-state plasma level. PLs were extracted from the brain and separated by thin layer chromatography and radioactivity was quantified by liquid scintillation counting. The net rate of entry of lysoPtdCho-DHA into the brain was between 59% and 86% lower than the net rate of entry of NE-DHA, depending on the PL class. The proportion of total PL radioactivity in the lysoPtdCho-14C-DHA group compared to the NE-14C-DHA group was significantly higher in choline glycerophospholipids (ChoGpl) (48% vs 28%, respectively) but lower in ethanolamine glycerophospholipids (EtnGpl) (32% vs 46%, respectively). In both groups, radioactivity was disproportionally high in phosphatidylinositol and ChoGpl but low in phosphatidylserine and EtnGpl compared to the corresponding DHA pool size. This suggests that DHA undergoes extensive PL remodeling after entry into the brain.

Whole-Body Docosahexaenoic Acid Synthesis-Secretion Rates in Rats Are Constant across a Large Range of Dietary α-Linolenic Acid Intakes.

BACKGROUND: Docosahexaenoic acid (DHA) is an ω-3 (n-3) polyunsaturated fatty acid (PUFA) thought to be important for brain function. Although the main dietary source of DHA is fish, DHA can also be synthesized from α-linolenic acid (ALA), which is derived from plants. Enzymes involved in DHA synthesis are also active toward ω-6 (n-6) PUFAs to synthesize docosapentaenoic acid n-6 (DPAn-6). It is unclear whether DHA synthesis from ALA is sufficient to maintain brain DHA. OBJECTIVE: The objective of this study was to determine how different amounts of dietary ALA would affect whole-body DHA and DPAn-6 synthesis rates. METHODS: Male Long-Evans rats were fed an ALA-deficient diet (ALA-D), an ALA-adequate (ALA-A) diet, or a high-ALA (ALA-H) diet for 8 wk from weaning. Dietary ALA concentrations were 0.07%, 3%, and 10% of the fatty acids, and ALA was the only dietary PUFA that differed between the diets. After 8 wk, steady-state stable isotope infusion of labeled ALA and linoleic acid (LA) was performed to determine the in vivo synthesis-secretion rates of DHA and DPAn-6. RESULTS: Rats fed the ALA-A diet had an ∼2-fold greater capacity to synthesize DHA than did rats fed the ALA-H and ALA-D diets, and a DHA synthesis rate that was similar to that of rats fed the ALA-H diet. However, rats fed the ALA-D diet had a 750% lower DHA synthesis rate than rats fed the ALA-A and ALA-H diets. Despite enrichment into arachidonic acid, we did not detect any labeled LA appearing as DPAn-6. CONCLUSIONS: Increasing dietary ALA from 3% to 10% of fatty acids did not increase DHA synthesis rates, because of a decreased capacity to synthesize DHA in rats fed the ALA-H diet. Tissue concentrations of DPAn-6 may be explained at least in part by longer plasma half-lives.

Neuromuscular adaptations to sprint interval training and the effect of mammalian omega-3 fatty acid supplementation.

