Immunoquantitative Real-Time PCR for Detection and Quantification of Staphylococcus aureus Enterotoxin B in Foods

A real-time immunoquantitative PCR (iqPCR) method for detection of Staphylococcus aureus enterotoxin B (SEB) was developed and evaluated using both pure cultures and foods. The assay consisted of immunocapture of SEB and real-time PCR amplification of the DNA probe linked to the detection antibody. iqPCR was compared to an in-house enzyme-linked immunosorbent assay (ELISA) using the same couple of capture-detection antibodies and to commercial kits for detection of S. aureus enterotoxins (SE). The iqPCR was approximately 1,000 times more sensitive (<10 pg ml⁻¹) than the in-house ELISA and had a dynamic range of approximately 10 pg ml⁻¹ to approximately 30,000 pg ml⁻¹. iqPCR was not inhibited by any of the foods tested and was able to detect SEB present in these foods. No cross-reactivity with SE other than SEB was observed. Application of iqPCR for detection of SEB in cultures of S. aureus revealed the onset of SEB production after 4 h of incubation at 22, 37, and 42°C, which was in the first half of the exponential growth phase. The total amounts of SEB produced by the two strains tested were larger at 42°C than at 37°C and were strain dependent.     

LC-MS/MS detection of peroxynitrite-derived 3-nitrotyrosine in rat microvessels

3-Nitrotyrosine (3NT) is used as a biomarker of nitrative pathology caused by peroxynitrite (PN), myeloperoxidase (MPO)-, and/or eosinophil peroxidase (EPO)-dependent nitrite oxidation. 3NT measurements in biological materials are usually based on either antibody staining, HPLC detection, or GC detection methodologies. In this report, a procedure is described for the measurement of 3NT and tyrosine (TYR) by LC-MS/MS that is simple, direct, and sensitive. Though highly specialized in its use as an assay, LC-MS/MS technology is available in many research centers in academia and industry. The critical assay for 3NT was linear below 100 ng/ml and the limit of detection was below 100 pg/ml. Regarding protein digested samples, we found that MRM was most selective with 133.1 m/z as the daughter ion. In comparison, LC-ECD was 100 times less sensitive. Basal levels of 3NT in extracted digests of rat brain homogenate were easily detected by LC-MS/MS, but were below detection by LC-ECD. The LC-MS/MS assay was used to detect 3NT in rat brain homogenate that was filtered through a 180 micron nylon mesh. Three fractions were collected and examined by phase contrast microscopy. The mass ratio (3NT/TYR) of 3NT in fractions of large vessel enrichment, microvessel enrichment, and vessel depletion was 0.6 ng/mg, 1.2 ng/mg, and 0.2 ng/mg, respectively. Ultimately, we found that the basal 3NT/TYR mass ratio as determined by LC-MS/MS was six times greater in microvessel-enriched brain tissue vs. tissue devoid of microvessels.     

Deoxygedunin,a Natural Product with Potent Neurotrophic Activity in Mice

Gedunin, a family of natural products from the Indian neem tree, possess a variety of biological activities. Here we report the discovery of deoxygedunin, which activates the mouse TrkB receptor and its downstream signaling cascades. Deoxygedunin is orally available and activates TrkB in mouse brain in a BDNF-independent way. Strikingly, it prevents the degeneration of vestibular ganglion in BDNF −/− pups. Moreover, deoxygedunin robustly protects rat neurons from cell death in a TrkB-dependent manner. Further, administration of deoxygedunin into mice displays potent neuroprotective, anti-depressant and learning enhancement effects, all of which are mediated by the TrkB receptor. Hence, deoxygedunin imitates BDNF''s biological activities through activating TrkB, providing a powerful therapeutic tool for treatment of various neurological diseases.     

