Stanley I. Rapoport, MD, Harvard University - US grants

IMAGING NEUROINFLAMMATION IN ALZHEIMER DISEASE Postmortem studies demonstrate neuroinflammatory markers in Alzheimer disease, but our ability to image neuroinflammation in humans in vivo is limited. Based on studies in a rat model of neuroinflammation, we predicted that brain uptake of intravenously injected radiolabeled arachidonic acid (AA) would be elevated in patients with Alzheimer disease. We confirmed this prediction using positron emission tomography (PET) in 8 mildly-severely demented Alzheimer disease patients compared with 8 aged-matched controls. AA incorporation was elevated in neocortical brain regions known to have high densities of senile neuritic plaques and activated microglia. Cerebral blood flow was reduced by comparison in these regions. Our PET method, after confirmation, might be used to examine progression of neuroinflammation in Alzheimer and other diseases in which it plays a role, and disease response to medication (Ref. 1). CHOLINERGIC MODULATION OF SYNAPTIC FUNCTION IN HEALTHY VOLUNTEERS Frontal cortex blood flow, measured using PET and 15O-water, was elevated in relation to working memory-task difficulty in young healthy volunteers, in relation to prolongation of reaction time. Administration of the anticholinesterase, physostigmine, prevented these changes. Thus, cholinergic modulation of synaptic transmission enhanced memory performance and reduced effortful synaptic recruitment in the frontal cortex. These changes may be related to the usefulness of anticholinesterase treatment in patients with Alzheimer disease (Ref. 2 and 3). IMAGING HUMAN BRAIN SIGNALING INVOLVING DOPAMINE We are conducting a PET protocol with the NIMH to image brain signal transduction via AA, related to dopaminergic transmission, in adults with Attention Deficit Hyperactivity Disorder (ADHD) and age-matched controls. Apomorphine, a dopamine D2/D3 receptor agonist, is administered to activate AA signaling via D2 receptors, as proven in preclinical studies. We hypothesize that this signaling will be disturbed in ADHD patients, based on genetic evidence of their altered dopamine receptor and transporter alleles. We have completed scans on 6 normal volunteers and are evaluating the results. REGIONAL DOCOSAHEXAENOIC ACID IMAGING IN THE HUMAN BRAIN DHA is a nutritionally essential polyunsaturated fatty acid in brain cell membranes and participates in many brain metabolic processes. Being able to image its consumption in human health and disease would be useful. We are conducting a PET protocol together with NIAAA investigator to quantify brain DHA signaling and consumption in healthy volunteers, based on our preclinical studies. For the entire brain, the mean rate of DHA incorporation from plasma equals 3.8 mg/day (Ref. 4). IMAGING HUMAN BRAIN SIGNALING INVOLVING DOPAMINE We initiated a PET protocol with the NIMH to image brain signal transduction via arachidonic acid, related to dopaminergic transmission, in adults with Attention Deficit Hyperactivity Disorder (ADHD) and age-matched controls. Apomorphine, a dopamine D2/D3 receptor agonist, is administered to activate arachidonic acid signaling via D2 receptors, as proven in preclinical studies. We hypothesize that this signaling will be disturbed in ADHD patients, based on genetic evidence of their altered dopamine receptor and transporter alleles. We have completed scans on 6 normal volunteers and are evaluating the results. REGIONAL DOCOSAHEXAENOIC ACID IMAGING IN THE HUMAN BRAIN Docosahexaenoic acid (DHA) is a nutritionally essential polyunsaturated fatty acid in brain cell membranes and participates in many brain metabolic processes. Being able to image its consumption in human health and disease would be useful. We are conducting a PET protocol together with NIAAA investigator to quantify brain DHA signaling and consumption in healthy volunteers, based on our preclinical studies. For the entire brain, the mean rate of DHA incorporation from plasma equaled 3.8 mg/day. We are preparing a manuscript on this research.

IMAGING ARACHIDONIC ACID-MEDIATED SIGNALING IN RODENT BRAIN.[unreadable] In rats pretreated with the non-selective cyclooxygenase (COX) inhibitor, flurbiprofen, and in mice in which cyclooxygenase (COX)-2 was genetically knocked out, the cholinergic-receptor initiated brain arachidonic acid (AA) signal, measured with our tracer method, was confirmed to represent AA release from brain membrane phospholipid by phospholipase A2, followed by AA metabolic loss almost entirely through the COX-2 pathway (Basselin et al. 2007).[unreadable] [unreadable] REDUCED ARACHIDONIC ACID SIGNALING BY CHRONIC AMPHETAMINE MAY BE RELATED TO POST-DRUG DEPRESSION IN HUMANS[unreadable] Withdrawal from amphetamine abuse causes depression in drug addicts. In rats, such withdrawal was shown to widely decrease brain AA signaling, an effect that may be related to the post-amphetamine depression noted in humans and in rats (Bhattacharjee et al. 2008a).[unreadable] [unreadable] APOMORPHINE STIMULATES ARACHIDONIC ACID SIGNALING VIA DOPAMINE D2-LIKE RECEPTORS[unreadable] Apomorphine, a mixed dopamine D1/D2 receptor agonist, is used to treat patients with Parkinson disease. In awake rats, acute apomorphine provoked a robust arachidonic acid (AA) signal in brain regions with dopamine receptors solely through the D2 receptors, as the signal could be blocked by pre-administration of raclopride, a D2/D3 receptor antagonist (Bhattacharjee et al. 2008b). This study supports our approved clinical protocol in which apomorphine is injected to image dopamine signaling with positron emission tomography.[unreadable] [unreadable] ELEVATED BRAIN ARACHIDONIC ACID SIGNALING IN SEROTONIN TRANSPORTER (5-HTT) DEFICIENT MICE[unreadable] Certain polymorphisms of the serotonin reuptake transporter (5-HTT) leading to its reduced function increase susceptibility to psychiatric disorders in human subjects. Heterozygous (5-HTT+/-) deficient mice, models for humans with these polymorphisms, had elevated brain serotonin concentrations and behavioral abnormalities. In these mice, baseline arachidonic acid (AA) signaling was elevated, as was brain cytosolic phospholipase A2 activity (which is coupled to post-synaptic serotonin receptors). These results suggest that AA signaling also would be elevated in patients with reduced-function polymorphisms. This could be explored using positron emission tomography.

