Author: The E.R.B.

Traumatic Brain Injury (TBI) and Concussion: Time for Brain Food

Presented July 7, 2017 in Snowbird, Utah at the 2017 Neurotrauma Conference. Abstract Published July issue of Journal of Neurotrauma.

Introduction: Given combat casualties, sports-related injuries, and motor vehicle accidents, traumatic brain injury (TBI) is the leading cause of death under age 44. TBI deficits linger in concentration, depression, sleep-wake cycle, behavior and motor skills. Basic science has long instructed that omega-3 fatty acids, specifically docosahexaenoic acid (DHA), improve cell membrane fluidity and signaling. B-complex vitamins provide nerve myelin substrate and neurotransmitter cofactors. The presence of all 20 amino acids is critical to cell membrane and neurotransmitter protein production.

Hypothesis: This systematic review hypothesizes that given ample substrates of DHA, B-complex vitamins, and elemental amino acids, the brain will promote tissue restoration.

Methods: A literature search of the PUBMED database identified current research using the keywords “omega-3-fatty acid”, “B-complex vitamin”, “elemental amino acids” with “TBI”, and “MTHFR C667T”.

Findings: DHA deficiency in the population may be as high as 80%. DHA having more double bonds than vegetable oil, provides greater brain tissue strength. Researchers have found that DHA increases cognitive ability, motor function, membrane fluidity, cell signaling, and is neural protective. DHA enhances neuronal growth, transmission speed, dendritic formation, and synaptic maintenance. The American Society for Parenteral and Enteral Nutrition now suggests fish oil for trauma patients. Studies have found B- complex vitamin deficiencies in spinal cord injury patients. B-complex vitamins improve sensorimotor skills, cognitive function, memory, vision, and DNA methylation. Folate’s role in DNA methylation may be reduced by genetic polymorphisms. Amino acids mediate cell damage, are neuro-protective, and decrease hippocampal deficits. Amino acids demonstrate increased cerebral blood flow and DNA methylation.

Conclusion: DHA, B-complex vitamins and elemental amino acids show evidence of improving TBI prognosis. These nutrients are a safe, effective manner to apply basic science to rebuild brain tissue and improve patient outlook.

Keywords: TBI, concussion, neuroprotective, DHA, B-complex, amino acids, MTHFR C677T

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Traumatic brain injury(TBI) causes approximately 52,000 deaths, 220 hospitalizations, and 85,000 permanent cases of debilitation each year in the U.S. “Approximately 15-20% of U.S. Soldiers in Iraq and Afghanistan experience TBI while deployed, making TBI one of the most common injuries among military personnel (Warden D, 2006).” “It is estimated that 1.6 – 3.8 million sports-related TBIs occur in the US annually (Hasadri et al., 2013).” In 2010, the economic cost of TBI was estimated at $76.5 Billion (Humphreys et al., 2013). Concussion or mild TBI (mTBI) has seen a 4.2 fold case increase since 1998 (Barrett et.al, 2014). Symptoms of mTBI include loss of consciousness, headache, mental fogginess, light sensitivity, and sleep disturbances for typically 7-10 days post injury. These symptoms may persist for months or longer. (Barrett et.al. 2014). “TBI is one of the most disabling injuries causing motor and sensory deficits, and leading to severe cognitive, emotional, and psychosocial impairment, crippling vital areas of higher functioning (Hasadri et al., 2013).” “Long term health disorders associated with TBI include post-traumatic stress disorder (PTSD), neurodegenerative diseases (Alzheimer’s disease and Parkinsonism), neurocognitive deficits, psychosocial health problems (e.g. Binge drinking, major depression, impairment of social functioning and ability to work, suicide), epilepsy, pain, and other alterations in personality or behavior (Omalu et al, 2006) (Guskiewicz KM, 2007)(Omalu BI, 2010).” The societal impact is staggering given brain injury’s excessive financial cost, short term disabilities, and long term propensity to cause mental disease. While there is no cure for TBI, the following scenario has lead to the exploration of a potential effective therapy.

A 14 y/o female, club soccer player suffered concussion. She continued to play through two full-length soccer games. In the days following, she suffered headaches and bright flashes of light in both visual fields. Loud sounds exacerbated the pain. She experienced complete exhaustion, lack of concentration, and difficulty sleeping. Sports activities caused headaches. She began missing school due to the chronic pain, causing her stellar grades to decline. This patient’s family traveled extensively the following year, paying for eight physician visits with no improvement in her condition. Despite their medical biochemistry coursework, none of these physicians offered a biochemistry based nutrient therapy that would replenish her injured brain tissue. As months passed, she discontinued sports and “lost hope in life”. To avoid social exclusion, she avoided public discussion of the pain. The following year, this patient learned about biochemical nutrients and began taking fish oil, B complex, and amino acids to rebuild brain tissue. Over the next month, her concussion symptoms diminished, allowing her to return to school and sports.

Results such as these, plus the fact there is no existing therapy for TBI, have prompted the research presented in this paper. This paper proposes that the brain macronutrients docosahexaenoic acid (DHA), B-complex vitamins, and amino acids, may be suited to replenish damaged brain tissue and lessen the impact of future injuries. Current literature is evaluated regarding the efficacy of utilizing these brain macronutrients to restore and fortify brain tissue, possibly minimizing future injuries. This paper is intended to bring heightened awareness to medical practitioners as to the importance of using these brain macronutrients in the treatment of TBI. To be sure these practitioners

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already have comprehensive training in biochemistry. Nevertheless, when medicine seeks restorative therapies, biochemistry is rarely applied. Albeit, the brain’s biochemical intricacies extend far beyond man’s comprehension. “The pathological sequelae of TBI are mediated by an interaction of acute and delayed molecular, cellular and physiological events that are both complex and multifaceted physiological complexity of brain (Flanagan et al. 2008).” “A successful TBI treatment may have to simultaneously attenuate many injury factors (Scrimgeour & Condlin, 2014).” Nutrients with their extensive functionality may indeed attenuate many complex factors, simultaneously. Providing rich macronutrient supplies would allow the brain to synergistically facilitate repair without second injury (IOM, 2011) and void of major contraindications/side effects. “The evidence supporting multiple dietary components to reduce TBI-associated neuroinflammation is strong” expectedly because “there may be considerable synergism between them”. “If used appropriately, there may not be a downside to using food agents for therapy (Scrimgeour & Condlin, 2014).” It is hoped that through the biochemical nutrient findings presented in this paper, medicine will better provide brain macronutrient substrates to synergistically and comprehensively revitalize pathways and repair brain tissue for the long term. If medicine will consider using macronutrient biochemistry such as omega-3 fatty acids, B-complex vitamins, and amino acids so diligently studied and taught, the long term therapeutic results may be astounding. It is in this hopeful vein that this paper considers fish oil, B complex vitamins and amino acid brain macronutrients as therapy for TBI.

Fish Oil (DHA/EPA) – Omega 3 fatty acids

The most important brain macronutrient is fish oil. Fish oil contains the omega-3 fatty acids docosahexaenoic acid (DHA) and eicosapentanoic acid (EPA). “DHA is a primary structural component of the mammalian cortex, comprising 50% of neuronal membrane phospholipids (Hasadri et al., 2013)(Singh et al., 2005).” While Western diets provide copious amounts of vegetable oil, “the CDC now estimates that up to 80% of the United States population is omega-3 fatty acid deficient (Scrimgeour & Condlin, 2014).” Studies report that omega-3 fatty acid deficiency for a single generation can decrease DHA content by up to 40% whereas severe DHA depletion (up to 80%) can be achieved after two or more consecutive generations (DeMar et al, 2006). Desai confirms that after three generations, “over 70% DHA depletion was observed in the cerebellum” (Desai A, et al, 2014). This widespread, generational deficiency makes fish oil and specifically DHA, the most important brain macronutrient to fortify.

The Western diet has made adequate dietary consumption of DHA and fish oil more challenging. Historically, the vegetable oil to fish oil dietary consumption ratios were closer to 1-2:1 (Scrimgeour & Condlin, 2014). This ratio mirrors DHA to vegetable oil concentration in the brain. However, in current diets, vegetable oil to fish oil consumption can be as high as 50:1 (Lewis et al., 2013). This ratio is likely due to increased red meat and vegetable oil consumption, and decreased fish consumption. While free range animals produce DHA, once these animals are caged and grain fed, their meat and eggs no longer contain DHA. Research finds that omega-3 fatty acids

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such as DHA “must be obtained from the diet, and are highly enriched in algal oil, krill, and cold water fish (Kris-Etherton et al, 2000)(Raper NR et al, 1992)(Tur JA, et al., 2012).” Often alpha linolenic acid(ALA) food sources such as “nuts, soybean, canola, and flax seed oils” are considered to be fortification of DHA levels. Unfortunately, insignificant amounts of ALA are converted to DHA, making these foods poor sources of DHA (Simopoulos AP, 2011). It is best to consume DHA foods directly or fortify the biochemical nutrient. The best foods are fresh fish such as salmon, tuna, and trout. DHA can also be found in fish skin, free range meat, cage free eggs, fortified milk, and fish oil.