Deuterated docosahexaenoic acid protects against oxidative stress and geographic atrophy‐like retinal degeneration in a mouse model with iron overload

Oxidative stress plays a central role in age‐related macular degeneration (AMD). Iron, a potent generator of hydroxyl radicals through the Fenton reaction, has been implicated in AMD. One easily oxidized molecule is docosahexaenoic acid (DHA), the most abundant polyunsaturated fatty acid in photoreceptor membranes. Oxidation of DHA produces toxic oxidation products including carboxyethylpyrrole (CEP) adducts, which are increased in the retinas of AMD patients. In this study, we hypothesized that deuterium substitution on the bis‐allylic sites of DHA in photoreceptor membranes could prevent iron‐induced retinal degeneration by inhibiting oxidative stress and lipid peroxidation. Mice were fed with either DHA deuterated at the oxidation‐prone positions (D‐DHA) or control natural DHA and then given an intravitreal injection of iron or control saline. Orally administered D‐DHA caused a dose‐dependent increase in D‐DHA levels in the neural retina and retinal pigment epithelium (RPE) as measured by mass spectrometry. At 1 week after iron injection, D‐DHA provided nearly complete protection against iron‐induced retinal autofluorescence and retinal degeneration, as determined by in vivo imaging, electroretinography, and histology. Iron injection resulted in carboxyethylpyrrole conjugate immunoreactivity in photoreceptors and RPE in mice fed with natural DHA but not D‐DHA. Quantitative PCR results were consistent with iron‐induced oxidative stress, inflammation, and retinal cell death in mice fed with natural DHA but not D‐DHA. Taken together, our findings suggest that DHA oxidation is central to the pathogenesis of iron‐induced retinal degeneration. They also provide preclinical evidence that dosing with D‐DHA could be a viable therapeutic strategy for retinal diseases involving oxidative stress.     

Limits of detection for the determination of mono- and dicarboxylic acids using gas and liquid chromatographic methods coupled with mass spectrometry

The chromatographic separation and instrumental limits of detection (LODs) were obtained for a broad range of C(1)-C(18) monocarboxylic (MCAs) and C(2)-C(14) dicarboxylic acids (DCAs) employing either chemical derivatization followed by gas chromatography-mass spectrometry and flame ionization detection (GC-MS/FID) or direct analysis with liquid chromatography high resolution MS and tandem MS (LC-MS). Suitability, efficiency and stability of reaction products for several derivatization agents used for esterification (BF(3)/butanol), and trimethysilylation, including trimethylsilyl-N-N-dimethylcarbamate (TMSDMC) and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) were evaluated. The lowest limits of detection for the majority of compounds below 10 pg (with the exception of acetic acid) were obtained for derivatization with BF(3)/butanol followed by GC-MS in the total ion current (TIC) mode. Further improvements were achieved when applying either selected ion monitoring (SIM), which decreased the LODs to 1-4 pg or a combination of SIM and TIC (SITI) (2-5 pg). GC-FID provided LODs comparable to those obtained by GC-MS TIC. Both trimethylsilylation (followed by GC-MS) and direct LC-MS/MS analysis yielded LODs of 5-40 pg for most of the acids. For volatile acids the LODs were higher, e.g., 25 and 590 ng for TMSDMC and BSTFA derivatized formic acid, respectively, whereas the LC-MS methods did not allow for the analysis of formic acid at all.     

Disruption of FADS2 gene in mice impairs male reproduction and causes dermal and intestinal ulceration

Delta-6 desaturase (D6D) catalyzes the first step in the synthesis of highly unsaturated fatty acids (HUFA) such as arachidonic (AA), docosapentaenoic (DPAn-6), and docosahexaenoic (DHA) acids, as well as the last desaturation of DPAn-6 and DHA. We created D6D-null mice (−/−), which enabled us to study HUFA deficiency without depleting their precursors. In −/−, no in vivo AA synthesis was detected after administration of [U-¹³C]linoleic acid (LA), indicating absence of D6D isozyme. Unexpectedly, all of the −/− developed ulcerative dermatitis when fed a purified diet lacking D6D products but containing ample LA. The −/− also exhibited splenomegaly and ulceration in duodenum and ileocecal junction. Male −/− lacked normal spermatozoa with a severe impairment of spermiogenesis. Tissue HUFAs in −/− declined differentially: liver AA and DHA by 95%, and a smaller decrease in brain and testes. Dietary AA completely prevented dermatitis and intestinal ulcers in −/−. DPAn-6 was absent in −/− brain under AA supplementation, indicating absence of D6D isozyme for DPAn-6 synthesis from AA. This study demonstrated a distinct advantage of the D6D-null mice (−/−) to elucidate (1) AA function without complication of LA deprivation and (2) DHA function in the nervous system without AA depletion or DPAn-6 replacement seen in traditional models.—Stroud, C. K., T. Y. Nara, M. Roqueta-Rivera, E. C. Radlowski, P. Lawrence, Y. Zhang, B. H. Cho, M. Segre, R. A. Hess, J. T. Brenna, W. M. Haschek, and M. T. Nakamura. Disruption of FADS2 gene in mice impairs male reproduction and causes dermal and intestinal ulceration.     

