**5. Diet, glutamate, neuroinflammation, and neurotoxicity**

#### **5.1 Monosodium glutamate (MSG): a glutamate receptor agonist**

Monosodium glutamate (MSG) is the sodium salt of *L*-glutamic acid. It is a natural dietary component found in dairy products as Roquefort and Parmesan cheese, and vegetables such as tomatoes, mushrooms, and broccoli. The unique taste of MSG, known as an essential component of the Asian cuisine, evoked its widespread use in restaurants and canned food all over the world to improve food palatability. The L-glutamic acid itself and its disodium salt have a milder taste. The average daily intake in humans ranges from 0.3 to 1.0 g [88]*.*

Despite being generally recognized as safe (GRAS) by the food safety regulatory agencies, animal and human studies continue to raise concerns about its potential toxicity. In 2006, the European Food Safety Association (EFSA) included MSG in the list of food additives for which established acceptable daily intake (ADI) was reassessed to be 30 mg/kg, considering its no-observed adverse effect level (NOAEL) that is 3200 mg/kg.

Focusing on its neurotoxicity, MSG has been alleged of causing stroke, epilepsy, schizophrenia, anxiety, depression, and AD [89]*,* all of which predispose to aggression. This food additive acts on Glu receptors, triggering an array of inflammatory events and oxidative stress [90], especially with chronic consumption of high doses [91]*.* By binding to hepatic Glu receptors, excess NH4 ions are produced, with the secondary generation of reactive oxygen species (ROS), and eventual hepatotoxicity [92], impairing MSG metabolism, leading to its blood accumulation, and increasing the likelihood of neurotoxicity. Downregulating mGluRs and NMDA receptors was one of the protective mechanisms exerted by curcumin against MSG neurotoxicity [93].

Multiple experiments tracked the behavioral and neurochemical events associated with MSG [94–99]. Notably, extrapolating animal studies employing the systemic route of administration to human practice may flaw results interpretation, bypassing the usual metabolic breakdown of oral MSG ingested in food [100]*.*

Interestingly, a positive link was detected between MSG and hemoglobin levels [101] and it was found to reduce the percentage of blood lymphocytes as well [102]*.* Moreover, the oxidative stress during MSG toxicity overwhelmed the Glu-derived antioxidants generated by erythrocytes [103].

### **5.2 Omega-3 (ω3) versus omega-6 (ω6) long-chain polyunsaturated fatty acids (LC-PUFA)**

Polyunsaturated fatty acids (PUFA) are those containing two or more carbon double bonds, classified as omega-3, -6, and -9*.* Among long-chain polyunsaturated fatty acids (LC-PUFA), omega-3 (ω3), and omega-6 (ω6) can be discriminated. While the literature recommended the addition of ω3 sources in the diet, they advised to limit the consumption of its nonidentical twin, ω6. A debatable issue was to whether focus on the relative ω6 to ω3 consumption, versus determining absolute figures for each [104]*.*

Despite being essential FA, ω6 PUFA have a narrow therapeutic window, requiring a rational dietary consumption to establish physiologic, rather than deleterious effects [105]*.* The major dietary ω6 is linoleic acid (LA), converted to other ω6 products as γ-linolenic acid and dihomo-γ-linolenic acid, and from which arachidonic acid (AA) is derived, yielding pro-inflammatory molecules and ROS. Rich sources of LA are vegetable oils such as corn, sunflower, soy, and canola oils, while AA is present in meat and eggs mainly [106]*.* The recommended daily intake of LA in adult men is 17 g/day, to be further reduced for adult women to 12 g/day [107]*.*

On the other side, the major component of ω-3 PUFA is the alpha-linolenic acid (ALA), contained in chia seeds, black walnuts, and soybean oil, and converted in the liver to docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA)*,* and decosapentaenoic acid (DPA) [108, 109]*;* the latter is a potential reservoir for DHA and EPA [110]. The consumption of fatty fish, such as salmon, herring, sardines, mackerel, and cod liver oil, or the substitution with fish oil, as rich sources of ω3 PUFA, was adopted to improve neurological functions, especially that relevant synthesizing enzymes are lacking [111] and the plant-based sources containing ALA are insufficient for humans [112], due to the incomplete hepatic conversion to EPA and DHA [113].