While fish oil contains both DHA and EPA, DHA has been found to be more important for maintaining cell membrane structure. As compared with vegetable oil, the omega-3 fatty acid DHA contains double bonds every third carbon, while the omega-6 vegetable oil contains only one double bond every sixth carbon. The DHA double bonds provide more strength, likely contributing to cell membrane structures which better withstand concussion. Research finds that DHA is important for “structural components of cell membranes, modulating membrane fluidity, cell wall thickness, cell signaling, and mitochondrial function”. DHA is “readily retained in neuronal plasma membranes … influencing the phospholipid content of plasma membrane (Dyall SC et al, 2008)(Salem N, et al., 2001).” “Membrane DHA in the brain provides resilience against propagation of injury-induced cellular damage and facilitates adaptive responses for recovery (Desai et al., 2014).” In neuronal membranes, DHA promotes growth (Cao, et al, 2005)(Robson et al., 2010), and within the body has an anti-inflammatory action (Scrimgeour & Condlin, 2014). Substantial rodent and human research findings support the importance of the brain macronutrient DHA to effect healing of TBI.

Rodent DHA Neuroprotective Findings and Safety

Studies indicate that TBI is a progressive event which causes axonal nerve injury and leads to the swelling and disconnect of the axon membrane in hours to days post injury. Brain damage causes a lack of transport and communication across axonal membranes, ultimately leading to cell death (Mills JD, et.al., 2011). “Membrane degrades phospholipids (Homayoun, et.al, 2000) resulting in disturbances in cellular membrane functions and contribution to secondary neuronal injury ” (Horrocks & Farooqui, 2004). DHA is an integral component of axonal and cell membrane structures, necessary to facilitate repair following TBI.

The findings from several rodent studies demonstrate that DHA in the diet improves membrane structure to enhance cell function and provides neuroprotection. UCLA researchers demonstrate that DHA in the diet improved molecular substrates of plasticity affected with brain concussive injury. “Diet plays a critical role to build resistance against the effects of TBI. Proper exposure to omega-3 fatty acids during gestation and throughout maturation of the central nervous system (CNS) is crucial for building neural resilience” (Ying et al., 2012). UCLA also found that the “combination of dietary DHA and voluntary exercise … counteracted the effects of TBI on cognitive function, … synaptic plasticity, and plasma membrane homeostasis.” “Exercise

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normalizes DHA content in the brain” and when combined with DHA supplementation has a “strong therapeutic potential for reducing TBI’s deleterious effects on membrane homeostasis, synaptic plasticity and cognition (Wu, et al., 2013).” Agrawal’s team from UCLA similarly found that “consumption of an omega-3 diet during early life influences the capacity of the adult brain to resist the effects of TBI”, thus giving credibility to DHA’s role in energy homeostasis, plasticity, mitochondrial biogenesis and control of oxidative damage (Agrawal, et al., 2014).

The Mayo Clinic reports that DHA is a highly neuroprotective and integral component for rebuilding brain cell membrane. It is the “longest unsaturated fatty acid found in biological membranes”. “The structure is tremendously flexible and …. versatile” requiring minimal energy for conformation changes (Stillwell & Wassall, 2003)(Stillwell et al., 2005). “DHA is capable of undergoing rapid interconversions… and is particularly enriched in membranes which require rapid vehicle formation and release, such as rod outer segments in the retina and neuronal synapses.” (Stillwell et al., 2005). The “omega-3 fatty acids are essential for maintaining membrane fluidity (Valentine & Valentine, 2004) which in turn impacts neuronal cell adhesion, axon guidance, synapse maintenance, dendritic formation, and the speed of neuronal transmission (Hering et al., 2003)(Innis SM, 2008).” Additionally, membrane Na+/K+ pump failure is a common finding in TBI. DHA may play a role in maintaining the Na+ K+ pump which accounts for 60% of the energy utilization in the brain (Turner et al, 2003). Omega 3 fatty acids were found in retinoid X receptors which affect neuronal growth and proliferation, differentiation of catecholaminergic neurons, and the hippocampus (Innis SM, 2007) (Lane & Bailey, 2005). Finally, omega 3 fatty acid deprivation played a role in “1) decreased body size of neurons in hippocampus, hypothalamus, and parietal cortex, 2) reduced complexity of dendritic arborization on cortical neurons and 3) significant deficits in spatial learning and memory (Ahmad et al., 2002)(Aid et al., 2003)(Chaion, 2006)(Innis et al., 2001)(Wainwright et al., 1998).”

Researchers at Loma Linda University found that DHA protected and restored neurons, resulting in significant improvement in the motor and sensory tract functions destroyed following spinal cord injury (Figueroa et al., 2013). Spinal cord tissue contains biochemical components analogous to cerebral tissue. Spinal cord injuries (SCI) have long caused debilitating injury to the sensory and motor neurons below the injury site. DHA supplemented for 8 weeks in female Sprague-Dawley rats mediated the chronic pain present with SCI. Researchers found that our Western diet may be hindering recovery from SCI, and that chronic DHA deficiency is associated with dysfunction (Figueroa et al., 2013). Their findings support dietary prophylaxis with DHA given distinctive improvements in nerve function that may facilitate recovery (Figueroa et.al., 2013).

An NIH study confirms DHA’s role in motor function by evaluating mice with DHA depletion. Researchers found that vestibulo-motor functioning in terms of acute motor deficits and fine motor control are impeded in DHA deficient mice (Desai et al., 2014). Acute motor deficits as determined by the rotarod were decreased by 50%. In one report, “when DHA was given within an hour of spinal cord injury (SCI), neuromotor

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function was maintained but the effect was lost when treatment was delayed four hours”. Further, DHA decreases neuronal damage by decreasing post-ischemic inflammation, and is neuroprotective (Hong et al., 2003) (Mukherjee et al., 2004).

Schober and colleagues from the University of Utah reported that “dietary DHA, given before cortical concussion impact (CCI) and continued daily, improved long-term rat pup outcomes after CCI. Specifically, DHA improved cognitive function, tissue sparing, and diffusion tensor imaging (DTI) indices of white matter injury after CCI, associated with decreased edema and oxidative stress.” “DHA decreased axonal injury in rat pups after CCI.” “DHA also modulated the cytokine response in a manner suggesting anti- inflammatory and neuroprotective activity.” In terms of safety and dosage, Schober’s results show that “DHA is safe and neuroprotective after experimental TBI in rat pups (Schober et al., 2016).” Researchers caution that their “diffusion tensor imaging (DTI) results are not directly comparable to humans because rat brains have significantly less white matter than do human brains (Kraft et al., 2012) (Schober et al., 2016).” Given the human brain’s increased surface area through gyri and sulci, these researchers expect that DHA would have a more dramatic effect on human brains than found with rodent brains. The dosage of DHA used was comparable to the 1.5-2 g/d used in clinical trials for adults and adolescents (Schober et al., 2016).

Thus, multiple medical centers have found DHA to be neuroprotective in rodent studies. This fatty acid’s versatile structure enables enriched membrane lipids to improve interconversion, fluidity, synapsis, transmission speed, neuronal size, plasticity and dendritic arborization. DHA decreases inflammation, oxidative damage, DTI damage, edema, and improves outcomes. These improved outcomes are evident through enhanced motor and sensory function, cognitive function, learning, and memory. DHA doses comparable to 1.5-2g/d in humans were found to be safe and strengthen neuroresilience in rodents.

Human DHA Neuroprotective Findings, Safety, and Recommendations

These prominent DHA rodent study results reducing the effect of TBI have been slow to transfer to human studies and recommendations. In 1997, the U.S. Food and Drug Administration confirmed that fish oil consumption at levels up to 3 g/day were generally recognized as safe in the Federal Registry. At the same time, the FDA determined that fish oil up to 4 g/day was safe for cardiovascular therapy (FDA, 1997). Since brain trauma commonly results in depression, human DHA studies began by evaluating depression as related to fish oil consumption. In 1998, retrospective studies found that depression rates were 50 times higher in countries with little seafood consumption (Hibbeln et al.,1998). By 2005, Daily Reference Intakes for DHA were not yet established by the United States Department of Agriculture (USDA). However in 2007, fish oil in the quantity of 2g/d were shown to reduce suicidal tendencies, depression, and the perception of stress (Hallahan B, et.al., 2007). In 2010, the U.S. Dietary Guidelines added “increase seafood consumption” to its recommendations.