DIET AND LIVER METABOLISM DETERMINE BRAIN METABOLISM OF NUTRITIONALLY ESSENTIAL POLYUNSATURATED FATTY ACIDS[unreadable] A coherent mathematical model, based on experimental studies in rodents, showed how brain composition and metabolism of the polyunsaturated fatty acids (PUFAs), docosahexaenoic acid (DHA) and arachidonic acid (AA), are regulated by their dietary intake and/or liver synthesis from their respective PUFAs, alpha-linolenic acid (alpha-LNA, 18:3n-3) and linoleic acid (LA, 18:2n-6). In the absence of dietary DHA, but with sufficient dietary alpha-LNA (4.5% of total fatty acids), the rat liver can maintain a normal brain DHA content by synthesizing 5-10 times more DHA than the brain consumes. Rates of incorporation of circulating DHA and AA into brain, determined with quantitative autoradiography in unanesthetized rodents and with positron emission tomography (PET) in humans, equal respective rates of consumption within brain. The adult human brain consumes AA and DHA at rates of 17.8 and 4.6 mg/day, respectively. AA consumption does not change significantly with human aging (Rapoport et al. 2007a, Rapoport et al. 2007b).[unreadable] [unreadable] DIETARY N-3 PUFA DEPRIVATION UPREGULATES ELONGASES AND DESATURASES IN RAT LIVER BUT NOT BRAIN[unreadable] Estimated synthesis-secretion coefficients of docosahexaenoic acid (DHA) from circulating unesterified alpha-linolenic acid (alpha-LNA) were higher in liver than brain in rats fed an adequate alpha-LNA diet, and were further upregulated in liver but not brain by 15 weeks of dietary n-3 polyunsaturated fatty acid (PUFA) deprivation. The diet induced elevation in the hepatic coefficients reflected increased mRNA and activity levels of enzymes that convert alpha-LNA to DHA, namely delta-5 and delta-6 desaturases and elongases 2 and 5. Thus, the rat liver responds to dietary n-3 PUFA deprivation by increasing transcription of enzymes that convert alpha-LNA to DHA, and upregulating its conversion capacity (Igarashi et al. 2007).[unreadable] [unreadable] HEART CAN'T SYNTHESIZE DOCOSAHEXAENOIC ACID FROM CIRCULATING ALPHA-LINOLENIC ACID BECAUSE IT LACKS ELONGASE-2[unreadable] Dietary supplementation with long-chain polyunsaturated fatty acids (PUFAs), including DHA, reduces the incidence of sudden cardiac death. The rat heart, however, cannot convert alpha-linolenic acid (18:3n-3) to longer-chain DHA (22:6n-3) because it lacks a critical enzyme, elongase 2. It must obtain its DHA entirely from dietary sources or by liver synthesis from alpha-LNA. Dietary n-3 PUFA deprivation in the rat reduces heart DHA and increases heart docosapentaenoic acid (22:5n-6), which may increase vulnerability to ischemia (Igarashi et al. 2008).[unreadable] [unreadable] FATTY ACID COMPOSITION OF WILD ANTHROPOID PRIMATE MILK[unreadable] Anthropoid primates vary in growth, development and brain size. In collaboration with scientists at the National Zoo, we found that milk from leaf eating species had a higher proportion of alpha-linolenic acid (18:3n-3) than did milk from omnivores. Mountain gorillas had a uniquely high proportion of milk arachidonic acid (20:4n-6). The differences were unrelated to brain size, and reflected species metabolic differences and/or differences in diet (Milligan et al. 2008).

ANTIMANIC THERAPIES TARGET BRAIN ARACHIDONIC ACID SIGNALING. [unreadable] Evidence from our laboratory, obtained by administering anti-bipolar agents and antidepressants chronically to rats so as to produce therapeutically relevant plasma concentrations, suggested that the common mechanism of action of the effective antimanic antibipolar drugs (lithium, carbamazepine and valproic acid) involves downregulation of the brain arachidonic acid cascade (AA, 20:4n-6) , and that the cascade is upregulated in the disease itself. This work was reviewed in detail, and the AA cascade hypothesis for anti-manic drug action was further elaborated (1).[unreadable] [unreadable] FLUOXETINE UPREGULATES THE BRAIN ARACHIDONIC ACID CASCADE IN RAT BRAIN. [unreadable] Some antidepressants, if given during the depressive phase of bipolar disorder, accelerate a switch back to mania. Relevant to these observations, we found that chronic (21 days followed by 3 days of washout) administration to rats of the selective serotonin reuptake inhibitor (SSRI) antidepressant, fluoxetine, upregulated incorporation and turnover of AA in brain phospholipids, as well as mRNA, protein and activity levels of AA-selective cytosolic phospholipase A2 (cPLA2), markers of the brain arachidonic acid cascade (2, 3). These results can be explained if bipolar mania involves an upregulated arachidonic acid cascade, as suggested by our multiple studies with anti-manic drugs (1), and if fluoxetine, in addition to having a selective antidepressant action, also upregulates this cascade. [unreadable] [unreadable] LAMOTRIGINE, APPROVED FOR TREATING BIPOLAR DEPRESSION, DOES NOT ALTER ARACHIDONIC ACID TURNOVER IN RAT BRAIN PHOSPHOLIPID. [unreadable] Lamotrigine is the only FDA-approved monotherapy for bipolar depression, and does not appear to increase switching to bipolar mania (see above). When given chronically to adult rats, unlike fluoxetine, lamotrigine did not change AA turnover in brain phospholipids. These results suggest that only antidepressants that increase the AA cascade promote switching to mania (4). [unreadable] [unreadable] TWO AGENTS APPROVED FOR TREATING BIPOLAR MANIA INCREASE MEMBRANE GRK3 IN RAT BRAIN.[unreadable] G-protein receptor kinases (GRKs) are a family of kinases involved in the desensitization of agonist-activated G-protein coupled receptors. We found that chronic administration to rats of lithium and carbamazepine (but not of valproic acid), mood stabilizers effective against bipolar mania, increased GRK3 expression in the membrane but not the cytosolic fraction of brain. This change is consistent with an effect of these agents on neuroreceptor mediated signal transduction (5). [unreadable] [unreadable] MOOD STABILIZERS DOWNREGULATE TRANSCRIPTION OF ENZYMES OF ARACHIDONIC CASCADE IN RAT BRAIN. [unreadable] We reported that chronically administered carbamazepine and valproic acid, to produce therapeutically relevant plasma concentrations, downregulate the brain AA cascade in rat brain. They do this in part by reducing mRNA levels of cyclooxygenase (COX)-2 and AA-selective cytosolic phospholipase A2 (cPLA2), respectively, in brain. In the current studies, we showed that these mRNA effects arose because carbamazepine reduced brain expression of the cPLA2 transcription factor, activator protein (AP)-2 in the first instance, and valproate reduced brain expression of the COX-2 transcription factor, NF-kappaB in the second instance (6, 7).[unreadable] [unreadable] AN ANIMAL MODEL OF AN UPREGULATED BRAIN ARACHIDONIC ACID CASCADE.[unreadable] Based on our reports that drugs effective against the mania of bipolar disorder downregulate brain AA turnover when given chronically to rats, as well as other markers of the AA cascade, we created an animal with an upregulated brain AA cascade as a potential model for such upregulation in bipolar disorder. Glutamatergic NMDA receptors are known to allow calcium into the cell to stimulate cytosolic cPLA2 and release AA from membrane phospholipid. We injected a subconvulsive dose in rats of N-methyl-D-aspartate (NMDA) daily for 21 days, and measured markers of the AA cascade in brain. Chronically injected rats had elevated expression of cPLA2 and its transcription factor, activator protein (AP)-2, whereas acutely NMDA injected rats did not have these changes. Thus, the chronic NMDA-treated rat is a usable model of an upregulated brain arachidonic acid cascade, which may characterize bipolar disorder as well, because of neuroinflammation, Alzheimer disease (3).

