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A quantitative method for measuring regional in vivo fatty-acid incorporation into and turnover within brain phospholipids: review and critical analysis

Docosahexaenoic acid (DHA, 22:6n-3) accretion in brain phospholipids is critical for maintaining the structural fluidity that permits proper assembly of protein complexes for signaling. Furthermore, membrane DHA can be released by phospholipase A₂ and act as a substrate for the synthesis of bioactive metabolites that regulate synaptogenesis, neurogenesis, inflammation, and oxidative stress. Thus, brain DHA is consumed through multiple pathways including mitochondrial β-oxidation, autoxidation to neuroprostanes, as well as enzymatic synthesis of bioactive metabolites including oxylipins, synaptamide, fatty-acid amides, and epoxides. By using models developed by Rapoport and colleagues, brain DHA loss has been estimated to be 0.07–0.26 μmol DHA/g brain/d. Since β-oxidation of DHA in the brain is relatively low, a large portion of brain DHA loss may be attributed to the synthesis of autoxidative and bioactive metabolites. In recent years, we have developed a novel application of compound specific isotope analysis to trace DHA metabolism. By the use of natural abundance in ¹³C-DHA in the food supply, we are able to trace brain phospholipid DHA loss in free-living mice with estimates ranging from 0.11 to 0.38 μmol DHA/g brain/d, in reasonable agreement with previous methods. This novel fatty acid metabolic tracing methodology should improve our understanding of the factors that regulate brain DHA metabolism.

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.

Mood stabilizers with disparate chemical structures are approved for treating bipolar disorder, but their mechanisms of action are not agreed on. However, when administered to unanesthetized rats at clinically relevant doses, they modulate neurotransmission involving arachidonic acid and brain activity of COX-2, which oxidizes arachidonic acid within the arachidonic acid metabolic cascade.

Inhibiting COX-2 directly might enhance mood stabilizer effects in bipolar disorder patients.

This paper reviews randomized controlled trials that showed that celecoxib, a selective COX-2 inhibitor, or low-dose aspirin, which inhibits COX-1 and inhibits/acetylates COX-2, reduced bipolar symptoms in patients on mood stabilizers. More convincing are two population based pharmacoepidemiological studies that each demonstrated that chronic low dose aspirin reduced bipolar severity markers in patients on mood stabilizers.

This clinical evidence is consistent with the hypothesis that low-dose chronic aspirin and celecoxib, which can inhibit COX-2 and enter brain, can be repurposed in bipolar disorder to enhance mood stabilizer effects on arachidonic acid metabolism and neurotransmission.

Studies in unanesthetized rats suggest that mood stabilizers approved for treating bipolar disorder downregulate brain arachidonic acid (AA) metabolism. AA plays a role in neurotransmission and neuroinflammation, among other processes. Other drugs that reduce brain AA metabolism may add to mood stabilizer action.

We reviewed randomized controlled trials (RCTs) and population studies to examine whether celecoxib, a selective cyclooxygenase (COX)-2 inhibitor, and acetylsalicylate (aspirin), a COX-1 and COX-2 inhibitor and acetylator, were useful in bipolar disorder patients on mood stabilizers. COX-1 and COX-2 metabolize AA to bioactive eicosanoids.

Celecoxib significantly enhanced mood stabilizer efficacy in two 6-week RCTs involving 86 manic bipolar inpatients, and in one 8-week RCT on 49 patients with treatment-resistant bipolar depression. With regard to aspirin, a Dutch pharmacoepidemiological study involving 5145 subjects taking lithium reported symptom reduction with added chronic low dose 30-80 mg/day aspirin, while a Danish study on 321,350 subjects taking chronic 75-150 mg/day aspirin found fewer manic episodes than in subjects not on aspirin. Finally, a recent 6-week RCT using low-dose aspirin and/or minocycline showed a specific positive effect of aspirin.

Efficacy of both celecoxib and aspirin as adjuncts to mood stabilizers in the treatment of bipolar disorder is consistent with the AA hypothesis for mood stabilizer action in that disorder.

The brain is highly enriched in the long-chain omega-3 (n-3) PUFA DHA. Due to the limited capacity for local DHA synthesis in the brain, it relies on a continual supply from the circulation to replenish metabolized DHA. Previous studies investigating brain DHA turnover and metabolism have relied on isotope tracers to determine brain fatty acid kinetics; however, this approach is cumbersome and costly. We applied natural abundance carbon isotope ratio analysis via high-precision gas chromatography combustion isotope ratio mass spectrometry, without the use of labeled tracers, to determine the half-life of brain DHA in mice following a dietary switch experiment. Mice fed diets containing either α-linolenic acid (ALA) or DHA as the sole dietary n-3 PUFA were switched onto diets containing ALA, DHA, or ALA + DHA at 6 weeks of age, while control mice were maintained on their respective background diet. We measured brain DHA carbon isotope ratios (reported as δ¹³C_DHA signatures) over a 168-day time course. Brain δ¹³C_DHA signatures of control mice maintained on background diets over the time course were stable (P > 0.05). Brain δ¹³C_DHA signatures of mice switched to the DHA or ALA + DHA diet from the ALA diet changed over time, yielding brain incorporation half-lives of 40 and 34 days, respectively. These half-lives determined by natural abundance carbon isotope ratio analysis were consistent with estimates from kinetic isotope tracer studies. Our results demonstrate the feasibility of natural abundance carbon isotope ratio analysis in the study of fatty acid metabolism without the use of isotopically labeled fatty acid tracers.

Despite being in routine for onco-diagnostics for years, the applicability of nucleosidic molecular imaging probes is severely restricted in neurological applications due to their low permeability across blood-brain-barrier (BBB). For extending nucleoside tracers utility for neuro-onco early diagnostics, suitable modification which enhances their BBB permeation needs investigation. Among various modifications, lipidization of nucleosides has been reported to enhance cellular permeability. Extending the concept, the aim was to exemplify the possibility of lipidized nucleosides as potential brain tracer with capability to cross intact BBB and evaluate as metal based neuro-imaging SPECT agent. Uridine based non-lipidic (NS_DAU) and di-C15-ketal appended lipidic (NL_DPU) ligands were conjugated to chelator, DTPA (DTPA-NS_DAU and DTPA-NL_DPU) using multi-step chemistry. The ligands were evaluated in parallel for comparative physical and biological parameters. Additionally, effects of enhanced lipophilicity on UV-absorption, acid strength, fluorescence and non-specific protein binding were evaluated. Fluorescence quenching of BSA indicated appreciable interaction of DTPA-NL_DPU with protein only above 10 mM without inducing conformational changes. In addition, DTPA-NL_DPU was found to be haematocompatible and cytocompatible with low dose-dependent toxicity in HEK-cells. The chelator DTPA was used for ^99mTc-complexation for SPECT imaging. Optimized ^99mTc-radiolabeling parameters resulted in quantitative (≥97%) labeling with good stability parameters in in-vitro serum and cysteine challenge studies. We demonstrate that the nucleolipid radiotracer (^99mTc-DTPA-NL_DPU) was successfully able to permeate the BBB with brain uptake of 0.2% ID/g in normal mice as compared to 0.06% ID/g uptake of ^99mTc-DTPA-NS_DAU at 5 min. Blood kinetics indicate biphasic profile and t1/2(distribution) 46 min for ^99mTc-DTPA-NL_DPU. The preferential accumulation of ^99mTc-DTPA-NL_DPU in brain tumor intracranial xenograft indicate the targeting capability of the nucleoside. We conclude that as first-of-its-kind, this work presents the potential of the biocompatible nucleolipidic system for brain targeting and early diagnostics.