Gaussian and linear deconvolution of LC-MS/MS chromatograms of the eight aminobutyric acid isomers

Isomeric molecules present a challenge for analytical resolution and quantification, even with MS-based detection. The eight aminobutyric acid (ABA) isomers are of interest for their various biological activities, particularly γ-aminobutyric acid (GABA) and the d- and l-isomers of β-aminoisobutyric acid (β-AIBA; BAIBA). This study aimed to investigate LC-MS/MS-based resolution of these ABA isomers as their Marfey's (Mar) reagent derivatives. HPLC was able to separate three Mar-ABA isomers l-β-ABA (l-BABA), and l- and d-α-ABA (AABA) completely, with three isomers (GABA, and d/l-BAIBA) in one chromatographic cluster, and two isomers (α-AIBA (AAIBA) and d-BABA) in a second cluster. Partially separated cluster components were deconvoluted using Gaussian peak fitting except for GABA and d-BAIBA. MS/MS detection of Marfey's derivatized ABA isomers provided six MS/MS fragments, with substantially different intensity profiles between structural isomers. This allowed linear deconvolution of ABA isomer peaks. Combining HPLC separation with linear and Gaussian deconvolution allowed resolution of all eight ABA isomers. Application to human serum found a substantial level of l-AABA (13 μM), an intermediate level of l-BAIBA (0.8 μM), and low but detectable levels (<0.2 μM) of GABA, l-BABA, AAIBA, d-BAIBA, and d-AABA. This approach should be useful for LC-MS/MS deconvolution of other challenging groups of isomeric molecules.     

Tissue-Specific Strategies of the Very-Long Chain Acyl-CoA Dehydrogenase-Deficient (VLCAD?/?) Mouse to Compensate a Defective Fatty Acid β-Oxidation

Very long-chain acyl-CoA dehydrogenase (VLCAD)-deficiency is the most common long-chain fatty acid oxidation disorder presenting with heterogeneous phenotypes. Similar to many patients with VLCADD, VLCAD-deficient mice (VLCAD^−/−) remain asymptomatic over a long period of time. In order to identify the involved compensatory mechanisms, wild-type and VLCAD^−/− mice were fed one year either with a normal diet or with a diet in which medium-chain triglycerides (MCT) replaced long-chain triglycerides, as approved intervention in VLCADD. The expression of the mitochondrial long-chain acyl-CoA dehydrogenase (LCAD) and medium-chain acyl-CoA dehydrogenase (MCAD) was quantified at mRNA and protein level in heart, liver and skeletal muscle. The oxidation capacity of the different tissues was measured by LC-MS/MS using acyl-CoA substrates with a chain length of 8 to 20 carbons. Moreover, in white skeletal muscle the role of glycolysis and concomitant muscle fibre adaptation was investigated. In one year old VLCAD^−/− mice MCAD and LCAD play an important role in order to compensate deficiency of VLCAD especially in the heart and in the liver. However, the white gastrocnemius muscle develops alternative compensatory mechanism based on a different substrate selection and increased glucose oxidation. Finally, the application of an MCT diet over one year has no effects on LCAD or MCAD expression. MCT results in the VLCAD^−/− mice only in a very modest improvement of medium-chain acyl-CoA oxidation capacity restricted to cardiac tissue. In conclusion, VLCAD^−/− mice develop tissue-specific strategies to compensate deficiency of VLCAD either by induction of other mitochondrial acyl-CoA dehydrogenases or by enhancement of glucose oxidation. In the muscle, there is evidence of a muscle fibre type adaptation with a predominance of glycolytic muscle fibres. Dietary modification as represented by an MCT-diet does not improve these strategies long-term.     