MOOD STABILIZERS USED FOR BIPOLAR DISORDER TARGET BRAIN ARACHIDONIC ACID CASCADE One approach to understanding and treating bipolar disorder (BD) may be to identify a common mechanism of action of the FDA-approved mood stabilizers used in its treatment. In two critical reviews, we proposed that the common mechanism is downregulation of the brain "arachidonic acid (AA) cascade." Thus, chronic administration to rats of lithium, carbamazepine, valproate, and lamotrigine downregulate AA turnover in brain phospholipids, prostaglandin E formation, and expression of AA cascade enzymes, including cytosolic phospholipase A2, cyclooxygenase-2 and acyl-CoA synthetase. The changes are selective for AA, as docosahexaenoic or palmitic acid metabolism is unaffected. Antidepressants that increased switching of bipolar depression to mania upregulated the rat brain AA cascade, consistent with the hypothesis. The conclusions are supported by our postmortem studies on the BD brain (Rapoport et al., 2009;Rao et al 2009). MANIA CAUSED BY CERTAIN ANTIDEPRESSANTS IN BIPOLAR DEPRESSED PATIENTS MAY BE RELATED TO INCREASED BRAIN ARACHIDONIC ACID METABOLISM Some antidepressants (e.g. fluoxetine, imipramine) when given to depressed bipolar disorder (BD) patients increase frequency of switching to mania, whereas others (e.g. bupropion) do not. Consistent with our hypothesis that BD, particularly the manic phase, is associated with upregulated brain AA metabolism, we showed in rats that chronic (but not acute) fluoxetine or imipramine increased brain AA metabolic markers, but bupropion did not (Lee et al. 2010). CHRONIC LITHIUM DAMPENS NEUROINFLAMMATION BY MODIFYING BRAIN ARACHIDONIC AND DOCOSAHEXAENOIC ACID METABOLISM Neuroinflammation characterizes many progressive human brain brain diseases, including Alzheimer disease, bipolar disorder and HIV-1 dementia that we are studying. Neuroinflammation involves activation of brain microglia, cytokine and nitric oxide release by them, and upregulation of brain arachidonic acid (AA) metabolism and metabolic enzymes. We reported that administration of lithium to rats in which neuroinflammation was induced by 6 days of brain infusion of bacterial lipopolysaccharide (LPS) suppressed the upregulated LPS-induced expression of AA metabolizing enzymes, AA metabolism, and synthesis of the pro-inflammatory AA metabolite, prostaglandin E2. Lithium also induced formation of 17-hydroxy docosahexaenoic acid (17-OH-DHA), a precursor of antiinflammatory neuroprotectins. 17-OH-DHA also can be formed after aspirin acetylates cyclooxygenase-2. Thus, an antiinflammatory synergy between lithium and aspirin is likely, and this is supported by our clinical epidemiological data (see below). Lithium's antiinflammatory action may account for its efficacy against bipolar disorder, and suggests that it would be of use in other neuroinflammatory diseases, such as Alzheimer disease and HIV-1 dementia (Basselin et al 2010). ASPIRIN ENHANCES LITHIUM EFFICACY IN BIPOLAR DISORDER: AN EPIDEMIOLOGICAL STUDY Using a pharmacological database, we confirmed our hypothesis that aspirin (see above) would enhance the bipolar effects of lithium, which was based on their common effects on suppressing brain arachidonic acid (AA) metabolism and increasing docosahexaenoic acid metabolism in rats. We used a pharmacological database with a pharmacoepidemiological analysis to show that low dose aspirin significantly reduced the number of medical events (dose increase or addition of antipsychotic agent) in patients treated with lithium, whereas other nonsteroidal antiinflammatory drugs and glucocorticoids did not. These findings support our proposal of upregulated brain AA metabolism in bipolar disorder, and suggest that lithium plus aspirin might be considered for this disease as will neuroinflammatory human brain diseases such as Alzheimer disease (Stolk et al. 2010).

ANIMAL MODEL OF EXCITOXICITY WITH NEUROINFLAMMATION[unreadable] Excitotoxicity is thought to contribute to brain damage in human diseases, including Alzheimer disease and bipolar disorder. We developed an animal model of excitotoxicity, the rat to which the glutamate receptor agonist, N-methyl-D-aspartate (NMDA), is administered daily for 21 days. The brain of this rat demonstrated upregulated markers of arachidonic acid metabolism, and increased neuroinflammatory markers (interleukin-1beta, tumor necrosis factor alpha, glial fibrillary acidic protein and inducible nitric oxide synthase). This model might be used to develop drugs that suppress the upregulated arachidonic metabolism of excitotoxicity-neuroinflammation, and to further understand pathological mechanisms (Lee et al. 2008, Chang et al. 2008).[unreadable] [unreadable] ARACHIDONIC AND DOCOSAHEXAENOIC ACID METABOLITES IN ISCHEMIC BRAIN[unreadable] Biologically active metabolites of arachidonic acid (AA) and docosahexaenoic acid (DHA) arise during brain ischemia and other brain insults, and can influence cell death and neuroprotection. We used reversed phase liquid chromatography/tandem mass spectrometry to quantify their production in rat brain subjected to cerebral ischemia and removed after head-focused microwave irradiation to stop postmortem metabolism. Brain AA, DHA and docosapentaenoic acid (n-6) concentrations were increased 18-, 5- and 4-fold compared to control values, respectively. Prostaglandin E2 and D2 were not detected in control brain. Concentrations of thromboxane B2, E2/D2-isoprostanes, 5-HETE, 5-oxo-ETE, and 12-HETE (other eicosanoids) were significantly elevated by ischemia, as was 17-hydroxy-DHA, the immediate precursor of neuroprotectin D1. These alterations in the balance of lipid mediators likely mediate brain injury and recovery (Farias et al. 2008).