The fatty acid composition of the brain consists of palmitate, AA, an ω6 PUFA, and DHA, as the major ω3 PUFA, other members of the latter group are present, but in very small quantities [114]. The brain depends on the uptake of ω3 PUFA from dietary or liver sources. Once absorbed from diet, ω3 PUFA are transported by lipoproteins and albumin to the blood stream [115]*.* In adult mice, blood and brain levels of ω3 PUFA (DHA and EPA) were dependent on dietary consumption [116]*.* Free fatty acid receptor (GPR40), which ligands include several medium and LC-FA, saturated or unsaturated, is ubiquitously expressed in the brain. If ω6 binds to GPR40, neurodegeneration follows. If the ligand is ω3, serum BDNF is increased with eventual synaptogenesis and neurogenesis [117]*.*

Poor nutrition has long been declared as one of the risk factors to antisocial personality disorder in adulthood [118] and increased aggression during childhood and adolescence [119]*.* A defective supply of DHA from ω3-PUFA, an integral part of astrocytic cell membrane, caused an impaired Glu clearance, with subsequent altered behavior in adulthood [120]*.* Several human studies adopted ω3 PUFA to hinder aggression [121–127]. Omega-3 deficiency favors the production of inflammatory cytokines, disturbing Glu homeostasis (**Figure 2**) [43, 128–132]. Inflammation was linked to aggressive behavior in lower mammals and humans [133, 134]. There seems to be a bidirectional interaction, so that aggression by itself can precipitate oxidative stress, as was demonstrated in birds subjected to a violent interaction [135].

Despite studies claiming the benefits of ω3 PUFA in neurological disorders, a lack of consistency remains. Moreover, most studies addressed ω3 PUFA without discrimination between individual constituents. Scarce work investigated ω3 short-chain PUFA claiming their additional neurological benefits [136]*;* however, insufficient data exist at the current time.

In blood, erythrocytes content of ω3 PUFA is dependent on either exchange with plasma lipoproteins, in the case of EPA, or erythrocytic turnover, in the case of DHA and DPA [137]*.* Surprisingly, giving EPA supplementation, but not DPA, was reflected at the level of erythrocytes. Other blood components seem to have a special *Perspective Chapter: Neurotoxins and Erythrocytes – A Double-headed Arrow DOI: http://dx.doi.org/10.5772/intechopen.108342*

#### **Figure 2.**

*Glutamate and neuroinflammatory mediators. Among inflammatory cytokines, tumor necrosis factor-alpha (TNF-α) upregulates 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl) propanoic acid (AMPA) receptors, on the expense of gamma-aminobutyric acid (GABA) downregulation***.** *The rapid rise of TNF-α from micromolar to millimolar levels can lead to sustained nuclear factor-kappa B (NF-Κb) activation and neurotoxicity***.** *A blunted TNF-α is not beneficial either, as by acting through its corresponding receptors (TNFR1 and TNFR2), it supports synaptic transmission by stimulating presynaptic N-methyl-D-aspartate (NMDA) and postsynaptic NMDA and AMPA activities, as well as assisting Glu transporters by regulating membrane trafficking of excitatory amino acid transporters (EAATs) and their recycling to the surface, added to conferring neuroprotection***.** *Interleukin-1 beta (IL-1β), activated by stress***,** *precipitates excitotoxicity by enhancing the postsynaptic NMDA pool and activity, by facilitating tyrosine kinase-mediated NMDA phosphorylation, increasing Ca2+ permeability. Interferon-gamma (IFN-γ), released from T-lymphocytes at high concentrations, can impair the sequestration of Glu by EAAT***,** *induces tryptophan catabolism through activation of IDO, generating Glu-like compounds as quinolinic acid and enhances AMPA-mediated neurotoxicity***.** *Interleukin-6 (IL-6) acts on soluble IL-6 receptor (IL-6R) to abrogate presynaptic Glu release and reduce AMPA and NMDA activities***.** *Cyclo-oxygenase-2 (COX-2) and prostaglandin E2 (PGE2) trigger the release of Ca2+ from intracellular stores***,** *causing reverse efflux of Glu by EAATs, followed by Glu spillover to bind to extra-synaptic NMDA***.**

affinity to supplements of different ω3 PUFA members. Blood levels and response to supplementation are subjected to multiple factors, related to genetics*,* gender*,* interindividual variability ranging from an 82% decrease to a 5000% increase, and the type of supplement used [138–141]*.*