PURPOSE: Sprint interval training (SIT) stimulates rapid metabolic adaptations within skeletal muscle but the nature of neuromuscular adaptions is unknown. Omega-3 polyunsaturated fatty acids (N-3 PUFA) are suggested to enhance neuromuscular adaptations to exercise. METHODS: We measured the neuromuscular adaptations to SIT (Study-1) and conducted a placebo-controlled randomized double blinded study to determine the effect of N-3 PUFA supplementation on neuromuscular adaptations to SIT (Study-2). In Study-1, seven active men (24.4 ± 2.6 years, VO2 peak 43.8 ± 8.7 ml kg min-1) completed 2-weeks of SIT with pre- and post-training 10 km cycling time trials (TT). In Study-2, 30 active men (24.5 ± 4.2 years, VO2 peak 41.0 ± 5.1 ml kg min-1) were randomly assigned to receive N-3 PUFA (2330 mg day-1) (n = 14) or olive oil (n = 16) during 2-weeks of SIT with pre- and post-training TTs. Four week post-training, a SIT session and TT were also performed. Change in neuromuscular function was assessed from resting twitches, quadriceps maximal voluntary contraction (MVC) force, and potentiated twitch force (Q tw). RESULTS: Study-1 showed that SIT did not elicit significant neuromuscular adaptations. Study-2 showed that N-3 PUFA supplementation had no significant effect on neuromuscular adaptations. Training caused lower MVC force [mean ± SD; N-3 PUFA -9 ± 11%, placebo -9 ± 13% (p < 0.05 time)] and Q tw peripheral fatigue [N-3 PUFA -10 ± 19%, placebo -14 ± 13% (p < 0.05 time)]. TT time was lower after training in all groups [Study-1 -10%, Study-2 N-3 PUFA -8%, placebo -12% (p < 0.05 time)]. CONCLUSION: Two weeks of SIT improved TT performance in the absence of measurable neuromuscular adaptations. N-3 PUFA supplementation had no significant effect on SIT training adaptations.

Tailored Extraction Procedure Is Required To Ensure Recovery of the Main Lipid Classes in Whole Blood When Profiling the Lipidome of Dried Blood Spots.

The use of dried blood spots has increased in research and clinical settings recently, particularly in field studies and screening, but comprehensive acyl-specific lipidomic profiling of dried blood spots has yet to be examined. An untargeted ultrahigh-performance liquid chromatography-tandem mass spectrometry method was adapted for the analysis of lipid extracts from human whole blood samples and dried blood spots collected on chromatography paper. Lipid recoveries were examined after different durations of exposure to extraction solvents (chloroform/methanol), physical disruption (homogenization or sonication) of the paper containing the dried blood spots, and acidification of extraction solvents. We demonstrated that comprehensive untargeted profiles can be obtained from dried blood spot samples that are comparable with whole blood for several species of lipids including phosphatidylcholine, lyso-phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, triacylglycerol, and cholesteryl ester. However, homogenization of the dried blood spots, followed by a 24 h exposure to solvents, and extraction with an acidic buffer (0.2 M NaHPO4 + 0.1 M hydrochloric acid) was required. Dried blood spots can be used for comprehensive, untargeted lipidomics of the most abundant lipid species in whole blood, but additional sample processing steps are required.

Sarcoplasmic Reticulum Phospholipid Fatty Acid Composition and Sarcolipin Content in Rat Skeletal Muscle.

In a previous study, we reported lower sarcoplasmic reticulum (SR) Ca(2+) pump ionophore ratios in rat soleus compared to red and white gastrocnemius (RG, WG) muscles which may be indicative of greater SR Ca(2+) permeability in soleus. Here we assessed the lipid composition of the SR membranes obtained from these muscles to determine if SR docosahexaenoic acid (DHA) content and fatty acid unsaturation could help to explain the previously observed differences in SR Ca(2+) permeability. Since we have shown previously that sarcolipin may also influence SR Ca(2+) permeability, we also examined the levels of sarcolipin in rat muscle. We found that SR membrane DHA content was significantly higher in soleus (5.3 ± 0.2 %) compared to RG (4.2 ± 0.2 %) and WG (3.3 ± 0.2 %). Likewise, total SR membrane unsaturation and unsaturation index (UI) were significantly higher in soleus (% unsaturation: 59.1 ± 2.4; UI: 362.9 ± 0.8) compared to RG (% unsaturation: 55.3 ± 1.0; UI: 320.9 ± 2.5) and WG (% unsaturation: 52.6 ± 1.1; UI: 310. ± 2.2). Sarcolipin protein was 17-fold more abundant in rat soleus compared to RG and was not detected in WG; however, comparisons between soleus, RG, and WG in sarcolipin-null mice revealed that, in the absence of sarcolipin, ionophore ratios are still lowest in soleus and highest in WG. Overall, our results suggest that SR membrane DHA content and unsaturation, and, in part, sarcolipin expression may contribute to SR Ca(2+) permeability and, in turn, may have implications in muscle-based metabolism and diet-induced obesity.