ALZHEIMER DISEASE DISTURBED CHOLINE PLASMALOGEN AND FATTY ACID CONCENTRATIONS IN ALZHEIMER DISEASE BRAIN. Alzheimer disease (AD) is a progressive neurodegenerative disorder characterized by senile (neuritic) plaques containing amyloid and neurofibrillary tangles, neuroinflammation, synaptic loss and overexpression of arachidonic acid (AA, 20:4n-6) metabolizing enzymes. Lipid concentration changes have been reported, but partially or as percent of total. We measured absolute lipid concentrations (per gram wet weight) in postmortem prefrontal cortex from 10 AD patients and 9 controls. Total brain lipid, phospholipid, cholesterol, and triglyceride concentrations did not differ significantly between groups. There was a significant 73% decrease in plasmalogen choline, but no difference in other phospholipids. Fatty acid concentrations in total phospholipid did not differ from control. Docosahexaenoic acid (DHA, 22:6n-3) was reduced in ethanolamine glycerophospholipid and choline glycerophospholipid, but increased in phosphatidylinositol. AA was reduced in choline glycerophospholipid, but increased in phosphatidylinositol, while docosatetraenoic acid (22:4n-6), an AA elongation product, was reduced in total lipid, cholesteryl ester and triglyceride. These changes, suggesting extensive membrane remodeling, may contribute to membrane instability and synaptic loss in AD and reflect neuroinflammation and other pathological processes (Igarashi et al., 2011). ALTERED NEUROINFLAMMATORY, ARACHIDONIC ACID CASCADE AND SYNAPTIC MARKERS IN POSTMORTEM ALZHEIMER DISEASE BRAIN. Alzheimers disease (AD) is the leading cause of dementia in the elderly. Our recent positron emission tomography imaging study demonstrated upregulated brain arachidonic acid (AA) metabolism in AD patients, suggestive of inflammation. To test this suggestion, we measured protein and mRNA levels of AA cascade, neuroinflammatory and synaptic markers in postmortem frontal cortex from 10 AD patients and 10 age-matched controls. Consistent with our hypothesis, AD frontal cortex showed significant increases in protein and mRNA levels of cPLA2-IVA, secretory sPLA2-IIA, cyclooxygenase-1 and -2, membrane prostaglandin synthase-1 and lipoxygenase- 12 and -15. Calcium-independent iPLA2-VIA and cytosolic PGE2 synthase were decreased. In addition, interleukin-1b, tumor necrosis factor-a, glial fibrillary acidic protein and CD11b were increased, and synaptophysin and drebrin, pre- and postsynaptic markers, were decreased. These results indicate that increased AA cascade and inflammatory markers could contribute to AD pathology. Altered brain AA cascade enzymes could be therapeutic targets for drug development (Rao et al., In press). DISTURBED NEUROTRANSMITTER TRANSPORTER EXPRESSION IN ALZHEIMER DISEASE BRAIN. An imbalance of neurotransmission has been proposed to cause the behavioral symptoms in Alzheimer disease (AD). Changes causing this imbalance are not clear. We tested whether it might involve changes neurotransmitter reuptake by vesicular glutamate transporters (VGLUTs), excitatory amino acid transporters (EAATs), the vesicular acetylcholine transporter (VAChT), the serotonin reuptake transporter (SERT), or the dopamine reuptake transporter (DAT). We tested this in postmortem prefrontal cortex from 10 AD patients and 10 non-AD controls. Compared with controls, protein and mRNA levels of VGLUTs, EAAT1-3, VAChT, and SERT were reduced significantly in AD. Expression of DAT and catechol O-methyltransferase was unchanged. Reduced VGLUTs and EAATs may alter glutamatergic recycling, and reduced SERT could exacerbate depression in AD. The reduced VAChT expression could contribute to the recognized cholinergic deficit in AD. Altered neurotransmitter transporters likely contribute to neurotransmission dysfunction in AD and are potential therapeutic targets (Chen et al., 2011). BIPOLAR DISORDER UNALTERED FATTY ACID COMPOSITION IN BIPOLAR DISORDER BRAIN. Docosahexaenoic (DHA) and arachidonic acid (AA) are critical to brain function and are concentrated in synaptic membrane phospholipids. However, in postmortem BD frontal cortex, we did not find a significant concentration difference for either fatty acid from control, whether concentration was measured per brain gram wet weight or as percent of the total fatty acid concentration. Since we reported altered AA metabolizing enzymes in the postmortem BD brain, we predict that measuring AA kinetics with our PET method would show brain abnormalities in patients (Igarashi et al., 2010). EXCITOTOXICITY AND NEUROINFLAMMATORY MARKERS IN FRONTAL CORTEX FROM BIPOLAR DISORDER PATIENTS. Cognitive decline, symptom worsening and brain atrophy in bipolar disorder (BD) indicate disease progression, which may involve excitotoxicity and neuroinflammation. To test this, we measured expression of excitotoxicity and neuroinflammatory markers in postmortem frontal cortex from 10 BD patients and 10 controls. Tissue was matched for age, postmortem interval and pH. There were significantly lower protein and mRNA levels of N-methyl-D-aspartate receptors, NR-1 and NR-3A, and higher protein and mRNA levels of interleukin (IL)-1beta, the IL-1 receptor (IL-1R), myeloid differentiation factor 88, nuclear factor-kappa B subunits, and astroglial and microglial markers (glial fibrillary acidic protein, inducible nitric oxide synthase, c-fos and CD11b) in the BD cortex. These data are consistent with excitotoxicity and neuroinflammation in BD, with activation of the IL-R cascade. They may account for disease progression and be a target for future therapy (Rao et al., 2010). APOPTOSIS AND SYNAPTIC LOSS IN BIPOLAR DISORDER BRAIN Bipolar disorder (BD) is associated with progressive brain atrophy and cognitive decline. We related these changes to cell death and synaptic loss, as we reported increased protein and mRNA levels of pro-apoptotic factors Bax, BAD, caspase-9 and caspase-3) and decreased levels of anti-apoptotic factors (BDNF and Bcl-2) and of synaptic markers (synaptophysin and drebrin) in BD compared with control postmortem prefrontal cortex. These changes are similar to changes in Alzheimer disease, suggesting a basis for common therapeutic approaches (Kim et al., 2010). ALTERED ARACHIDONIC ACID CASCADE ENZYMES IN BIPOLAR DISORDER BRAIN. Mood stabilizers that are approved for treating bipolar disorder (BD), when given chronically to rats, decrease expression of markers of the brain arachidonic acid (AA) metabolic cascade, and reduce excitotoxicity and neuroinflammation-induced upregulation of these markers. We therefore hypothesized that that AA metabolic markers are upregulated in the BD brain, and measured these markers in postmortem frontal cortex from 10 BD patients and 10 age-matched controls. Protein and mRNA levels of AA-selective cytosolic phospholipase A2 (cPLA2) IVA, secretory sPLA2 IIA, cyclooxygenase (COX)-2, and membrane prostaglandin E synthase (mPGES) were elevated, whereas levels of COX-1 and cytosolic PGES (cPGES) were reduced relative to controls, in BD cortex. These results confirm that enzymes associated with AA release from membrane phospholipid and with its downstream metabolism are upregulated in BD, and may explain why mood stabilizers that downregulate enzymes in animal model are clinically effective. An upregulated cascade should be considered as a target for drug development and for neuroimaging in BD (Kim et al., 2011). NEUROINFLAMMATION AND EXCITOTOXICITY IN BIPOLAR DISORDER. In a review of bipolar disorder (BD), we indicated how disease progression is related to neuroinflammation and excitotoxicity, and an upregulated brain arachidonic acid (AA) cascade. As similar changes occur in Alzheimer disease, antiinflammatory-antiexcitotoxicity therapies might be considered for both disorders (Rao et al, 2010).