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In 2011, the Institute of Medicine (IOM) published an extensive report entitled “Nutrition and Traumatic Brain Injury: Improving Acute and Subacute Health Outcomes in Military Personnel”, which among other brain nutrients, reviewed current literature on DHA. The commission reported that 80 percent of the fatty acids consumed in the U.S. were omega-6 or linoleic acids at a rate of 17g/day, however these fatty acids do not convert well to DHA in the body. The IOM reported human studies showing that DHA could reduce inflammation in TBI within 60 minutes of infusion (IOM, 2011). The IOM noted that while intravenous fish oil formulations are available in Europe they have not yet been approved by the FDA in the U.S. The IOM recommended that patients in the early phase of severe TBI be provided with continuous enteral feeding formulas containing fish oil (IOM, 2011). Appreciably, this IOM report recommended that more DHA animal studies and human clinical trials be conducted for TBI.

Additionally in 2011, Dr. Michael D. Lewis and Dr. Joseph R. Hibbein of the Uniformed Services University in Bethesda, Maryland sought to compare suicide rates with DHA levels. A markedly higher risk of suicide is often associated with individuals who have suffered TBI (Nowrangi et al., 2014). In a retrospective, large random study, researchers assayed serum samples from 800 military personnel who had committed suicide (Lewis et al., 2011). Subjects were randomly matched with 800 controls for age, collection date, sex, rank and year of incident with sera within 12 months of their matched case (Lewis et al., 2011). Samples from both subjects and controls were assayed for DHA composition by robotic direct methylation coupled with fast gas-liquid chromatography to account for possible degradation. Researchers found that there was a 62% greater risk of suicide death among men with lower serum DHA levels (Lewis et al., 2011).

In 2014, the Academy of Nutrition and Dietetics (AND) issued a position paper on “Dietary Fatty Acids for Healthy Adults”. The Academy’s Evidence Analysis Library examined 14 studies, whereupon 6 studies showed that DHA ingestion demonstrated a decreased risk of cognitive decline (AND, 2014). In this position paper, the Academy discusses the need for 20-35% of the diet to be comprised of a variety of fatty acids, stating the mean daily intake of DHA was only 80mg for men and 60mg for women (AND, 2014) far below the recommended research dosages. DHA’s double bond structure, inflammation modulation, and neuroprotective properties are discussed. The AND paper also notes that DHA is found in fatty fish, seafood, salmon, sardines, tuna, herring, trout, seal meat, and marine or algal sources (AND, 2014). Most importantly, AND writes that DHA should be considered essential, because less than 1% of alpha- linolenic acid (ALA) reliably converts to DHA (Davis, 2003)(Burdge, 2004). If fish is not consumed, biochemical nutrients can be purchased. Nutrients are made from fish oils such as anchovy, salmon, cod liver, krill and squid oils (AND, 2014). Additionally, vegetarian sources of algae are available and genetically engineered nutrients are being developed. Multiple agencies were cited in this AND position paper, including the American Psychiatric Association (APA), which stated that lower levels of DHA have been observed in individuals with cognitive decline and Alzheimer’s Disease (AND, 2014). The APA recommends fish twice a week for an average 450 to 500 mg of EPA and DHA per day (AND, 2014).

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Per the 2014 National Institutes of Health (NIH) review paper, “The American Heart Association has … established intakes of 1.0 g of EPA and DHA from fish or fish oils for patients with cardiovascular disease, and supplements of 2-4g for subjects with high blood triglycerides (Harris et al., 2008). Most clinical studies of DHA have used a dose of 2-6 g/d, and no consistent adverse events have been observed in humans consuming from less than 1.0 up to 7.5 g/day of DHA (Lien EL, 2009).” Additionally the NIH reports that “multiple clinical trials have shown that high-dose fish oil consumption is safe, even in patients receiving other agents that may increase the risk of bleeding, such as aspirin and warfarin (Maresta et al., 2002)(Filion et al., 2010)(Marik et al., 2009).” “Doses up to 6g/d do not have deleterious effects on platelet aggregation or other clotting parameters in normal persons, and fish oil does not augment aspirin- induced inhibition of blood clotting (Lien EL, 2009).” “Platelet function is … inhibited by DHA consumption in persons with type 2 diabetes … but may be beneficial to these persons (Woodman et al., 2003).” “It may be prudent to discontinue high-dose supplementation in the setting of a patient with TBI who presents with polytrauma (e.g an acute bleeding illness or at high risk for hemorrhagic stroke) or, as is frequently recommended with aspirin, warfarin, and clopidogrel, before planned invasive procedures with the highest risk of bleeding complications (Kris-Etherton et al., 2003) (Bays, 2007)(Caio et al., 2005)(Covington, 2004).” It is important for consumers to become aware of the efficacy of fish oil for TBI, its high availability, and low cost.

The military, seeking to heal traumatized soldiers, has recognized the benefits of DHA in healing brain trauma. In the November 2014 issue of Military Medicine, Julian Bailes MD and Vimal Patel PhD report that “military members have a higher risk of repetitive injury due to explosive devices, resulting in a “unprecedented rate of non-penetrating head injury” (Bailes and Patel, 2014). Mitigation or prevention can be accomplished through DHA to improve the neuroprotective effect when high doses (40 mg/kg) are given (Bailes and Patel, 2014). In this review article, Bailes and Patel explain that more recently, TBI damage has been found to include neurofibrillary tangles and the build up of tau protein. Long term, chronic traumatic encephalopathy may develop (Bailes and Patel, 2014). Researchers describe how the highly flexible, long chain DHA fatty acid creates thin phospholipids which pack well within the cell membrane, and create a more permeable phospholipid more suitable to membrane proteins, transport, signaling, and enzymes. They state that DHA decreases b-amyloid plaque buildup, reduces neuronal apoptosis, and may act as a prophylactic against cerebral concussion (Bailes and Patel, 2014). Bailes and Patel discuss the potential presence of mercury in fish. They recognize that given the safety profile, general health benefits, purity, availability and affordability of DHA, both athletes and military populations with high exposure to repetitive brain impacts are at risk unless they ingest adequate levels of DHA (Bailes and Patel, 2014).

A 2015 retrospective human study by Cedars-Sinai reviewed 240 TBI patients, and compared patients receiving immune enhancing nutrition (IEN) containing DHA with standard formula. Researchers found that while sepsis rates decreased and prealbumin levels were higher in IEN patients, the IEN patients spent more time in ICU and on the ventilator. IEN nutritional support was Pivot 1.5 containing 1.3 g/L DHA and 1500

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calories (Painter et al., 2015). Retrospectively, this low level of DHA did not reduce ICU stay. Neurological function testing was not conducted.

As of January 2016, the American Society for Parenteral and Enteral Nutrition (ASPEN) and the Society of Critical Care Medicine (SCCM) now recommend that “immune enhancing enteral formulations with omega-3 fatty acids should be used in critically ill surgical patients including trauma (McClave et al., 2009)(McClave et al., 2016).” Orally administered omega-3-fatty acids may take days to weeks to incorporate into cellular membranes … therefore IV administration … could be more suitable … following TBI (Hasadri et al., 2013).” Recently, Nestle’s Impact Peptide 1.5 enteral nutrition formula providing 4.9 g DHA/EPA per liter (Nestle, 2017) was specifically formulated for trauma patients, and SMOF lipid provides enteral nutrition with DHA and EPA omega-3s (SMOF, 2017).

In 2016, Schoeber reports DHA “has an excellent safety record in non-TBI pediatric studies that span the developmental spectrum between infancy and adolescence (Colombo et al, 2013)(Fleith et al., 2005)(Stonehouse et al., 2014). These “results complement those of Russell and colleagues (Russel et al., 2014) in showing that DHA is safe and neuroprotective after experimental TBI in rat pups.” Schoeber describes the NIH case study which reports a motor vehicle accident patient, with an initial GCS of 3, remaining on Nordic Natural Ultimate Omega delivering 9,756mg EPA and 5,756 DHA via PEG for over one year who experienced no side effects (Lewis et al., 2013), further demonstrating fish oil’s high safety profile.