Ascorbic Acid-Dependent Regulation of Redox Levels of Chlorogenic Acid and its Isomers in the Apoplast of Leaves of Nicotiana tabacum L.

There is a question whether ascorbic acid (AA) can control redoxlevels of phenolics in the apoplast. The present study was designedto answer this question. AA, dehydroascorbic acid (DHA), chlorogenicacid (CGA) and its two structural isomers were present in theapoplast of leaves of tobacco (Nicotiana tabacum L. cv. BelW3).The levels of AA plus DHA (AA + DHA) and the ratios of AA to(AA + DHA) decreased while the levels of CGA plus its isomersincreased during leaf aging. o-Quinones of CGA plus its isomerswere found in the apoplast only in aged leaves of which apoplasticlevel of AA was nearly zero. In addition, activity of apoplasticperoxidase that could oxidize CGA and its isomers increasedduring leaf aging. From the observations, it is concluded thatAA can regulate the accumulation of the o-quinones of CGA andits isomers in the apoplast. Based on the conclusion, it isproposed that soluble peroxidase in the apoplast has two functions,namely, (i) scavenging of H₂O₂ and/or regulation of the levelof apoplastic H₂O₂ in the presence of AA, and (ii) accumulationof oxidation products of the phenolics in the absence of AA. (Received January 30, 1998; Accepted April 7, 1998)     

Imaging decreased brain docosahexaenoic acid metabolism and signaling in iPLA2�� (VIA)-deficient mice

Ca²⁺-independent phospholipase A₂β (iPLA₂β) 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 iPLA₂β-deficient mice without overt neuropathology. Saline or the cholinergic muscarinic M_1,3,5 receptor agonist arecoline (30 mg/kg) was administered to unanesthetized iPLA₂β^−/−, iPLA₂β^+/−, and iPLA₂β^+/+ mice, and [1-¹⁴C]DHA was infused intravenously. DHA incorporation coefficients k* and rates J_in, representing DHA metabolism, were determined using quantitative autoradiography in 81 brain regions. iPLA₂β^−/− or iPLA₂β^+/− compared with iPLA₂β^+/+ mice showed widespread and significant baseline reductions in k* and J_in for DHA. Arecoline increased both parameters in brain regions of iPLA₂β^+/+ mice but quantitatively less so in iPLA₂β^−/− and iPLA₂β^+/− mice. Consistent with iPLA₂β’s reported ability to selectively hydrolyze DHA from phospholipid in vitro, iPLA₂β deficiency reduces brain DHA metabolism and signaling in vivo at baseline and following M_1,3,5 receptor activation. Positron emission tomography might be used to image disturbed brain DHA metabolism in patients with PLA2G6 mutations.     

In vitro replication and thermodynamic studies of methylation and oxidation modifications of 6-thioguanine

The cytotoxic effects of thiopurine drugs are mostly exerted through the formation of thioguanine nucleotide and its subsequent incorporation into DNA. The 6-thioguanine (6-TG) in DNA can be converted to S⁶-methylthio-2-aminopurine (2-AP-6-SCH₃) and 2-aminopurine-6-sulfonic acid (2-AP-6-SO₃H) upon reaction with S-adenosyl-L-methionine and irradiation with UVA light, respectively. Here we prepared oligodeoxynucleotides (ODNs) harboring a 6-TG, 2-AP-6-SCH₃ or 2-AP-6-SO₃H at a defined site and examined, by using LC-MS/MS, the in vitro replication of these substrates with yeast polymerase η and Klenow fragment (KF⁻). Our results revealed that 2-AP-6-SCH₃ could be bypassed by KF⁻, with significant misincorporation of thymine opposite the lesion. The 2-AP-6-SO₃H, however, blocked markedly the nucleotide insertion by KF⁻. Yeast pol η could bypass all three modified nucleosides; although dCMP was inserted preferentially, we found substantial misincorporation of dTMP and dAMP opposite 2-AP-6-SCH₃ and 2-AP-6-SO₃H, respectively. Moreover, both KF⁻ and yeast pol η induced a considerable amount of -2 frameshift products from the replication of 2-AP-6-SCH₃- and 2-AP-6-SO₃H-bearing substrates. Our results also underscored the importance of measuring the relative ionization efficiencies of replication products in the accurate quantification of these products by LC-MS/MS. Moreover, thermodynamic studies revealed that 2-AP-6-SCH₃ and 2-AP-6-SO₃H could cause more destabilization to duplex DNA than 6-TG. Taken together, the results from this study shed important new light on the biological implications of the two metabolites of 6-TG.     