MOOD STABILIZERS TARGET THE BRAIN ARACHIDONIC ACID CASCADE[unreadable] Administration to rats at therapeutically relevant doses of each of the FDA-approved mood stabilizers -- lithium, valproate, carbamazepine, and lamotrigine -- downregulated brain arachidonic acid (AA) metabolic markers but not docosahexaenoic acid markers. The first three agents, which are preferred for treating bipolar mania, decreased turnover of AA in brain phospholipids, expression of cyclooxygenase (COX)-2 and AA-selective cytosolic phospholipase A2 or acyl-CoA synthetase, and the concentration of prostaglandin E2. Lamotrigine, preferred for bipolar depression, downregulated COX-2 transcription and activity. These studies lend further support to our hypothesis that the brain AA cascade is the common target of effective mood stabilizers, and suggest that measuring AA cascade markers in the rat can be used to screen for new, less-toxic, bipolar disorder agents (Rao et al. 2008; Lee et al. 2007, Lee et al. 2008).[unreadable] [unreadable] MOOD STABILIZERS UPREGULATE EXPRESSION OF BRAIN NEUROTROPHIC FACTORS[unreadable] Brain derived neurotrophic factor (BDNF) and B-cell lymphoma-2 (Bcl-2) are involved in neuronal signaling, cell survival and plasticity. Decreased brain levels of these factors occur in bipolar disorder. We showed that chronic administration to rats of lithium, valproic acid, carbamazepine, or lamotrigine, to produce therapeutically relevant plasma concentrations, increased BDNF and Bcl-2 levels in the cytosolic fraction of the frontal cortex. This common effect of the mood stabilizers on brain neuroprotective factors may be mediated by downregulation of arachidonic acid metabolism (Chang et al. In press).

IN VIVO IMAGING OF BRAIN SIGNAL TRANSDUCTION AND METABOLISM VIA ARACHIDONIC AND DOCOSAHEXAENOIC ACIDIN ANIMALS AND HUMANS. The polyunsaturated fatty acids (PUFAs), arachidonic acid (AA, 20:4 n-6) and docosahexaenoic acid (DHA, 22:6 n-3), important second messengers in brain, are released from membrane phospholipid following receptor-mediated activation of specific PLA2 enzymes. We developed an in vivo method in rodents using quantitative autoradiography to image PUFA incorporation into brain from plasma, and showed that AA and DHA incorporation rates equal their rates of metabolic consumption by brain. We employ our imaging method in rodents to demonstrate signaling effects of mood stabilizers on brain AA/DHA incorporation during neurotransmission by muscarinic M(1,3,5), serotonergic 5-HT(2A/2C), dopaminergic D(2)-like (D(2), D(3), D(4)) or glutamatergic N-methyl-D-aspartic acid (NMDA) receptors, and effects of inhibition of acetylcholinesterase, of selective serotonin and dopamine reuptake transporter inhibitors, of neuroinflammation (HIV-1 and lipopolysaccharide) and excitotoxicity, and in genetically modified rodents. The method has been extended for the use with positron emission tomography (PET), and can be employed to determine how human brain AA/DHA signaling and consumption are influenced by diet, aging, disease and genetics. TRANSLATIONAL STUDIES ON REGULATION OF BRAIN DOCOSAHEXAENOIC ACID METABOLISM IN VIVO. One goal in the field of brain polyunsaturated fatty acid (PUFA) metabolism is to translate studies conducted in vitro and in animal models to the clinical setting. Doing so can elucidate the roles of PUFAs in the human brain, and effects of diet, drugs, disease and genetics on this role. In a review, we discussed new in vivo radiotracer kinetic and neuroimaging techniques that allow us to do this, with a focus on docosahexaenoic acid (DHA). We illustrated how brain PUFA metabolism is influenced by graded reductions in dietary n-3 PUFA content in unanesthetized rats, and how kinetic tracer techniques in rodents have helped to identify mechanisms of action of mood stabilizers used in bipolar disorder, how DHA participates in neurotransmission, and how brain DHA metabolism is regulated by calcium-independent iPLA(2)beta. In humans, regional rates of brain DHA metabolism can be quantitatively imaged with positron emission tomography following intravenous injection of 1-(11)CDHA. (1) MECHANISMS UNDERLYING THE IN VIVO DOCOSAHEXAENOIC ACID SIGNAL. It is established from in vitro studies that docosahexaenoic acid (DHA) can be hydrolyzed from the sn-2 position of phospholipids by a calcium-independent iPLA2. iPLA2 has been identified in post-synaptic sites, but its coupling to the activation of specific receptors in vivo is not fully understood. We confirmed independence of the DHA signal of extracellular derived calcium, since NMDA administration, which activates ionotropic synaptic receptors did not give a DHA signal (although it produced an AA signal mediated by entry into the cell of calcium dependent cPLA2). This suggests that the DHA signal arises when iPLA2 is activated indirectly from intracellular calcium stores, through the intervention of phospholipase C. In addition ,we are finding that arecoline, which activates G-protein coupled muscarinic receptors, did give an in vivo DHA signal, whereas a signal was not produced by a G-protein coupled D2 receptor agonist (both muscarinic and D2 receptor activations produce an AA signal).

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Ca(2+)-independent phospholipase A(2)[unreadable] (iPLA(2)[unreadable]) selectively hydrolyzes docosahexaenoic acid (DHA, 22:6n-3) in vitro from phospholipid. Mutations in the PLA2G6 gene encoding this enzyme occur in patients with idiopathic neurodegeneration plus brain iron accumulation and dystonia-parkinsonism without iron accumulation, whereas mice lacking PLA2G6 show neurological dysfunction and neuropathology after 13 months. We hypothesized that brain DHA metabolism and signaling would be reduced in 4-month-old iPLA(2)[unreadable]-deficient mice without overt neuropathology.