In summary, “our knowledge of the pathophysiology of cerebral concussion has undertaken significant advances in the last decade (Bailes and Patel, 2014).” DHA has been established as the most important brain macronutrient given that it comprises approximately 30% of brain tissue. DHA’s double bonds provide additional strength to brain tissue which enhances cell membrane fluidity, signaling, neuronal growth, dendritic formation and synaptic maintenance. Improving these axonal and cell membrane properties results in cognitive and motor improvements. Following the recommendations of 2011 IOM report, ASPEN and SCCM now recommend fish oil for trauma patients. Nestle has produced Impact Peptide 1.5 with 4.9g of fish oil per liter, and SMOF lipid is now available with an w-3 profile as parenteral nutrition. One gram per day is generally the RDA for DHA and EPA. Therapeutic dosages of DHA and EPA often provide 2-3g/day. These researchers have shown that the DHA found in fish oil is a safe brain macronutrient which improves prognosis of TBI in both rodents and humans.

B Complex

TBI is the leading cause of death among the population under age 44 given combat casualties, sports-related injuries, and motor vehicle accidents. B complex vitamins are the second macronutrient vital to maintaining and restoring brain tissue damaged by TBI. B complex vitamins are important for nerve production and used as cofactors for

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protein synthesis including DNA/RNA synthesis and neurotransmitter formation. TBI causes motor and cognitive dysfunction, memory problems, scar tissue buildup, apoptosis, and balance problems. While few therapeutic B complex studies have yet to be reported for TBI, individual B vitamin studies have reported the restoration of brain tissue and neurological function following TBI.

Riboflavin

Riboflavin (B2) is vital to cellular respiration. Riboflavin enables energy production from the electron transport chain through flavin adenine dinucleotide. Additionally, riboflavin has vital roles in the production of glutathione and DNA synthesis. Given TBI, Haar found the benefits of riboflavin “lead to substantial functional recovery in sensorimotor function and working spatial memory, less edema, less reactive astrocytes and smaller lesions” in rodents (Haar, 2015). Additionally, Hoane found that riboflavin decreased behavioral impairment, lesion size, reactive astrocytes and cortical edema (Hoane, 2005). He determined that riboflavin likely functions as a free radical scavenger. Hoane treated 41 rodents with either vitamin B2 or saline. Treatment was administered 15 minutes and 24 hours post injury. The rats were examined on a variety of tests to measure sensorimotor performance and cognitive ability in the Morris water maze.” Results showed that riboflavin “significantly reduced behavioral impairments observed on the bilateral tactile removal test, through improved acquisition of both reference and working memory tests”. “B2 showed a reduction in the size of the lesion, reduced the number of glial fibrillary acidic protein positive (GFAP+) astrocytes (indicating healthier tissue), and significantly reduced cortical edema.” Hoane indicates that riboflavin likely functions through the reduction of oxidative damage (Hoane, 2005).

Niacin

Niacin (B3) produces nicotinamide adenine dinucleotide which like riboflavin has a critical role in cellular respiration produced through the electron transport chain. Niacin additionally participates in cholesterol, fatty acid and protein catabolism. Following TBI, Hoane found that functional recovery in rodents can be improved with niacin treatment. “Nicotinamide supplementation was shown to improve sensory, motor and cognitive function following frontal lobe injury (Hoane et al., 2003).” In this cortical contusion model injury, 30 male Sprague-Dawley rats, 3 months old were utilized. Following injury, 9 rats were given niacin (500mg/kg) and 9 control rats were given saline solution (1ml/kg) 15 minutes and 24 hours post injury. Hoane (2003) found that rats administered niacin post cortical contusion “significantly lessened the behavioral impairments observed following injury and led to a long-lasting improvement in functional recovery. The data from the bilateral tactile removal test showed that administration of niacin following injury … prevented the occurrence of a working memory deficit in the Morris Water Maze (MWM) model. The acquisition of a reference memory task in the MWM was significantly improved compared to saline-treated rats following injury.” Niacin did not improve skilled use.

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“The administration of niacin following injury significantly reduced the number of GFAP+ reactive astrocytes (GFAP+ astrocytes are thought to correlate with scar tissue production and lesion size.)” Niacin’s ability to prevent depletion of nicotinamide adenine dinucleotide (NAD+) and ATP are the likely mechanisms which improve TBI (Hoane et al., 2003). Hoane’s researchers have consistently shown positive results through a number of rodent studies. Hoane studies have demonstrated “reduced apoptosis and improved blood-brain-barrier (BBB) function (Hoane et al., 2006)”. In 2011, however, Swan found “no improvement at a standard [niacin] dose and increased impairment at higher doses (Swan et al., 2011).”

Pyridoxine

Pyridoxine (B6) is a cofactor for amino acid reactions including the production of neurotransmitters such as gamma amino butryric acid (GABA), the most prevalent inhibitory neurotransmitter in the body, as well as for the dopamine pathway, critical to motor movement and adrenaline production. While there are few research studies therapeutically utilizing pyridoxal phosphate for TBI, one important study evaluates the effect of high homocysteine (Hcy) levels on trauma outcomes. Pyridoxine (B6) is a key enzyme in the conversion of the deleterious homocysteine to methionine. In this study, there was a significant correlation between plasma Hcy levels and severity of trauma and prognosis in patients with TBI.” This prospective study examined the “relationship between Hcy plasma levels and the outcome of patients with traumatic brain injury (TBI).” “Outcome was evaluated according to the Glasgow Outcome Scale score at the time of discharge from the hospital and 6 months after hospitalization. The case group comprised of 150 patients with TBI (men, 54.7%; mean age, 55.90 years ± 12.31), and a control group of 150 healthy individuals (men, 52%; mean age, 49.56 years ± 15.64) were studied. The mean ± SD plasma Hcy level in the TBI group (20.91 μmol/L ± 15.56) was significantly higher than plasma Hcy level in the control group (7.45 μmol/L ± 13.54, P = 0.000). There was a significant relationship between Hcy plasma levels, Glasgow Coma Scale score, and computed tomography findings. Also, there was a significant difference in mean Hcy plasma between patients who died as a result of TBI and patients who were still alive at the end of the study period (Rahmani et al., 2016).” Additionally, high pyridoxine levels have been found to cause peripheral abnormalities and Schaeffer found in mice that high doses may cause central nervous system abnormalities (Schaeffer, 1993).” Should B complex be given on a regular basis, pyridoxine levels should be monitored.

Folate

Folate (B9) is likely the most critical of the B complex vitamins as deficiency during pregnancy may result in neural tube and heart defects. Folate is critical in DNA and RNA synthesis, as well as amino acid metabolism. One study gives extensive support to folate’s role in CNS axonal repair. In Naim’s piglet model, folate was found to improve cognitive and neurobehavior after TBI (Naim et al., 2011). “Piglets have a similar cortical grey-white differentiation gyral pattern and physiologic response to TBI in humans”. In this study, half the piglets (n=15) “received inertial rotation of the head to

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induce TBI” while 15 were uninjured. Seven piglets were given folic acid (80ug/kg) 15 minutes post injury and for the following 6 days, while eight piglets were given saline. “Piglets were assessed on memory, learning, behavior and problem solving ability.” “Cognitive Composite Dysfunction scores …were based on a variety of tests including open-field behavior, a mirror test, glass barrier task, food cover task, balance beam performance, and maze test.” Results demonstrate “that there was significant improvement in functional ability of the injured piglets given folic acid between day 1 and day 4, post injury.” Results showed more rapid completion of the balance beam task, improved motor function, increased visual problem solving with folate supplementation. Folate, however, was not found to reduce lesion size.

This study gives additional credence to folate’s role in DNA repair in that the mechanism was identified. Haar’s researchers found that folate can prevent DNA de-methylation. These researchers utilized a spinal cord injury model in rodents. They determined that when spinal cord injury occurs, DNA is de-methylated. Folate is attracted to the injury site via receptor expression and can prevent this de-methylation. There is a correlation between receptor expression, DNA methylation and regeneration of axons, thereby acknowledging that reduced folate has these pro-regenerative effects.

Folate Polymorphism

New genetic evidence supports the existence of polymorphisms in the gene producing the enzyme responsible for folate (B9) metabolism. This frequent polymorphism affects the methyl-tetrahydrofolate reductase gene. This enzyme, methylene-tetrahydrofolate (MTHFR) which requires folate (B9) is responsible for re-methylation of the neurotoxic homocysteine into methionine (Paul et al., 2004). The polymorphism can significantly reduce production of new DNA, making new tissue formation following TBI difficult (Paul et al, 2004). Individuals with this polymorphism may require additional folate to effect repair.

