Imaging plasma docosahexaenoic acid (dha) incorporation into the brain in vivo , as a biomarker of brain DHA: Metabolism and neurotransmission

Inastudyinvolv-Abstract: Docosahexaenoic acid (DHA) is critical for normal brain structure and function, and its brain concentration depends on dietary DHA content and hepatic conversion from its dietary derived n-3 precursor, a -linolenic acid ( a -LNA). We developed an in vivo method in rats using quantitative autoradiography to image incorporation into brain of unesteriﬁed plasma DHA, and showed that the incorporation rate equals the rate of brain metabolic DHA consumption. Thus, quantitative imaging of DHA incorporation from plasma into brain can be used as a biomarker of brain DHA metabolism and neurotransmission. The method has been extended to humans with the use of positron emission tomography (PET). Furthermore, imaging in unanesthetized rats using DHA incorporation as a biomarker in response to N-methyl-D-aspartate (NMDA) administration conﬁrms that regional DHA signaling is independent of extracellular calcium, and likely mediated by a calcium-independent phospholipase A 2 (iPLA 2 ). Studies in mice in which iPLA 2 -VIA ( b ) was knocked out conﬁrmed that this enzyme is critical for baseline and muscarinic cholinergic signaling involving DHA.

Multiple rodent studies have demonstrated that prolonged dietary n-3 PUFA deprivation, from 15 weeks to as long 3 generations, results in reduced brain DHA content within individual phospholipids, associated with increased brain concentrations of docosapentaenoic acid (DPA)n-6 (22:5n-6) largely derived from liver biosynthesis (Salem et al., 2001). These studies generally involved euthanization and direct chemical analysis of brain lipid content, limiting our ability to interpret in vivo brain DHA metabolism and kinetics. In view of conflicting evidence that low dietary intake of DHA-containing fish products is correlated with multiple human brain diseases, including Alzheimer disease, and that dietary DHA supplementation may be helpful in some of these conditions (Conquer et al., 2000;Quinn et al., 2010), it would be useful to have an in vivo biomarker of regional brain DHA consumption that could be used in awake animals and in humans to test the efficacy of DHA supplementation and other experimental or clinical conditions. Further, it would be useful to understand DHA participation in signal transduction, thus if DHA incorporation into brain might be used as a biomarker of regional brain metabolism and neurotransmission in health and disease.
Neither arachidonic acid (AA, 20:4n-6) nor DHA can be synthesized de novo from 2 carbon chains in vertebrates (Holman, 1986). Both long-chain PUFAs enter the brain from the circulation, but quantitation of their rates of entry as well as of their disposition within brain have remained controversial. In a study involv-Abstract: Docosahexaenoic acid (DHA) is critical for normal brain structure and function, and its brain concentration depends on dietary DHA content and hepatic conversion from its dietary derived n-3 precursor, a-linolenic acid (a-LNA). We developed an in vivo method in rats using quantitative autoradiography to image incorporation into brain of unesterified plasma DHA, and showed that the incorporation rate equals the rate of brain metabolic DHA consumption. Thus, quantitative imaging of DHA incorporation from plasma into brain can be used as a biomarker of brain DHA metabolism and neurotransmission. The method has been extended to humans with the use of positron emission tomography (PET). Furthermore, imaging in unanesthetized rats using DHA incorporation as a biomarker in response to N-methyl-D-aspartate (NMDA) administration confirms that regional DHA signaling is independent of extracellular calcium, and likely mediated by a calcium-independent phospholipase A 2 (iPLA 2 ). Studies in mice in which iPLA 2 -VIA (b) was knocked out confirmed that this enzyme is critical for baseline and muscarinic cholinergic signaling involving DHA. ing feeding a radiolabeled fatty acid to rats (Purdon et al., 1997), we showed that only after its hydrolysis from circulating lipoprotein was its brain uptake measurable, and at a rate equivalent to the rate following injecting the unesterified fatty acid intravenously, thus arguing for uptake of the unesterified fatty acid. This finding was supported by studies in mice genetically lacking lipoprotein receptors (Chen et al., 2008). Calculations of on-off kinetics of unesterified fatty acids bound to serum albumin indicate that about 5% of the fatty acid is removed from albumin as blood passes through brain (Robinson et al., 1992).
It is possible to calculate and image the rate of incorporation of unesterified unlabeled DHA in a single study in an unanesthetized rodent, by infusing radiolabeled [1-14 C]DHA intravenously and measuring regional incorporation coefficients k* into brain using quantitative autoradiography, then multiplying the unlabeled concentration of unesterified plasma DHA by the incorporation coefficient to calculate the incorporation rate, J in , of unlabeled DHA, where the asterisk identifies labeled plasma or brain concentrations of DHA (Robinson et al., 1992).