LIPOPOLYSACCHARIDE INDUCED NEUROINFLAMMATION SELECTIVE INVOLVEMENT OF ARACHIDONIC ACID IN NEUROINFLAMMATION In a rat model of neuroinflammation, produced by a 6-day intracerebral ventricular infusion of bacterial lipopolysaccharide (LPS), we reported marked disturbances in brain arachidonic acid (AA, 20:4n-6) metabolism. In the present study, we demonstrated that parameters of brain docosahexaenoic acid (DHA, 22:6n-3) metabolism were unaffected in this model. Selective targeting of brain AA metabolism with non-steroidal antiinflammatory or other drugs should be considered for treating human brain diseases associated with neuroinflammation (Rosenberger et al., 2010). ANTI-INFLAMMATORY EFFECT OF ASPIRIN ON BRAIN ARACHIDONIC ACID METABOLITES Pro-inflammatory and anti-inflammatory mediators derived from arachidonic acid (AA) modulate peripheral inflammation and its resolution. Aspirin (ASA) is a non-steroidal anti-inflammatory drug (NSAID) that switches AA metabolism from prostaglandin E (PGE) and thromboxane B (TXB) to lipoxin A (LXA) and 15-epi-LXA. It is unknown whether chronic therapeutic doses of ASA are anti-inflammatory in the brain. We hypothesized that ASA would dampen increases in brain concentrations of AA metabolites in a rat model of neuroinflammation, produced by a 6-day intracerebroventricular infusion of bacterial lipopolysaccharide (LPS). In rats infused with LPS (0.5 ng/h) and given ASA-free water to drink, concentrations in high-energy microwaved brain of PGE, TXB and leukotriene B (LTB) were elevated. In rats infused with artificial cerebrospinal fluid, 6 weeks of treatment with a low (10 mg/kg/day) or high (100 mg/kg/day) ASA dose in drinking water decreased brain PGE, but increased LTB, LXA and 15-epi-LXA concentrations. Both doses attenuated the LPS effects on PGE, and TXB. The increments in LXA and 15-epi-LXA caused by high-dose ASA were significantly greater in LPS-infused rats. The ability of ASA to increase anti-inflammatory LXA and 15-epi-LXA and reduce pro-inflammatory PGE and TXB suggests considering aspirin further for treating clinical neuroinflammation (Basselin et al., 2011a). LITHIUM MODIFIES BRAIN ARACHIDONIC AND DOCOSAHEXAENOIC METABOLISM IN RAT LIPOPOLYSACCHARIDE MODEL OF NEUROINFLAMMATION. Neuroinflammation, caused by 6 days of intracerebroventricular infusion of a low dose of lipopolysaccharide (LPS;0.5 ng/h), stimulates brain arachidonic acid (AA) metabolism in rats, but 6 weeks of lithium pretreatment reduces this effect. To further understand this action of lithium, we measured concentrations metabolites generated from AA and docosahexaenoic acid (DHA), in high-energy microwaved rat brain using LC/MS/MS and two doses of LPS. In rats fed a lithium-free diet, low (0.5 ng/h)- or high (250 ng/h)-dose LPS compared with artificial cerebrospinal fluid increased brain unesterified AA and prostaglandin E2 concentrations and activities of AA-selective Ca2+-dependent cytosolic phospholipase A2 (cPLA2)-IV and secretory sPLA2. LiCl feeding prevented these increments. Lithium increased brain concentrations of lipoxygenase-derived AA metabolites, 5- hydroxyeicosatetraenoic acid (HETE), 5-oxo-eicosatetranoic acid, and 17-hydroxy-DHA by 1.8-, 4.3- and 1.9-fold compared with control diet. Lithium also increased 15-HETE in high-dose LPS-infused rats. This study demonstrated, for the first time, that lithium can increase brain 17-hydroxy-DHA formation, a precursor of antiinflammatory resolvins, indicating a new antiinflammatory therapeutic action of lithium (Basselin et al., 2010). PARKINSON DISEASE MODEL UPREGULATED BRAIN ARACHIDONIC ACID ENZYMES IN RAT MODEL OF UNILATERAL PARKINSON DISEASE We had reported that arachidonic acid (AA) signaling is upregulated in the caudate-putamen and frontal cortex of unilaterally 6-hydroxydopamine (6-OHDA) lesioned rats, a model for asymmetrical Parkinson disease. In the present study, we demonstrated that this upregulation was associated with increased expression of two enzymes involved in AA metabolism, cytosolic phospholipase A2 (cPLA2) and cyclooxygenase (COX)-2, ipsilateral to the lesion in the caudate putamen and frontal cortex. This confirms that the tonically increased ipsilateral AA signal in the lesioned rat corresponds to upregulated cPLA2 and COX-2 expression within the AA metabolic cascade;such changes, if present in Parkinson disease, may contribute to symptoms and pathology of this disorder (Lee et al., 2010). HIV-1 DEMENTIA MODEL IMAGING NEUROINFLAMMATION WITH ARACHIDONIC ACID IN HIV-1 TRANSGENIC RAT Human immunodeficiency virus (HIV)-1 associated infection involves entry of virus-bearing monocytes into brain, followed by microglial activation, neuroinflammation, and upregulated arachidonic acid (AA) metabolic enzymes. The HIV-1 transgenic (Tg) rat, a noninfectious HIV-1 model, shows neurologic and behavioral abnormalities after 5 months of age. We used our in vivo imaging method with quantitative autoradiography to demonstrate that brain AA metabolism was elevated in 6-7 month old unanesthetized HIV-1 Tg rats. Brain activities of cytosolic phospholipase A2 (cPLA2-IV), secretory sPLA2, and calcium independent iPLA2-VI, which release AA and docosahexaenoic acid from membrane phospholipids, and concentrations of proinflammatory prostaglandin E2 and leukotriene B4, also were elevated, consistent with neuroinflammation and increased AA metabolism (Basselin et al., 2011b). We now plan to use our clinical method of positron emission tomography with 1-11CAA, to test whether brain AA metabolism is upregulated in HIV-1-infected patients as a marker of neuroinflammation (AG000148). INCREASED INFLAMMATORY AND ARACHIDONIC ACID CASCADE MARKERS, AND REDUCED SYNAPTIC PROTEINS, IN BRAIN OF HIV-1 TRANSGENIC RAT Cognitive impairment has been reported in human immune deficiency virus-1 (HIV-1-) infected patients and in the HIV-1 transgenic (Tg) rat. We hypothesized that this impairment in the rat could be linked to neuroinflammation, disturbed brain arachidonic acid (AA) metabolism, and synapto-dendritic injury. To test this, we measured protein and mRNA levels of markers of neuroinflammation and the AA cascade, as well as pro-apoptotic factors and synaptic proteins, in brain from 7- to 9-month-old HIV-1 Tg and control rats. Compared with control brain, HIV-1 Tg rat brain showed immunoreactivity to glycoprotein 120 and tat HIV-1 viral proteins, and significantly higher protein and mRNA levels of (1) the inflammatory cytokines interleukin-1and tumor necrosis factor alpha, (2) the activated microglial/macrophage marker CD11b, (3) AA cascade enzymes: AA-selective Ca2+-dependent cytosolic phospholipase A2 (cPLA2)-IVA, secretory sPLA2-IIA, cyclooxygenase (COX)-2, membrane prostaglandin E2 synthase, 5-lipoxygenase (LOX) and 15-LOX, cytochrome p450 epoxygenase, and (4) transcription factor NF-Bp50 DNA binding activity. HIV-1 Tg rat brain also exhibited decreased levels of brain-derived neurotrophic factor and drebrin, a marker of post-synaptic excitatory dendritic spines. In summary, HIV-1 Tg rats show elevated brain markers of neuroinflammation and AA metabolism, with a deficit in several synaptic proteins. These changes are associated with viral proteins and may contribute to cognitive impairment. The HIV-1 Tg rat may be a useful model for understanding progression and treatment of cognitive impairment in HIV-1 patients (Rao et al., In press).