There are approximately 40 single nucleotide polymorphisms (SNP) identified with this MTHFR gene. The C677T SNP is located on the short p arm of the first chromosome at locus 36.3 (Lizer et al., 2011)(Bhatia & Singh, 2015). The cytosine base errantly codes for thymine, resulting in the amino acid product of valine instead of alanine (Paul et al., 2004). Patients with genetic profiles containing this SNP have reduced functionality of the MTHFR enzyme by perhaps 50%. The thermolabile genotype may have 64% reduction in enzyme activity (Paul et al., 2004). This is the major pathway for methionine production. Methionine is the lead nucleotide on each new strand of DNA, and methionine maintains adrenal neurotransmitter production. Healing the brain requires new DNA production. Lack of a fully functioning MTHFR gene may result in a plethora of neurological disease including Parkinson’s Disease, neural tube defects (NTDs), and depression (Paul et al., 2004) which are common TBI sequelae.

Nazki’s study reports that heterozygotes (C677CT) have 65% and homozygotes (C677TT) have 30% of the MTHFR enzymatic activity to convert

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methylenetetrahydrofolate to methytetrahydrofolate (Nazki et al., 2014). There are a number of mechanisms by which the C677T allele reduces MTHFR action (Paul et al., 2004). 5-methyltetrahydrofolate dominates circulation and re-methylates homocysteine (Hcy) into methionine (Bhatia & Singh, 2015). Methionine is a major methyl donor. Low methionine, thus low S-adenylmethionine (SAMe) causes dysregulation of neurotransmitter production (Paul et al., 2004). Catecholamine production and membrane fluidity are reduced, and neuronal transmission becomes inadequate (Bhatia & Singh, 2015). Glutathione production is reduced increasing oxidative stress. Methylation is required for serotonin, DNA, and RNA synthesis (Bhatia & Singh, 2015). Without methylation DNA strands become weakened and mutagenic (Nazki et al., 2014). Additionally, with the C677T SNP’s reduced conversion of Hcy to methionine, Hcy levels elevate (Delport et al., 2014). Hcy is neurotoxic at high levels thus promoting neuronal damage and increased production of reactive oxygen species. Hcy may directly break DNA strands and cause apoptosis (Bhatia & Singh, 2015), and the lack of regulated Hcy levels may cause depression and neurotransmitter imbalance (Bhatia & Singh, 2015).

Given the prevalence of this SNP, researchers have addressed whether supplementation of the B complex vitamins including folate can impact the successful conversion of homocysteine to methionine, compensating for the genetic MTHFR C667T defect. One study treated 282 major depressive disorder (MDD) subjects with either reduced B vitamins/micronutrients or placebo, Mech showed an 82.4% reduction in Hcy levels, and 42% complete remission with B complex vitamins (Mech & Farah, 2016). Achour randomized 132 hemodialysis patients and supplemented with both folate and cobalamin (Anchour et al., 2016). His team found that Hcy in MTHFR patients could be significantly reduced. Should supplementation fail, genetic testing could be completed to identify an MTHFR polymorphism. Patients with the C667T allele may benefit from the highly bioavailable L-methylfolate (Lizer et al., 2011).
Furthermore, B vitamins are best delivered in a complex. Mech supplemented B complex vitamins and normalized several systemwide pathways (Mech & Farah, 2016).

Cobalamin

Cobalamin (B12) has a function in producing DNA, RNA, and red blood cells. This vitamin has a role in creating nerve tissue and in relieving neuropathy. Cobalamin makes methionine, critical for methylation DNA and neurotransmitter production. One human cross-sectional study was conducted by Veterans Affairs to determine the prevalence of cobalamin deficiency in subjects with spinal cord injury (SCI). SCI studies are important correlates to brain trauma given the extension of brain meningeal tissue into the spinal cord. This cobalamin study by Petchkrua is entitled “Prevalence of vitamin B12 deficiency in spinal cord injury.” Medical records were reviewed retrospectively with prospective blood collection. Cobalamin deficiency is known to impair DNA synthesis and cause “white matter demyelination of spinal cord and cerebral cortex, and distal peripheral nerve” damage. “Common clinical findings [of deficiency] are paresthesias and numbness, gait ataxia, depressed mood, and memory impairment (Petchkrua et al., 2003).” “These symptoms can be reversed with parenteral or high-

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dose oral cobalamin supplements, but can worsen and become irreversible if not treated early.” In this study 105 men (mean age 54.1 years) with SCI mostly due to trauma were assessed. Fasting blood samples were utilized to evaluate nutrient levels including cobalamin, folate, and methylmalonic acid. Researchers concluded that “13% of patients with SCI …. had high MMA or low cobalamin levels. Neuropsychiatric symptoms possibly due to cobalamin deficiency were seen in half of these patients.” “Vitamin B12 deficiency was more common in persons with complete SCI or subacute degeneration of the spinal cord.” “Given the possible risk or irreversible neuropsychiatric deficits, such as weakness or dementia, and given the relatively low cost of screening and the low cost and high efficacy of high-dose oral B12 replacement, clinicians should consider screening and early treatment of B12 deficiency (Petchkrua et al., 2003).”

B complex vitamins are critical to brain tissue function and restoration. B complex is water soluble and thus must be replaced in the diet daily. B complex vitamins are critical to function with amino acids in protein synthesis. Human studies have demonstrated dietary B complex deficiencies given injury and disease conditions. A research study on spinal cord injury (SCI) conducted with funding from the Canadian Institute of Health Research, by Walters et al is entitled “Evidence of dietary inadequacy in adults with chronic spinal cord injury” (Walters et al, 2009). Researchers found that B complex vitamins were deficient in women and men who suffered from chronic SCI. This study utilized 24 hour food frequency questionnaires in comparison with the Daily Recommended Intakes (DRIs). Subjects had experienced spinal cord trauma at least 12 months prior, and used assistance for mobility. Dietary recalls were conducted in the home where recipes, food items, and brands could be verified by the double-pass method. Baseline food frequency questionnaires were collected from 77 subjects initially and from 68 subjects at 6 months. Approximately, 50% of the participants regularly ingested supplements.

Walters’ results showed that men (n=63) had only 22% and women (n = 14) 14% of the DRI intake of thiamine. Men had a 5% of DRI intake of riboflavin and a 24% of DRI intake of pyridoxal phosphate. Folate intakes were 75% of DRI for men and 79% of DRI for women. Cobalamin intake was 6% of DRI for men and 29% of DRI for women.

Prior to this study “little had been known about dietary intake or adequacy among people with SCI.” These researchers recognized that “the DRIs are intended for healthy- able bodied persons, and meeting the recommended intakes for nutrients does not necessarily provide enough for individuals with acute or chronic disease.” Most multi- vitamin supplements do not appear to contain all B complex vitamins in adequate amounts.

B complex vitamins provide vital cofactors to facilitate brain healing. Riboflavin (B2) improves sensorimotor function, memory and decreases reactive astrocytes, edema, lesion size, and behavioral impairments. Niacin (B3) provides ATP and NAD for cell energy, improves sensory, motor, cognitive, behavioral, memory and decreases reactive astrocytes under TBI conditions. Pyridoxine (B6) decreases deleterious Hcy levels toxic to brain tissue. Both pyridoxine (B6) and folate (B9) are involved with producing methionine required for neurotransmitter pathways, transmethylation, transsulfuration,

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and DNA synthesis. Cobalamin (B12) was found to be deficient in SCI patients, and cobalamin supplementation reverses paresthesias, numbness, gait disorders, memory and mood disorders commonly found with cobalamin deficiency. Under injury conditions, B vitamin DRIs intended for healthy persons are not likely sufficient. B complex vitamins are water soluble and should either be replaced daily in the diet or supplemented. Given their prevalence in brain tissue, B vitamins are an important macronutrient to effect TBI repair in conjunction with DHA and amino acids.

Amino Acids

TBI brings lingering deficits in concentration, depression, sleep-wake cycle, behavior, and motor skills. In addition, is the concern of long term neurological sequelae of chronic traumatic encephalopathy, dementia, and Alzheimer’s disease. The “resting metabolic expenditure of the severely injured brain is almost 40% higher”. “Insufficient availability of essential amino acids cannot only negatively influence cell survival and function in the injured brain, but also can impair the function of other organs.” (Dash et al., 2016). The third most important brain macronutrients are the amino acids. Researchers have demonstrated amino acid deficits following TBI, as well as benefits of amino acid therapy post TBI.

Amino Acid Deficits with TBI.