Simultaneous analysis of angiotensin peptides by LC-MS and LC-MS/MS: metabolism by bovine adrenal endothelial cells

Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods were developed to simultaneously determine the concentrations of angiotensin (Ang) II, Ang 1-7, Ang III, and Ang IV in biological samples. The samples were extracted with C18 solid-phase extraction cartridges and separated by a reverse-phase C18 column using acetonitrile in water with 0.1% formic acid as a mobile phase. Ang peptides were ionized by electrospray and detected by triple quadrupole MS in the positive ion mode. (M+3H)(3+) and (M+2H)(2+) ions were chosen as the detected ions in the single ion recording (SIR) mode for LC-MS. The limits of detection (signal/noise [S/N]=3) using SIR are 1 pg for Ang IV and 5 pg for Ang 1-7, Ang III, and Ang II. Multiple reaction monitoring (MRM) mode was used for LC-MS/MS. The limits of detection (S/N =3) using MRM are 20 pg for Ang IV and 25 pg for Ang 1-7, Ang III, and Ang II. These methods were applied to analyze Ang peptides in bovine adrenal microvascular endothelial cells. The results show that Ang II is metabolized by endothelial cells to Ang 1-7, Ang III, and Ang IV, with Ang 1-7 being the major metabolite.     

Neurofurans, novel indices of oxidant stress derived from docosahexaenoic acid

Isoeicosanoids are free radical-catalyzed isomers of the enzymatic products of arachidonic acid. They are formed in situ in cell membranes, are cleaved, circulate, and are excreted in urine. Isomers of prostaglandin F(2alpha), the F(2)-isoprostanes, have emerged as sensitive indices of lipid peroxidation in vivo. Analogous compounds formed from docosahexaenoic acid (DHA) are termed neuroprostanes and are more abundant than isoprostanes (iPs) in brain. Isofurans are another class of isoeicosanoids characterized by a substituted tetrahydrofuran ring. They are preferentially formed, relative to iPs, under conditions of elevated oxygen tension. Here, we report the discovery of neurofurans (nFs), the analogous family of compounds formed from DHA. Formation of nFs is characterized by mass spectrometry and confirmed by oxidation of DHA in vitro and following CCl(4) administration in liver in vivo. It is demonstrated that the levels of nFs are elevated in the brain cortex of a mouse model of Alzheimer disease and are depressed in mouse brain cortex by deletion of p47(phox), an essential component of the phagocyte NADPH oxidase. Measurement of the nFs may ultimately prove useful in diagnosis, timing, and selection of dose in the treatment and chemoprevention of neurodegenerative disease.     

Conversion of Glycerol to Dihydroxyacetone by Immobilized Whole Cells of Acetobacter xylinum

Enzymatic production of dihydroxyacetone (DHA) was studied by immobilization of the whole cells of acetic acid bacteria capable of oxidizing glycerol to DHA. Acetobacter xylinum A-9 cells immobilized in a polyacrylamide gel were selected as the most favorable enzyme preparation. The enzymatic properties of immobilized cells converting glycerol to DHA were investigated and compared with those of intact cells. The optimum pH for the immobilized cells was broad (4.0 to 5.5), whereas the intact cells had a narrow pH optimum at 5.5. The thermal stability of the immobilized cells was somewhat higher than that of the intact cells. Apparent K_m values for glycerol with both intact and immobilized cells were about equal, 6.3 × 10⁻² to 6.5 × 10⁻² M. The complete conversion of glycerol to DHA was achieved within 40 h under optimum conditions, and pure crystalline DHA was readily isolated from the reaction mixture with over 80% yield.     