Doing this gives a whole brain incorporation rate of DHA equal to about 0.19 mmol/gram brain per day in the adult rat (Contreras et al., 2000). Confirmation that this calculated incorporation rate from a single study equals the rate of whole brain DHA consumption was derived from separate studies in multiple rats in which [4,5-3 H]DHA was injected into the cerebral ventricles and brain DHA radioactivity and concentrations were followed in animals killed animals from 0 to 60 days thereafter. A whole brain half-life of 33 days was calculated for DHA, giving a daily rate of DHA consumption of 0.25 mmol/g/day (DeMar et al., 2004), equivalent to the single injection value considering the variance of the data. The efficiency and simplicity of the single injection measurement, using quantitative autoradiography, makes it ideal for measuring whole brain DHA consumption.
Additional measurements with intracerebroventricular [4,5-3 H]DHA showed that 15 weeks of dietary n-3 PUFA deprivation prolonged the DHA half-life in brain to 90 days and reduced brain DHA consumption to 0.06 mmol/g/day, while downregulating expression of calcium-independent phospholipase A 2 (iPLA 2 )-VIA (b) (Rao et al., 2007). In vitro studies indicate that this enzyme is selective for the hydrolysis of DHA from membrane phospholipid (DeMar et al., 2004;Garcia and Kim, 1997;Strokin et al., 2004). The observed downregulation of enzyme expression with would be expected to help to preserve brain DHA. As DHA metabolites, many of which are antiinflammatory and include neuroprotectins and resolvins, are the major pathways of DHA metabolic loss in brain (Bazan, 2009), these results provided a basis for the reduced resistance to neuroinflammation and cognitive dysfunction in animals subjected to dietary n-3 PUFA deprivation (DeMar et al., 2006;Farooqui et al., 2007).
The equivalence between J in for DHA calculated from an intravenous infusion injection at a single time point, and the DHA consumption rate calculated by intracerebroventricular injection followed by sampling brain from multiple animals over a 60-day period, indicates that the single time point measurement represents a biomarker of brain DHA consumption. Accordingly, we synthesized positron-labeled [1-11 C]DHA and conducted studies using positron emission tomography (PET) to quantitatively image incorporation of unesterified plasma DHA into the brain of adult healthy human volunteers (Channing and Simpson, 1993;Umhau et al., 2009). Values of incorporation coefficients k* for DHA were higher in gray than white matter brain regions. For the entire human brain, the net DHA incorporation rate J in , the product of k* and the unesterified plasma DHA concentration, equaled 3.8AE1.7 mg/ day ( figure 1). This net rate, approximating the net rate of DHA consumption by brain, is less than the suggested human diet DHA supplementation 200 mg per day of DHA (Kris-Etherton et al., 2000).
In an as yet unpublished study (Kim et al., submitted), we subjected rats post-weaning for 15 weeks to DHA-free diets having graded reductions in a-LNA content below a dietary ''sufficient'' level containing 4.6% a-LNA (DeMar et al., 2004). While plasma DHA fell in rough proportion to the reduction in dietary a-LNA, the brain DHA concentration surprisingly was maintained down to 1.7% dietary a-LNA, suggesting that brain DHA does not track plasma DHA until quite large reductions in plasma concentration arise. In this regard, plasma DHA is reduced by 50% in vegetarians compared with omnivores (Rosell et al., 2005), despite there being no difference in overall mortality or mortality from any general cause between the two groups (Key et al., 2009). Together, the rodent and human data suggests that using the blood DHA concentration as a biomarker of brain DHA integrity may be incorrect. Thus, it would be of importance to determine whether brain consumption of DHA, measured with our intravenous infusion method in rodents or humans, compared with the brain concentration of DHA, measured by direct determination in rodents, is the major determinant of brain functional integrity by measuring consumption in relation to plasma DHA content in rats or in humans with the intravenous infusion method.
One approach that as has not been exploited sufficiently is the use of radiolabeled DHA to image its role in regional brain signal transduction and neuroplasticity using quantitative autoradiography in vivo. For example, 3 months after removing one eye in a rat, DHA incorporation from plasma into the contralateral brain regions, e.g. superficial gray matter of the superior colliculus and dorsal lateral geniculate nucleus, that normally were innervated by the eye that was removed, was significantly reduced, emphasizing a role for DHA in signaling and neuroplasticity that deserves to be exploited (Wakabayashi et al., 1995). DHA incorporation also was altered in an L1210 leukemia cells implanted in rat brain, suggesting a role for DHA in tumor metabolism (Nariai et al., 1994).