Several studies have evaluated amino acid deficits as related to TBI. Researchers from the University of Texas evaluated 17 moderate to severe TBI patients living in long term care facilities. TBI patients had individual and essential amino acid levels significantly lower than 24 controls. Researchers suggest these findings demonstrate that TBI is a chronic disease state, where low amino acid levels persist long term (Durham et al., 2017).

Italian researchers suggest that hypoacidemia is an important force acting upon the traumatized brain. They evaluated concentrations of free amino acids post mTBI and sTBI. Modest changes in the levels of arginine, glycine, and GABA were found within the first week post mTBI. While sTBI patients experienced significantly longer lasting modifications of “glutamate, glutamine, aspartate, GABA, serine, glycine, alanine, arginine, taurine, methionine, tyrosine and phenylalanine.” Additionally, the Glu-Gln/ GABA and methyl cycles were disturbed (Amorini et al., 2017).

This Italian research team demonstrated that two months post TBI all essential amino acids (EAA) and most nonessential amino acid levels are reduced (Aquilani et al., 2003). Stroke victims were also found to have low plasma concentrations of amino acids (Aquilani et al., 2014). A Shriner’s research team from Texas looked at plasma amino acid concentrations in six male, sTBI patients. These were motor vehicle accident victims with a mean GCS of 5, but were generally able to perform basic living activities. TBI patient amino acid concentrations were 12% lower than controls. Even given a variety of diet, most amino acid levels remain stable in normal controls. Most of

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the reduction in TBI patient amino acids was due to valine which was 33% lower than controls. A 7g EAA drink was given to the six patients and six healthy controls. Total EAA increased after the drink, however, non-EAA production increased more in controls than TBI patients. Arginine and glutamine increased to higher levels in controls. Thus, TBI patients were not producing non-EAA. This report points to the need for TBI patients to maintain amino acid levels, particular valine long term (Borsheim et al., 2007).

The BCAAs isoleucine, leucine, and valine are critical to brain function in that they control protein synthesis. (Vuille-Dit-Billie, et al., 2012). Jeter found that mTBI patients had lower plasma branched-chained amino acids (BCAA) levels and sTBI patients had significantly lower plasma BCAA levels (Jeter et al., 2013). Following fluid percussion injury in adult, male mice, glutamate and GABA levels were not altered, however valine (50.8%), isoleucine (21.2%), and leucine (52.3%) were reduced. Compared with healthy controls, critically ill, sTBI patients demonstrate venous isoleucine, leucine, and valine levels which were 35% lower than controls during the first two weeks post TBI. However, tryptophan, phenylalanine, and tyrosine levels were increased by 19% in the weeks following TBI. Subjects received enteral nutrition in a dose dependent manner, which elevated but did not normalize plasma BCAA levels. Tryptophan, tyrosine, isoleucine, leucine and valine amino acid levels correlated with enteral nutrition received, while phenylalanine levels did not.

These studies have identified the essential, non-essential, and branched-chained amino acid group deficits in the TBI patient. Additional deficits exist for the TBI patient with individual amino acid levels. Deficits of the following individual amino acids may impede critical neurotransmitter pathway operations within the brain.

Methionine Deficit.

Methionine is the first amino acid in each new strand of DNA. Methionine is one of the body’s few methyl donors providing substrate to the transmethylation and transsulfuration pathways. The transmethylation provides methylation for DNA and histone production to package DNA, as well as phosphotidylcholine synthesis. Phosphotidylcholine is an integral cell membrane lipid, important for signaling and cell- cell communication. S-adenylmethionine (SAM), which is produced in the transsulfuration pathway, “is one of the most commonly used molecules in living organisms.” (Dash et al., 2016). SAM supplementation directly correlates with Hamilton Depression Rating scores. Levels of SAM products such as choline and other methionine metabolite levels were diminished with TBI (Dash et al., 2016).

University of Pennsylvania researchers found that “both mild and severe TBI causes significant reductions in plasma methionine levels (Dash et al., 2016).”

Additionally, a single nucleotide polymorphism (SNP) exists in catechol-o- methyltransferase (COMT) which metabolizes catecholamine neurotransmitters catalyzed by methionine. Given reduced degradation of the dopamine pathway metabolites, this SNP may influence nonverbal cognitive function post TBI.

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Researchers found that both mTBI and sTBI patients with the COMT Met158 allele have better prognoses, and the allele may be neuroprotective (Winkler et.al., 2017)(Winkler et al., 2016). Post TBI, this SNP discovery identifies how methionine epigenetic anomalies affect the efficacy of the dopamine pathway, in conjunction with methionine deficits being deleterious to the dopamine pathway, transmethylation, and transsulfuration.

Tyrosine and Tryptophan Deficit.

The amino acids tyrosine and tryptophan are critical to brain function. Tyrosine works with methionine in the dopamine pathway to provide dopamine, norepinephrine and epinephrine, important catecholamines for neural function. COMT degrades the final product epinephrine in this dopamine pathway. Tryptophan creates serotonin through 5- HTP which supplies the gut and brain. Tyrosine and tryptophan are essential in the diet. While small amounts of tyrosine are formed from phenylalanine, the conversion rate is low. Nutritional intake may be insufficient to correct plasma abnormalities in a catabolic state.

An investigation was undertaken to monitor levels of tyrosine, tryptophan and BCAAs post TBI. Ten men (15-60y/o) with severe TBI were admitted in a stupor due to traffic collisions causing diffuse brain damage. The best Glasgow Coma Scale was 8. Six healthy men were selected as controls. Total amino acid plasma and tryptophan levels of TBI patients and controls were similar. TBI patients had a significantly lower tyrosine level and reduced plasma availability of BCAAs, methionine and phenylalanine. Up to 110 days post trauma, patients continued to have low tyrosine and EAA plasma concentrations, but normal tryptophan. Aquilani found that subjects with sTBI showed reduced levels of tyrosine, tryptophan and other EAAs at admission and discharge.

In rodents, low tyrosine resulted in low catecholamine production reducing cognitive activity, motor performance, and memory (Aquilani et al., 2003). Reducing mice dietary intake of tyrosine by 40% reduces maze performance. Tyrosine and Tryptophan deficits are apparent with TBI and may impair cognitive, motor and memory function.

GABA-Glutamyl Deficit.

Following injury, de novo synthesis of GABA and glutamine are impaired in the hippocampus. Under gas chromatography mass spectroscopy (GC-MS) examination, TBI hippocampal brain slices as compared with control slices showed a 61.4% reduction in glutamate, 86.6% reduction in GABA, and a 92.2% reduction in aspartate. Specific enzymes required to produce these amino acids were found to be reduced (Cole et al., 2010).

“The balance of glutamatergic and GABAergic tone is crucial to normal neurologic function (Guerriero et al., 2015).” Glutamate is the primary excitatory neurotransmitter in the brain, while gamma-amino-butyric-acid (GABA) is the predominant inhibitory

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neurotransmitter. TBI causes brain cell membrane to swell and eventually burst. The Na+/K+ pump fails and chemicals including stored neurotransmitters such as GABA and glutamate are released into the damaged tissue. Injury disturbs the delicate cortex excitability:inhibitory neurotransmitter ratio of GABA and glutamate within the brain. Glutamate is interconverted to glutamine and GABA in the presence of vitamin B6.

Citric acid cycle intermediates are involved in GABA production, making production more complex. Complicated pathways such as balancing these GABA and glutamine neurotransmitter levels may be easily disrupted within the injured brain.

The brain stores GABA and glutamate in neurotransmitter pools to prevent neurotransmitter level disruptions. However, these pools are likely damaged during injury. TBI increases glutamate levels, perhaps through release of pooled glutamate or through amino acid remodeling, since glutamate is the end product of most amino acid breakdown. High levels of glutamate appear to be indicators of poor prognosis. Additionally, the disruption of GABA production by the loss of GABA neurons and increased nutrient requirements appears to be deleterious. As GABA neurons are lost, long term decreased inhibition may contribute to neurocognitive impairments, behavioral impairments, and eventually seizures. “As the [glutamate] excitability: [GABA] inhibitory ratio shifts towards excitability, neuronal injury, dysfunction, and apoptosis increases, resulting in an increase in morbidity (Guerriero et al, 2015).”

Amino Acid Interventions.

Neuron and brain function require an ample pool of free amino acids to utilize as precursors to form neurotransmitters “involved in motor, cognitive, neuroendocrine, and behavioral functions (Aquilani, 2003).” These metabolic and synaptic amino acid pools may require replenishment following TBI. Post TBI, the non-essential amino acids may not be produced (Borsheim et al, 2007), and the storage and production level of amino acid derivatives may be decreased (Jeter et al., 2013).