I. MOOD STABILIZERS LITHIUM AND THE OTHER MOOD STABILIZERS EFFECTIVE IN BIPOLAR DISORDER TARGET THE RAT BRAIN ARACHIDONIC ACID CASCADE. A critical review evaluated the arachidonic acid (AA, 20:4n-6) cascade hypothesis for actions of lithium and other FDA-approved mood stabilizers in bipolar disorder (BD). The hypothesis is based on evidence in unanesthetized rats that chronically administered lithium, carbamazepine, valproate, or lamotrigine each downregulated brain AA metabolism, and is consistent with upregulated AA cascade markers in post-mortem BD brain. In the rats, each mood stabilizer reduced AA turnover in brain phospholipids, cyclooxygenase-2 expression, and prostaglandin E2 concentration. Lithium and carbamazepine also reduced expression of cytosolic phospholipase A2 (cPLA2) IVA, which releases AA from membrane phospholipids, whereas valproate uncompetitively inhibited in vitro acyl-CoA synthetase-4, which recycles AA into phospholipid. Topiramate and gabapentin, which are ineffective in BD, changed rat brain AA metabolism minimally. On the other hand, the atypical antipsychotics olanzapine and clozapine, which show efficacy in BD, decreased rat brain AA metabolism by reducing plasma AA availability. Each of the four approved mood stabilizers also dampened brain AA signaling during glutamatergic NMDA and dopaminergic D2 receptor activation, while lithium enhanced the signal during cholinergic muscarinic receptor activation. In BD patients, such signaling effects might normalize the neurotransmission imbalance proposed to cause disease symptoms. Additionally, the antidepressants fluoxetine and imipramine, which switch BD depression to mania, increased AA turnover and cPLA2 IVA expression in rat brain, suggesting that brain AA metabolism is higher in BD mania than depression. The AA hypothesis for mood stabilizer action is consistent with our reports that low-dose aspirin reduced morbidity in patients taking lithium, and that high n-3 and/or low n-6 polyunsaturated fatty acid diets, which in rats reduce brain AA metabolism, were effective in BD and migraine patients. (3) II. TESTING NONTERATOGENIC SUBSTITUTES FOR VALPROATE IN TREATING BIPOLAR DISORDER VALNOCTAMIDE, A NON-TERATOGENIC AMIDE DERIVATIVE OF VALPROIC ACID, INHIBITS ARACHIDONIC ACID ACTIVATION IN VITRO BY RECOMBINANT ACYL-COA SYNTHETASE-4. Valproic acid (VPA), a mood stabilizer used in bipolar disorder (BD), uncompetitively inhibits acylation of arachidonic acid (AA) by recombinant AA-selective acyl-CoA synthetase 4 (Acsl4) at an enzyme inhibition constant (Ki ) of 25 mM. Inhibition may account for VPA's therapeutic effect against BD. However, VPA is teratogenic. We tested whether valnoctamide (VCD), a non-teratogenic amide derivative of a VPA chiral isomer, which had antimanic potency in a phase III BD trial, also inhibits recombinant Acsl4. Rat Acsl4-flag protein was expressed in Escherichia coli. Using Michaelis-Menten kinetics, we showed that activation of AA to AA-CoA by Acsl4 was inhibited uncompetitively by VCD, with a Ki of 6.38 mM. at a lower Ki than VPA, VCD and other non-teratogenic Acsl4 inhibitors might be considered further for treating BD. (1) DOES CHRONIC VALNOCTAMIDE INHIBIT ARACHIDONIC ACID TURNOVER IN BRAIN OF UNANESTHETIZED RAT? As a follow up, we are testing whether valnoctamide (VCD), which inhibits Acsl4 in vitro, also reduces AA turnover in unanesthetized rats, as does valproate and lithium. If so, we would apply for a use patent for it as a non-teratogenic analog of valproate for less toxic treatment of bipolar disorder. III. DRUG EFFECTS ON BRAIN ARACHIDONATE METABOLISM TRANSIENT POSTNATAL FLUOXETINE DECREASES BRAIN ARACHIDONIC ACID METABOLISM AND CYTOCHROME P450 4A IN ADULT MICE. Fetal and perinatal exposure to selective serotonin (5-HT) reuptake inhibitors (SSRIs) have been reported to alter childhood behavior, while transient postnatal exposure in rodents alters behavior and decreases brain 5-HT in adulthood. In this study, we tested whether transient postnatal exposure to fluoxetine also alters brain arachidonic acid (AA) metabolism in adult mice. Brain AA incorporation coefficients k* and rates Jin were imaged following intravenous 1-14CAA infusion of unanesthetized adult mice that had been injected daily with fluoxetine (10mg/kg i.p.) or saline during postnatal days P4-P21. Brain AA metabolic enzymes and other relevant markers also were measured. On neuroimaging, k* and Jin were decreased widely in early fluoxetine- compared to saline-treated adult mice. AA-selective cPLA2 activity was unchanged, while Ca(2+)-independent iPLA2 activity was increased. Importantly, there was a significant 74% reduced protein level of cytochrome P450 (CYP) 4A, which can convert AA to bioactive 20-hydroxyeicosatetraenoic acid (HETE). These changes might contribute to the reported altered behavior following early SSRIs in rodents or humans. (3). TRANSIENT POSTNATAL FLUOXETINE REDUCES CYTOCHROME P450 4A METABOLITES IN ADULT MICE. As a follow up study, we are using ELISA to examine arachidonic acid (AA) metabolites in brains of adult mice treated postnatally at P4-P21 with fluoxetine. We are finding reduced concentrations of cytochrome P450 4A metabolites, as predicted by that study, namely bioactive 20-hydroxyeicosatetraenoic acid (HETE) and 15-epi-(lipoxin) LXA4. Behavioral effects in adulthood of early fluoxetine exposure may be mediated in part by disturbed metabolism of AA through the cytochrome P450 pathway. Research is in progress. LOW-DOSE ASPIRIN DAMPENDS INFLAMMATION-INDUCED INCREMENTS IN BRAIN ARACHIDONIC ACID METABOLITES IN OLD HIV-1 TRANSGENIC RAT. Even HIV-1 patients treated with antiretroviral therapy are at risk of developing HIV-1-associated neurocognitive disorder (HAND). Older HIV-1 transgenic rats that develop behavioral changes and show neuroinflammation, neuronal loss, and increased brain arachidonic acid (AA) metabolism, are a model for HAND. We are showing that chronic low-dose aspirin (equivalent to a human low dose) reduces upregulated brain AA metabolism in HIV-1 transgenic rats. Wildtype and HIV-1 transgenic rats, aged 7-9 months, were treated for an additional 42 days with 10 mg/kg/day (equivalent to human low-dose) aspirin in drinking water, then were subjected to head-focused microwave fixation. ELISA measurements showed that brain 15-epi-lipoxin A4 and 8-isoprostane concentrations were significantly higher in the HIV-1 transgenic than wildtype rats. These differences were insignificant following aspirin. Aspirin also reduced brain prostaglandin E2 and leukotriene B4 concentrations in HIV-1 Tg but not wildtype rats. Thromboxane B2, 15-HETE, lipoxin A4 and resolvin D1 concentrations were unrelated to genotype or treatment. Treatment with low-dose aspirin reduces AA-metabolite markers of inflammation and oxidative stress in HIV-1 Tg brain, and might be considered in clinical trials in HIV-1 patients with HAND. Work submitted for publication.