Many years ago, low blood ω3 was detectable in impulsive offenders [142]. Blood levels of ω3 were negatively correlated to behavioral indices of aggression [143]. Blood samples were recommended when dealing with ω3 supplementation, being better predictors of aggression, that can discriminate responders from no- or low-responders and can tackle interactions with other nutrients [127]. Recently, a total daily dose of 960 mg DHA and EPA was provided to adult male prisoners in a correctional center as fish oil capsules reduced their aggression, most of them were nonaggressive at baseline [144]*.* In this trial, non-fasted blood samples were withdrawn, and plasma was separated from packed erythrocytes. Then, erythrocytes were prepared for fatty acid analysis [145]*.* Individual fatty acid analysis was done, then ω3 index was calculated as the sum of EPA and DHA, to be expressed as the mol percent of total erythrocyte fatty acids [146]*.* Participants with an index of 6% or higher were unlikely to benefit from the supplements due to a potential ceiling effect [147]*.*

Unfortunately, results correlating blood levels of ω3 PUFA to the brain levels showed inconsistencies, limiting their applicability as surrogate biomarkers for brain disorders, at least for the time being. In fact, ω3 and ω6 PUFA complement to maintain constant levels of unsaturated membrane phospholipids, so that they compensate for each other [148].

#### **6. Blood–brain bridge, rather than barrier**

The brain is no more that sealed-off structure from the rest of the body, as detected in mice lacking immune cells and depicting difficulty in social behavior [149]*.* Instead of crossing the brain, immune cells signal through cytokines, so that knocking out cytokine receptors on the neurons can disturb social behavior in laboratory animals [150]*.* In turn, the brain areas involved in positive emotions and motivation can alter immune responses in inflammatory and oncogenic disorders [151]*.* Although, in healthy humans, limiting the amount of Glu that crosses the BBB [152] protects brain Glu levels from fluctuations of blood Glu [99]*.* Glutamate can breach such restrictive entry by enhancing the blood-brain permeability, while triggering cerebral vasodilatation [153]*.*

In vascular injury of the brain, whether ischemic or hemorrhagic, the concomitant sizeable rise of blood and brain Glu occurs [154]*.* Such elevations were also noticed in many neurological disorders, including AD, epilepsy, and schizophrenia [155]*.* Following traumatic brain injury, the rise of brain Glu persists for months or even years thereafter*.* Such BBB disruption, not only allows blood Glu to reach the brain, but prevents the escape of cerebral Glu to the bloodstream.

In primary hypertension, the increased arterial content of Glu was linked to the higher Glu entry into the brain [156]*.* Similarly, systemic injection of Glu exacerbated brain damage [157]*.* Conversely, medications that lower blood Glu can assist Glu efflux from the brain [158]*.* So, restoring Glu level in both blood and brain to normal levels is required to reestablish the brain–blood Glu homeostasis. In their review, Gruenbaum et al. [159] highlighted the disruption of Glu efflux, breaking the integrity of the BBB, suggesting the feasibility of blood Glu scavengers in the treatment of depression following stroke.

One applied entity is the stress-induced aggression. During an anger attack, blood perfusion is increased, contrasted by cerebral hypoperfusion in between attacks, owing to stress-induce cerebral vasoconstriction [160]*.* Chronic stress causes disorganized BBB integrity, permitting the influx of mediators from peripheral blood, causing oxidative stress and neuroinflammation [161]**.** Altering the blood–brain Glu balance can excite excess Glu exit from the brain. To revert aggression and other subsequent psychological issues, oxaloacetate (OxAc) [162]*,* the substrate of the enzyme glutamate-oxaloacetate transaminase 1 (GOT), that consumes Glu to render OxAc, was given to reduce blood Glu level.