Determination of 4,4′-methylenediphenyldianiline (MDA) and identification of isomers in technical-grade MDA in hydrolysed plasma and urine from workers exposed to methylene diphenyldiisocyanate by gas chromatography-mass spectrometry

Gas chromatography-mass spectrometry using chemical ionization with ammonia as reagent gas monitoring both positive and negative ions was applied. Negative-ion monitoring using ammonia and the pentafluoropropionic anhydride (PFPA) derivatives were chosen owing to low detection limits and good separation for the isomers studied. Technical-grade methylenediphenyldiioscyanate (MDI) was analysed and three isomers, 4,4′−, 2.4′− and 2,2−methylenediphenyldianiline (MDA), were determined in addition to methylated MDA. Plasma and urine from an exposed worker were hydrolysed and analysed and the MDA isomers were identified in the biological samples.     

Cis astaxanthin and especially 9-cis astaxanthin exhibits a higher antioxidant activity in vitro compared to the all-trans isomer

In recent years, a number of studies have implicated the potent antioxidant property of astaxanthin in various experimental systems; however, these studies employed only the all-trans isomer. On the other hand, it has been reported that all-trans natural astaxanthin is readily isomerized to cis-trans, especially 9-cis and 13-cis isomers, under certain conditions by chemical analysis; however, the biological activities of the cis isomers of astaxanthin are little known. In the present study, we investigated the antioxidant activity of 9-cis and 13-cis astaxanthin compared to the all-trans isomer in vitro. In a stable radical DPPH scavenging activity test and in rat microsome and rabbit erythrocyte ghost membrane lipid peroxidation systems induced by AAPH and t-BuOOH, respectively, the results apparently showed that cis-astaxanthin, especially 9-cis astaxanthin, exhibited a higher antioxidant effect than the all-trans isomer. In addition, during polyunsaturated fatty acid (PUFA) oxidation, both DHA and linoleic acid hydroperoxides formation were markedly inhibited by astaxanthin isomers addition in the order 9-cis >13-cis >all-trans. Furthermore, 9-cis also exhibited the most effective inhibition of the generation of ROS induced by 6-hydroxydopamine (6-OHDA) in human neuroblastoma SH-SY5Y cells among the astaxanthin isomers, as well as on the degradation of collagen type II induced by DHA and linoleic acid hydroperoxides. The above-mentioned results suggest, for the first time, that cis isomer astaxanthin, especially 9-cis astaxanthin, has a much higher antioxidant potency than that of the all-trans isomer.     

Docosahexaenoic Acid Decreases Pro-Inflammatory Mediators in an In Vitro Murine Adipocyte Macrophage Co-Culture Model

Paracrine interactions between adipocytes and macrophages contribute to chronic inflammation in obese adipose tissue. Dietary strategies to mitigate such inflammation include long-chain polyunsaturated fatty acids, docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids, which act through PPARγ-dependent and independent pathways. We utilized an in vitro co-culture model designed to mimic the ratio of macrophages:adipocytes in obese adipose tissue, whereby murine 3T3-L1 adipocytes were cultured with RAW 264.7 macrophages in direct contact, or separated by a trans-well membrane (contact-independent mechanism), with 125 µM of albumin-complexed DHA, EPA, palmitic acid (PA), or albumin alone (control). Thus, we studied the effect of physical cell contact versus the presence of soluble factors, with or without a PPARγ antagonist (T0070907) in order to elucidate putative mechanisms. After 12 hr, DHA was the most anti-inflammatory, decreasing MCP1 and IL-6 secretion in the contact system (−57%, −63%, respectively, p≤0.05) with similar effects in the trans-well system. The trans-well system allowed for isolation of cell types for inflammatory mediator analysis. DHA decreased mRNA expression (p<0.05) of Mcp1 (−7.1 fold) and increased expression of the negative regulator, Mcp1-IP (+1.5 fold). In macrophages, DHA decreased mRNA expression of pro-inflammatory M1 polarization markers (p≤0.05), Nos2 (iNOS; −7 fold), Tnfα (−4.2 fold) and Nfκb (−2.3 fold), while increasing anti-inflammatory Tgfβ1 (+1.7 fold). Interestingly, the PPARγ antagonist co-administered with DHA or EPA in co-culture reduced (p≤0.05) adiponectin cellular protein, without modulating other cytokines (protein or mRNA). Overall, our findings suggest that DHA may lessen the degree of MCP1 and IL-6 secreted from adipocytes, and may reduce the degree of M1 polarization of macrophages recruited to adipose tissue, thereby decreasing the intensity of pro-inflammatory cross-talk between adipocytes and macrophages in obese adipose tissue.     