LOWERING DIETARY LINOLEIC ACID REDUCES BIOACTIVE OXIDIZED LINOLEIC ACID METABOLITES IN HUMANS. Linoleic acid (LA) is the most abundant polyunsaturated fatty acid in human diets, a major component of human tissues, and the direct precursor to the bioactive oxidized LA metabolites (OXLAMs), 9- and 13 hydroxy-octadecadienoic acid (9- and 13-HODE) and 9- and 13-oxo-octadecadienoic acid (9- and 13-oxoODE). These four OXLAMs have been mechanistically linked to pathological conditions ranging from cardiovascular disease to chronic pain. Plasma OXLAMs, which are elevated in Alzheimer's dementia and non-alcoholic steatohepatitis, have been proposed as biomarkers useful for indicating the presence and severity of both conditions. Because mammals lack the enzymatic machinery needed for de novo LA synthesis, the abundance of LA and OXLAMs in mammalian tissues may be modifiable via diet. To examine this issue in humans, we measured circulating LA and OXLAMs before and after a 12-week LA lowering dietary intervention in chronic headache patients. Lowering dietary LA significantly reduced the abundance of plasma OXLAMs, and reduced the LA content of multiple circulating lipid fractions that may serve as precursor pools for endogenous OXLAM synthesis. These results show that lowering dietary LA can reduce the synthesis and/or accumulation of oxidized LA derivatives that have been implicated in a variety of pathological conditions. Future studies evaluating the clinical implications of diet-induced OXLAM reductions are warranted. (1). DIETARY OMEGA-6 FATTY ACID LOWERING INCREASES THE BIOAVAILABILITY OF PLASMA OMEGA-3 FATTY ACIDS IN HUMAN PLASMA LIPID POOLS. Dietary linoleic acid (LA, 18:2n-6) lowering in rats reduces n-6 polyunsaturated fatty acid (PUFA) plasma concentrations and increases n-3 PUFA (eicosapentaenoic (EPA) and docosahexaenoic acid (DHA)) concentrations, thus bioavailability for maintaining brain and heart integrity. Objective: To evaluate whether dietary n-6 PUFA lowering, with or without increases in dietary n-3 PUFAs, reduces plasma n-6 PUFA and increases n-3 PUFA concentrations, and to compare magnitude of changes between groups in human subjects. Design: Subjects were randomized to: (1) average n-3, low n-6 (L6) diet; or (2) high n-3, low n-6 LA (H3-L6) diet for 12 weeks. Esterified and unesterified plasma fatty acids were quantified at baseline and after 12 weeks on diet. Results: Compared to baseline, the L6 diet reduced esterified LA and increased esterified n-3 PUFA concentrations, but did not significantly change arachidonic acid (AA, 20:4n-6) concentration. Only the unesterified EPA concentration was increased significantly. The H3-L6 diet decreased esterified LA and AA concentrations, and produced more marked increases in the n-3 PUFA esterified and unesterified concentrations. Conclusions: Dietary n-6 PUFA lowering for 12 weeks reduced LA and increased n-3 PUFA concentrations in plasma, without altering the plasma AA concentration. A concurrent increase in dietary n-3 PUFA for 12 weeks further increased n-3 PUFA plasma concentrations, and also reduced AA. Dietary n-6 lowering can increase circulating n-3 PUFA bioavailability particularly when combined with dietary n-3 PUFAs, for maintaining brain and heart integrity. Research is being prepared for submission. IDENTIFICATION OF OXIDIZED LINOLEIC ACID METABOLITES (OXLAMS) IN PLASMA BY QUADRUPOLE TIME-OF-FLIGHT MASS SPECTROMETRY. Linoleic acid (LA) and LA-esters are the precursors of LA hydroperoxides, which are readily converted to 9- and 13-hydroxy-octadecadienoic acid (HODE) and 9- and 13-oxo-octadecadienoic acid (oxo ODE) metabolites in vivo. These oxidized LA metabolites (OXLAMs) have been implicated in a variety of pathological conditions. Therefore, their accurate measurement may provide mechanistic insights into disease pathogenesis. We published a novel quadrupole time-of-flight mass spectrometry (Q-TOFMS) method for quantitation and identification of target OXLAMs in plasma, using rat plasma. In this method, the esterified OXLAMs were base-hydrolyzed and followed by liquid-liquid extraction. Quantitative analyses were based on one-point standard addition with isotope dilution. The Q-TOFMS data of target metabolites were acquired and multiple reaction monitoring extracted-ion chromatograms were generated post-acquisition with a 10 ppm extraction window. The limit of quantitation was 9.7-35.9 nmol/L depending on the metabolite. The method was reproducible with a coefficient of variation of <18.5%. Mean concentrations of target metabolites in rat plasma were 57.8, 123.2, 218.1 and 57.8 nmol/L for 9-HODE, 13-HODE, 9-oxoODE and 13-oxoODE, respectively. Plasma levels of total OXLAMs were 456.9 nmol/L, which correlated well with published concentrations obtained by gas chromatography/mass spectrometry (GC/MS). The concentrations were also obtained utilizing a standard addition curve approach. The calibration curves were linear with correlation coefficients of >0.991. Concentrations of 9-HODE, 13-HODE, 9-oxoODE and 13-oxoODE were 84.0, 138.6, 263.0 and 69.5 nmol/L, respectively, which were consistent with the results obtained from one-point standard addition. Target metabolites were simultaneously characterized based on the accurate Q-TOFMS data. This is the first study of secondary LA metabolites using Q-TOFMS. (2). QUANTIFYING HEPATIC SECRETION OF DOCOSAHEXAENOIC ACID (DHA). DHA is a polyunsaturated a fatty acid enriched in brain tissue that has important structural and signaling implications for addictive disorders and neurological function. Rodent data and human trials suggest that dietary omega-6 linoleic acid interferes with liver synthesis-secretion of omega-3 DHA. Therefore high omega-6 linoleic acid diets may contribute to deficits in omega-3 DHA, leading to suboptimal neurological function. A reduction in dietary omega-6 linoleic acid may increase liver synthesis-secretion and tissue accumulation of omega-3 DHA from its dietary precursor omega-3 alpha-linolenic acid (LNA). To test this, we plan to use the BPMS established infusion method and model to quantify liver synthesis-secretion rates of long-chain omega-3 polyunsaturated fatty acids, (omega-3 EPA and DHA) from circulating unesterified omega-3 LNA in humans consuming an average US diet for 12 weeks. We plan to initiate an IRB approved protocol that requires a 22-hour hospital admission to the NIAAA inpatient unit, including an 8-hour infusion of d5-LNA with serial blood draws and LC/GC analysis. The primary outcome will be the liver synthesis-secretion rate of docosahexaenoic acid (DHA) from infused d5-LNA. PUFAS IN BIPOLAR DISORDER. Several publication indicate a distortion of plasma concentrations of n-3 and n-6 polyunsaturated fatty acids and their metabolites in bipolar disorder, possibly related to general inflammatory risk. We are initiating a collaboration with Dr. Erika Saunders at Penn State School of Medicine to measure concentrations of these compounds in patient with bipolar disorder in relation to diet and drug history.