### **7. Tips for erythrocyte glutamate assay in CNS disorders**

In the brain, Glu is taken up from the extracellular to the intracellular domain of neurons and astrocytes by bidirectional transport mechanisms that, not only maintains low/high extracellular/intracellular levels, but also acts as a source of extracellular Glu when low [163]*,* through stimulating Glu release [164]. Similarly, the Glu active transport in erythrocytes maintains a high erythrocyte/plasma (E/P) concentration and a low plasma concentration.

In children with migraine, erythrocytic Glu was employed to mirror a centrally enhanced cellular uptake of this amino acid. In this setting, measuring plasma and erythrocytic Glu revealed a significant decrease in plasma, with a higher E/P concentration which was suggested as a reflection to mishandled CNS Glu turnover [165]. In contrast to the pediatric age group, adult migraineurs experienced elevated plasma and platelets Glu when measured during the attack-free periods [166]*.* Recently, stress, an aggression trigger, was documented to affect blood Glu levels [167].

A blood assay of Glu should be obtained after an overnight fast, to enhance specificity, avoiding misinterpretation due to nutritional factors, unless dietary management is planned. A preferable practice would be to monitor plasma Glu at the fixed time of the day, if multiple testing is needed, as plasma Glu might fluctuate along the day [168]*.* For better and more accurate interpretation, multiple factors that can modify blood Glu should be kept in mind, apart from nutritional status mentioned earlier, age, gender [169], body temperature [170]*,* and even blood sampling sites seem confounding factors [171]*.*

Normal Glu in plasma and whole blood is 50–100 and 150–300 μmol/l, respectively [59]*.* In the whole brain, Glu concentration is 12 μmol/g [172]*.* The free amino acids concentration can be calculated using whole blood and plasma concentrations [173]*.*

The inverse relationship between plasma Glu and nitrogen hemostasis implicates that plasma urea and ammonia nitrogen should be assessed as well.

It is worthwhile to measure more than one inflammatory marker (C-RP, TNF-α, IL6, IFN-γ, and IL-1β) to identify patients who are likely to respond to Glu-targeted therapies, since inflammation seems an incident predisposing to Glu excitotoxicity. This was corroborated when the elevated inflammatory markers in blood predicted the favorable antidepressant response to the noncompetitive NMDA antagonist, ketamine [174]*.* Also, the administration of the inflammatory cytokine and interferon (IFN)-α induced a high plasma TNF-α [175]*.* Moreover, the higher plasma CRP level in depressed patients was correlated to a higher brain Glu [176]*.* As implicated in neuropsychiatric disorders, IL-6 promotes hepatic acute phase proteins, while processing neuroinflammation in the brain [177]*.*

No study tracking the patients' behavior to neuropsychiatric medications or dietary manipulations has targeted both the blood level of Glu and the inflammatory markers. So, elaborative research work is indispensable to elucidate the benefits of blood assays in prediction and management panels.

## **8. Conclusions**

Glutamate is one of the key mediators involved in aggressive behavior. In neuropsychiatric disorders, blood or erythrocytic Glu level mirrored brain Glu fluctuations. Anemia was demonstrated to affect brain Glu level, meanwhile precipitating aggression. Lowering blood Glu increased Glu clearance from the brain. Dietary manipulation was successful in controlling aggression, as inflammation and oxidative stress have been implicated in altered brain Glu and aggression. Hence, blood or erythrocytic assay of Glu might help the diagnosis and prognosis of aggression, as well as planning corresponding therapeutic strategies ranging from simple dietary manipulation, up to complex pharmacologic treatments. The more advances in the scientific research, knowledge, and testing techniques, the more explicit will be the

dynamics of behavioral issues, the more feasible and successful will be the diagnostic, preventive, and therapeutic interventions. Nonetheless, Glu is not the only culprit in aggression, other explored neurotransmitters and inflammatory markers can be assayed and targeted as well, to obtain a panel of laboratory markers and plan several therapeutic alternatives using these mediators, hoping to prevent an outraged ideation from proceeding to a devastating aggression.