Shen found in a systematic review of five databases from 2005 to 2015 that antioxidant therapies such as amino acid replacement were generally beneficial in TBI patients. He notes that it is secondary TBI damage that causes the most injury through hematomas, edema, rising intracranial pressure (ICP) and decreased cerebral blood flow (CBF). Free radical damage such as mitochondrial dysfunction and apoptosis also occurs. The indication is that with secondary damage some element of prevention and/or restoration can occur. This review encourages providers that “by incorporating antioxidant therapies into practice, clinicians can help attenuate the oxidative post traumatic brain damage and optimize patients’ recovery (Shen et al., 2016).”

Providing amino acids for the brain to rebuild and reconstruct post injury is important for restoration of macronutrient levels and brain function. In Dal Negro’s study, essential amino acid (EAA) supplementation versus placebo was compared in severe COPD patients. These patients are important to EAA brain trauma studies as they showed altered cognition. Eighty-eight subjects were randomized to received a 4g oral mixture dose of essential amino acids or placebo for 12 weeks. Cognitive function was

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measured by the Mini Mental State Examination one week before EAA administration, then 4 and 12 weeks following EAA administration. This study found improved cognition, muscle metabolism, physical activity in the EAA supplemented cohort (Dal Negro et al., 2012). Additionally, the effect of supplemental EAAs vs. placebo was assessed in 41 elderly, coronary artery disease patients (mean age 79.8 yrs). Oral supplementation of EAAs was found to improve nutritional status, muscle strength, depressive symptoms, physical performance, and quality of life (Rondanelli et al., 2011)

Vuille-Dit-Bille considered aeromatic amino acid (AAA) and BCAA amino acid levels fortified through enteral nutrition as related to ICP and jugular venous oxygen saturation. Excitotoxicity has commonly been deleterious for TBI concomitantly with increased edema and cerebral pressure. Researchers compared retrospective analyses with 19 severe TBI patients under pharmacological coma with 44 healthy volunteers. Plasma TBI BCAA levels were significantly decreased in the first week, then trended higher with leucine and valine remaining lower than controls. Tyrosine, phenylalanine, and tryptophan levels gradually increased in TBI patients. Phenylalanine was associated with lower cerebral oxygen consumption and lower ICP, whereas BCAAs increased ICP and cerebral oxygen consumption (Vuille-Dit-Bille et al., 2012).

BCAA Intervention.

Some amino acid studies have considered intervention with the BCAAs of valine, isoleucine, and leucine. BCAA supplementation improved cognitive performance in several rodent and human research reports. Dietary BCAAs have been found to promote cognitive improvement by restoration of hippocampal function, where cognitive deficits often manifest following TBI. Additionally, BCAAs are found to help maintain synaptic and metabolic pools of GABA and glutamate. Rodents were treated with BCAA drinking water for 5 days which restored BCAA concentration levels similar to sham animals. Mice consuming BCAA drinking water showed no change from control mice to conditioned fear response as utilized to identify hippocampal cognitive impairment, and injury induced hippocampal deficits and cognitive impairment was reversed in study mice. “All brain injured mice that consumed BCAAs demonstrated cognitive impairment with a simultaneous restoration of net synaptic efficacy”. Additionally, BCAA applied brain slices demonstrated restoration of net synaptic efficacy.

Aquilani’s study of 40 male patients with severe TBI showed cognitive improvement with BCAA therapy. This study provided the intervention of 19.6g/day BCAA intravenously. Aquilani analyzed cognitive recovery, plasma BCAA concentrations, and plasma tyrosine and tryptophan levels. Patients receiving BCAA therapy significantly elevated their BCAA and tyrosine plasma levels. Tryptophan levels were higher in controls. Fifteen days post admission, while both patients and controls had improvement in the Disability Rating System scores, only BCAA supplemented patients had significant cognitive improvement (Aquilani et al., 2005).

In addition to improving cognitive deficits, BCAA supplemented in the drinking water of mice decreased sleep difficulties and increased wakefulness. Post TBI, 72% of patients

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experienced sleep difficulties. These sleep and wakefulness improvements were accomplished by “dietary amino acids [which] have [been] shown to act directly on orexin neurons to modulate membrane excitability” and increase orexin neuron stability (Lim et al., 2013). In eleven studies reviewed by Sharma, BCAA supplementation was found to have a neuroprotective role when low BCAA levels were restored. No study reported adverse effects with BCAA supplementation. In one case, a 60g dose was utilized (Fenstrom JD, 2005). This review article supports the beneficial effect of BCAA therapy for severe TBI (Sharma B et al., 2017).

In addition to cognitive and sleep improvements given BCAA, further studies found BCAA therapy to be neurorestorative (Cole et al., 2010). “BCAAs are essential amino acids and are precursors for de novo synthesis of GABA and glutamate, and intermediates for the citric acid cycle.” Supplemental BCAAs restored hippocampal network excitability, and wakefulness as evidence by EEG rhythms. They likely accomplish this task through glutamate and GABA pool restoration. Freezing response was restored analogous to naive animals indicating cognitive restoration. Hippocampal network excitability became normal. To determine BCAA treatment dose and duration, researchers fed FPI mice with 100mM BCAA drinking water ad libitum or 0.26 g/kg via oral gavage. Hippocampal performance and cognitive impairment improved after 5 and 10 days of therapy. Study findings report that a minimum of 5 days of BCAA treatment is required to restore normal fear conditioning. Functional deficits return if therapy is discontinued after 5 days (Elkind et al., 2015).

These findings demonstrating generally low amino acid levels post TBI are of critical importance given the need for adequate amino acid substrate to produce new proteins to heal TBI. The finding of Borsheim that non-essential amino acids were not being produced is significant to heal TBI. Elemental formulas containing all 20 amino acids are available. Supplemental protein drinks often provide 20 essential and non-essential amino acids to boost protein production in malnourished patients. The Institute of Medicine found that when sufficient elemental nutrient substrate was provided to brain trauma patients that second injury was reduced (IOM, 2011). Providing sufficient amino acid nutrition is now possible using supplemental enteral and parental formulas. Albeit, the benefits of intervening with these elemental amino acids must become better recognized for brain trauma patients.

Tyrosine and Tryptophan Intervention

TBI catabolism causes massive amino acid loss and skeletal muscle degradation. Given TBI damage, its likely that nutritional intake does not address plasma insufficiencies, and catabolic needs dwarf nutrient supplies. In rodents, researchers found that 45 minutes post tyrosine administration, brain tyrosine levels increased by 81% and DOPA levels increased 13%. Tyrosine improves behavioral anomalies, cognitive deficits, and working memory. Increasing tyrosine intake to the hypothalamus and cerebral cortex reversed maze impairment and improved behavioral deficits in both animal and humans. In humans, tyrosine affects memory, mood and performance due to hypoxia. Oral tyrosine increased dopa in CSF of Parkinson’s Disease (PD) patients (Aquilani et

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al., 2003). Prior human investigations with tyrosine have shown behavioral, cognitive, and stress response improvements post TBI. Tryptophan was found to improve myoclonus, reduce sleep latency, mania and depression (Aquilani et al., 2003). These nutrients are important in the diet to ensure proper function of the tyrosine-dopamine and tryptophan-serotonin neurotransmitter pathways and are critical to restoring brain function.

Cysteine and Glutamine Intervention

Cysteine is another important amino acid providing neuroprotective effects given TBI. N-acetyl-cysteine(NAC) improves behavioral, auditory and vestibular abilities following brain injury. Eakin’s study with male Sprague-Dawley rats “documents the efficacy of NAC in reversing or preventing cognitive abnormalities in rodent models of mild to moderate TBI” (Eakin et al., 2014). This study included a cognitive assessment using the novel object recognition and Y maze (spatial memory) behavioral tests. Early administration of NAC to TBI rodents eliminated cognitive Morris water maze deficits. More platform crossings were seen in rodents treated with NAC and sham rodents as compared with injured non-treated rodents. “Treatment with NAC reversed behavioral deficits associated with TBI.” NAC is likely neuroprotective working through the mechanism of having anti-oxidant, anti-inflammatory, and mitochondrial enhancing effects. The potent anti-oxidant glutathione which reduces oxidative stress is produced from cysteine. Memory and motivation regulation may be associated with cysteine.