Dietary Docosahexaenoic Acid Supplementation Modulates Hippocampal Development in the Pemt?/? Mouse

The development of fetal brain is influenced by nutrients such as docosahexaenoic acid (DHA, 22:6) and choline. Phosphatidylethanolamine-N-methyltransferase (PEMT) catalyzes the biosynthesis of phosphatidylcholine from phosphatidylethanolamine enriched in DHA and many humans have functional genetic polymorphisms in the PEMT gene. Previously, it was reported that Pemt^−/− mice have altered hippocampal development. The present study explores whether abnormal phosphatidylcholine biosynthesis causes altered incorporation of DHA into membranes, thereby influencing brain development, and determines whether supplemental dietary DHA can reverse some of these changes. Pregnant C57BL/6 wild type (WT) and Pemt^−/− mice were fed a control diet, or a diet supplemented with 3 g/kg of DHA, from gestational day 11 to 17. Brains from embryonic day 17 fetuses derived from Pemt^−/− dams fed the control diet had 25–50% less phospholipid-DHA as compared with WT (p < 0.05). Also, they had 60% more neural progenitor cell proliferation (p < 0.05), 60% more neuronal apoptosis (p < 0.01), and 30% less calretinin expression (p < 0.05; a marker of neuronal differentiation) in the hippocampus compared with WT. The DHA-supplemented diet increased fetal brain Pemt^−/− phospholipid-DHA to WT levels, and abrogated the neural progenitor cell proliferation and apoptosis differences. Although this diet did not change proliferation in the WT group, it halved the rate of apoptosis (p < 0.05). In both genotypes, the DHA-supplemented diet increased calretinin expression 2-fold (p < 0.05). These results suggest that the changes in hippocampal development in the Pemt^−/− mouse could be mediated by altered DHA incorporation into membrane phospholipids, and that maternal dietary DHA can influence fetal brain development.     

Inhibition of astroglial glutamate transport by polyunsaturated fatty acids: Evidence for a signalling role of docosahexaenoic acid

Brain cells are especially rich in polyunsaturated fatty acids (PUFA), mainly the n-3 PUFA docosahexaenoic acid (DHA) and the n-6 PUFA arachidonic acid (AA). They are released from membranes by PLA2 during neurotransmission, and may regulate glutamate uptake by astroglia, involved in controlling glutamatergic transmission. AA has been shown to inhibit glutamate transport in several model systems, but the contribution of DHA is less clear and has not been evaluated in astrocytes. Because the high DHA content of brain membranes is essential for brain function, we investigated the role of DHA in the regulation of astroglial glutamate transport.We evaluated the actions of DHA and AA using cultured rat astrocytes and suspensions of rat brain membranes (P1 fractions). DHA reduced d-[³H]aspartate uptake by cultured astrocytes and cortical membrane suspensions, while AA did not. This also occurred in astrocytes enriched with α-tocopherol, indicating that it was not due to peroxidation products. The reduction of d-[³H]aspartate uptake by DHA did not involve any change in the concentrations of membrane-associated astroglial glutamate transporters (GLAST and GLT-1), suggesting that DHA reduced the activity of the transporters. In contrast with the inhibition induced by free-DHA, we found no effect of membrane-bound DHA on d-[³H]aspartate uptake. Indeed, the uptake was similar in astrocytes with varying amount of DHA in their membrane (induced by long-term supplementation with DHA or AA). Therefore, DHA reduces glutamate uptake through a signal-like effect but not through changes in the PUFA composition of the astrocyte membranes. Also, reactive astrocytes, induced by a medium supplement (G5), were insensitive to DHA. This suggests that DHA regulates synaptic glutamate under basal condition but does not impair glutamate scavenging under reactive conditions.These results indicate that DHA slows astroglial glutamate transport via a specific signal-like effect, and may thus be a physiological synaptic regulator.