Recognizing the in vitro enzyme selectivity of cPLA 2 and iPLA 2 for AA and DHA, respectively, we confirmed their in vivo dependencies on extracellularderived calcium in unanesthetized rats by showing that NMDA administration increased incorporation of intravenously injected radiolabeled AA but not of radiolabeled DHA into the brain  (figure 2). The results suggest that greater AA than DHA release during glutamate-induced excitotoxicity could cause brain cell damage since high concentrations of AA and its metabolites are considered to be neurotoxic and proinflammatory (Bazan et al., 1981).
We also confirmed a role for iPLA 2 -VIA (iPLA 2 b) in brain DHA signaling in vivo DeGeorge et al., 1991), consistent with evidence that this enzyme can be activated by calcium derived from intracellular calcium stores of the endoplasmic reticulum, thereby displacing it from calmodulin (Rosa and Rapoport, 2009). Mutations in the PLA2G6 gene encoding the enzyme occur in patients with idiopathic neuro-degeneration plus brain iron accumulation and dystonia-parkinsonism without iron accumulation (Kurian et al., 2008), whereas mice lacking PLA2G6 show 3.8 ± 1.7 (SD) mg/brain/day neurological dysfunction and significant neuropathology after 13 but not 4 months of age. We hypothesized that brain DHA metabolism and signaling in response to the cholinergic muscarinic M 1,3,5 agonist, arecoline (DeGeorge et al., 1991) would be reduced in 4month-old iPLA 2 b-deficient mice without overt neuropathology. Saline or arecoline (30 mg/kg) was administered to unanesthetized (homozygous, heterozygous or wildtype mice iPLA 2 b (-/-), (+/-), or (+/+) mice), and [1-14 C]DHA was infused intravenously. DHA incorporation coefficients and rates representing DHA metabolism were determined using quantitative autoradiography in 81 brain regions. iPLA 2 b(-/-) and (+/-) mice compared with iPLAb (+/+) mice showed widespread and significant baseline reductions in k* and J in for DHA. Arecoline increased both parameters in brain regions of iPLA 2 b(+/+) mice but quantitatively less so in iPLA 2 b(-/-) and iPLA 2 b (+/-) mice . Consistent with iPLA 2 b's reported ability to selectively hydrolyze DHA from phospholipid in vitro, a genetic iPLA 2 b deficiency reduced brain DHA metabolism and signaling in vivo at baseline and following M 1,3,5 receptor activation by arecoline. Positron emission tomography might be used to image disturbed brain DHA metabolism in patients with PLA2G6 mutations.

Conclusion
Brain imaging of DHA incorporation (consumption) as a biomarker of DHA metabolism following a single intravenous injection of radiolabeled DHA may prove useful in studying the role of DHA in health and disease, and in investigating the influence of diet, in animal and humans. As noted above, in a study in which plasma DHA was reduced in relation to reduced dietary a-LNA in rats subjected to 15 weeks of a DHA-free diet containing different a-LNA content starting with 4.6% DHA (adequate diet), brain DHA did not follow plasma DHA but fell only after dietary DHA had declined by more than half, to 1.7% a-LNA (Kim et al., submitted). Thus brain DHA did not track plasma DHA when the latter was reduced markedly by diet. A similar lack of tracking may occur in humans a well.
Another important issue is that brain DHA content and metabolism depend not only on diet, but also on the ability of the liver to synthesize DHA from circulating a-LNA, making it critical to be able to assess liver synthesis under different dietary conditions. To address this issue, we have developed a method involving a constant intravenous infusion of heavy isotopically labeled precursor [U-13 C]a-LNA for 2 h in rats on a DHA containing diet, while measuring labeled and unlabeled n-3 PUFA in arterial plasma using negative chemical ionization GC-MS (Gao et al., 2009b). Newly synthesized esterified [ 13 C]DHA, [ 13 C]EPA, and [ 13 C]DPA (22:5n-3) appeared in arterial plasma after 60 min of infusion, then their concentrations rose in an S-shaped manner. Esterified concentration Â plasma volume data were fit with a sigmoidal equation, whose peak first derivatives provided synthesis rates of unlabeled EPA, DPA, and DHA. The DHA synthesis rate exceeded the published daily rat brain DHA consumption rate by 30-fold, suggesting that liver synthesis from a-LNA could maintain brain DHA homeostasis were DHA absent from the diet. Conversion rate from infused isotopically labeled EPA also were measured (Gao et al., 2009b). More recently, we showed that the synthesis rates from a-LNA were markedly elevated when DHA was absent from the diet, as was expression of appropriate liver elongases and desaturases (Gao et al., unpublished). This stable isotope infusion method could be used to quantify whole-body DHA synthesis rates in humans in relation to consumption by brain and other organs, and fill out the equation that whole body DHA is the sum of dietary and hepatic inputs.
The ability to image labeled DHA incorporation into brain with quantitative autoradiography in rodents or with PET in humans provides an opportunity as a biomarker that remains to be exploited under different experimental and clinical conditions.