Cryopreservation prevents iron-initiated highly unsaturated fatty acid loss during storage of human blood on chromatography paper at -20°C.

BACKGROUND: Fingertip prick whole blood collection on chromatography paper is amenable to high-throughput fatty acid (FA) profiling for large clinical and field studies. However, sample storage is problematic because highly unsaturated FAs (HUFAs) in erythrocytes rapidly degrade in samples stored at -20°C. OBJECTIVE: The aim of the current study was to determine the mechanism of HUFA degradation and to develop prevention protocols. METHODS: Free fatty acid (FFA) standards and whole blood reference material from a single participant were used to examine sample storage at -20°C for up to 90 d in triplicate. Iron chelation with deferoxamine (0-5000 µg), antioxidant protection with butylated hydroxytoluene (50 µg), cryopreservation with glycerol, and blood drying were examined using whole blood on chromatography strips. Biological replicate blood samples from additional participants (n = 6) with a range of ω-3 (n-3) HUFA concentrations were similarly assessed. RESULTS: FFAs were relatively stable when stored on chromatography strips at -20°C. Glycerol treatment prevented HUFA degradation in whole blood reference material for 30 d (45 ± 0.4 to 46.8 ± 0.1, means ± SDs) compared to untreated saline controls (45.9 ± 1.0 to 6.8 ± 0.2). Pretreatment of paper for blood spots with deferoxamine and drying blood before storage slowed, but not entirely prevented, HUFA degradation over 30 d to 22% and 19% below baseline, respectively, compared to 86-92% in the controls. Protection against HUFA degradation with blood drying and glycerol treatment was confirmed in the biological replicate study and confirmed by prevention of cell lysis. CONCLUSIONS: HUFA degradation during storage at -20°C appears to be due to hemolysis and subsequent iron-initiated peroxidation. This degradation may be prevented by glycerol, iron chelation, and/or dried blood spotting. A more thorough understanding of methods to prevent degradation during storage is critical with increasing use of FA profiling in large clinical studies.

The percentage of DHA in erythrocytes can detect non-adherence to advice to increase EPA and DHA intakes.

Characterisation of long-term adherence to EPA and DHA intakes through biomarkers and dietary assessments has implications for interpreting the findings of long-term intervention studies. Adherence to dietary advice targeting an EPA+DHA intake of 1 g/d was examined over 1 year. Men and women (n 45) received dietary advice to increase EPA and DHA intakes from seafood, nutraceutical (fish oil) or functional food sources, while a fourth group received combined advice. Blood biomarkers and dietary intakes of EPA and DHA were evaluated at baseline and post-intervention at weeks 4, 8, 12, 24 and 52. Assessment by 3 d diet records indicated that EPA+DHA intakes increased relative to baseline in weeks 4-52 following the seafood, nutraceutical and combined advice (advice group × time effect, P= 0·03). The percentage of DHA in plasma and whole blood and the percentage of EPA in erythrocytes, plasma and whole blood were higher in weeks 4-52 when compared with the corresponding baseline measurement. In contrast, the percentage of DHA in erythrocytes increased to a maximum at week 12 and returned to baseline levels in weeks 24 and 52 (time effect, P< 0·01). Measurement of the percentage of DHA in erythrocytes indicates that adherence was sustained during the first 12 weeks following the dietary advice, while other blood measurements of the percentage of EPA and DHA and dietary assessment suggest short-term increases in EPA+DHA intakes immediately before weeks 24 and 52. The percentage of DHA in erythrocytes characterises adherence to EPA and DHA intakes in long-term interventions.