Neuroprotective effects are apparent with cysteine derivative, N-acetyl-cysteine amide (NACA) administration in this next study. Given cysteine’s low bioavailability, NACA is utilized with its higher permeability. Gunther studied male Sprague-Dawley rats with focal penetrating TBI. They were administered intraperitoneally 300mg/kg NACA, immediately post TBI and 4 hours post TBI. At 24 hours, neural degeneration (Fluoro- Jade) was reduced by 35.0%, apoptosis (TUNEL staining) reduced by 38.7%, and the antioxidant manganese superoxide dismutase increased by 35.9%. NACA treatment reduced degenerating neurons and apoptosis (Gunther et al., 2015).

The neuronal uptake of the amino acid cysteine is regulated by a transporter gene which Choi, evaluated. His study found that the cysteine transporter EAAC1 is “an important antioxidant system for reducing TBI-induced neuronal death.” “NAC significantly prevented neuronal death and reactive oxygen species production in the hippocampus” and reduced zinc’s accumulation (Choi et al., 2016). Finally, in one study using injured hippocampal slices, glutamine was found to reverse dentate gyrus hyperexcitability and injury-induced net synaptic imbalance in a restorative effect (Cole et al., 2010).

Taurine Intervention.

The most abundant free amino acid in the brain is taurine. “Taurine is involved in neuroprotection and regeneration after injury in the nervous system”. “Studies have

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demonstrated that taurine plays a critical role in the aspects of detoxification, membrane stabilization, osmoregulation, neurotransmission, calcium homeostasis and antioxidant (Huxtable 1992; Schaffer et al. 2000; Georgia et al. 2003; El Idrissi 2008).” “Taurine has been helpful for TBI recovery (Curtis and Epstein 2014)”. “Sun demonstrated that treatment with taurine markedly reduce neurological deficits, lessened brain swelling, attenuated cell death, and decreased the infarct volume 72h after ischemia in a rat model of stroke (Sun et al, 2012)(Wang et al., 2016).”

The protective effects of taurine were evaluated in this next investigation. The left cortex of adult, male Sprague-Dawley rats was damaged by fluid percussion injury (FPI). Taurine 200mg/kg was given by tail IV once daily for 7 days post TBI. CBF in the left cortex significantly improved within 30 minutes post injury and more so after 7 days of treatment. The ultrastructure of nerve cells almost returned to normal. Edema was further alleviated and ICP elevated. Data from this study suggested that taurine could exert a similar effect as anticoagulant therapy and improve the post TBI hypercoagulable state. “Mitochondria abnormalities cause progression in neuronal injury post TBI. Taurine treatment for 7 days alleviated the swelling of mitochondria and other organelles.” This study showed improved efficacy of the mitochondrial respiratory complexes, and improved their enzymatic activity. Taurine lowered the apoptosis rate, prevented calcium overload, and prevented endoplasmic reticulum stress. Taurine appears to exert a protective function in numerous pathways specifically by reducing cortex and hippocampal neuron damage (Wang et al., 2016).

Many enteral/parenteral nutrition formulas supply the eight essential amino acids in the form of peptides or protein. These proteins and peptides require digestion before they are available in the blood stream for use. Elemental amino acid formulas are available, however the use of these formulas for brain trauma may not yet be widespread. Rondanelli found that free amino acid ingestion increased plasma essential amino acid (EAA) levels more quickly than dietary protein ingestion. Free amino acids were readily usable versus proteins requiring digestion prior to new tissue incorporation. As amino acid plasma levels increased, extra-intestinal tissue/organ uptake increased. Additionally, free amino acids were found to disappear more quickly, suggesting increased uptake and usability. Nine voluntary elderly subjects and nine younger subjects were fed in a random alternate manner either free essential amino acids diluted with 100ml water or dietary protein components. EAA levels peaked in younger subjects at 30 minutes whereas, EAA levels peaked in older subjects at 90 minutes. Free EAAs were found to provide a greater stimulus to replenish cells (Rondanelli et al., 2017).

Enteral and parental formulas supply essential amino acids, however, Borsheim found that the non-essential amino acids are not produced given TBI. The simultaneous presence of all 20 amino acids is required for construction of new DNA and cell protein structures. The TBI hyper-metabolic state may require the increased efficiency of having these 20 free amino acids simultaneously available in the blood supply for efficient new protein construction. Rondanelli found that amino acid derivatives may not be produced given TBI (Rondanelli et al., 2017). Pathways and/or communication may be damaged

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post TBI. Thus, derivatives such as GABA and taurine must be individually supplied to receive benefit. Finally, as discussed previously, B complex vitamins are required as cofactors to metabolize amino acids. Patients ingesting nutrition formulas supplying amino acids would benefit from the addition of B complex vitamins.

In conclusion, there currently is no cure for TBI. Amino acids, B-complex vitamins and DHA are three critical macronutrients necessary to replenish the damaged brain and mitigate injury. These macronutrients function akin to the bricks, mortar, and rebar to reconstruct a damaged wall. Given sufficient individual substrates, the brain is able to immediately begin repair of TBI, repairing tissue with substrate analogous to the nutrients found in original brain tissue. Working with macronutrient biochemistry to provide original nutrients to effect repair is more likely to restore original function to cerebral tissue. It is hopeful that the detailed research presented in this report will heighten the awareness of medical practitioners as to the efficacy of biochemical nutrients improving TBI prognosis. According to the research findings presented in this report, the three important, basic macronutrients of DHA, B-complex and amino acids may provide long term improvement in TBI prognosis. We currently have no cure for TBI. Given the increased incidence, staggering cost, disabling injuries, long term mental decline, and epidemic status of TBI, patients are desperately in need of health care providers to offer a simple biochemical therapy to fortify and rebuild their brain tissue. The brain macronutrients of DHA, B-complex vitamins and elemental amino acids may be the long term solution.

DHA References:

Warden D. (2006). Military TBI during the Iraq and Afghanistan wars. J Head Trauma Rehabil 21, 398–402.

Hasadri L, Wang BH, Lee JV, Erdman JW, Liano DA, Barbey AK, Wszalek T, Sharrock MF, Wang HJ. Omega-3 fatty acids as a putative treatment for traumatic brain injury. J Neurotrauma. 2013 Jun 1;30(11):897-906.doi:10.1089/neu.2012.2672.Epub 2013 Jun 5.

Humphreys, I., Wood, R.L., Phillips, C.J., and Macey, S. (2013). The costs of traumatic brain injury: a literature review. Clinicoecon. Outcomes Res. 5, 281–287.

Barrett EC, McBurney MI, Ciappio ED. w-3 fatty acid supplementation as a potential therapeutic aid for the recovery from mild traumatic brain injury/concussion. Adv Nutr. 2014 May 14;5(3):268-77. doi: 10.3945/an.113.005280. Print 2014 May.

Omalu BI, DeKosky ST, Hamilton RL, et al. (2006). Chronic trau- matic encephalopathy in a national football league player: Part II. Neurosurgery 59, 1086–1092; discussion 92–93.

Guskiewicz KM, Marshall SW, Bailes J, et al. (2007). Recurrent concussion and risk of depression in retired professional football players. Med Sci Sports Exerc 39, 903–909.

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Omalu BI, Bailes J, Hammers JL, and Fitzsimmons RP. (2010). Chronic traumatic encephalopathy, suicides and parasuicides in professional American athletes: The role of the forensic pathologist. Am J Forensic Med Pathol 31, 130–132.

Flanagan, S.R., Cantor, J.B., and Ashman, T.A. Traumatic brain injury: future assessment tools and treatment prospects. (2008). Neuropsychiatr. Dis. Treat. 4, 877– 892

Scrimgeour AG, Condlin ML (2014). Nutritional Treatment for Traumatic Brain Injury. Journal of Neurotrauma 31:989-999. (NIH-R)

Institute of Medicine, April 22, 2011. Nutrition and Traumatic Brain Injury: Improving Acute and Subacute Health Outcomes in Military Personnel.

DeMar JC Jr. Ma K, Bell JM, Igarashi M, Greenstein D, et al. (2006). One generation of n-3 polyunsaturated fatty acid deprivation increases depression and aggression test scores in rats. J. Lipid Res 47:172-180.

Desai A, Kerala K, Hee-Yong K. (2014). Depletion of brain docosahexaenoic acid impairs recovery from traumatic brain injury. PLOS one, volume 9, issue 1.

Lewis M, Ghassemi MS, Hibbein MD. (2013). Therapeutic use of omega-3 fatty acids in severe head trauma. Am J Emerg Med 31(1): 273.e5-273.e8. Doi:10.1016/j.ajem. 2012.05.014

Kris-Etherton PM, Taylor DS, Yu-Poth S, et al. (2000). Poly- unsaturated fatty acids in the food chain in the United States. Am J Clin Nutr 71, 179S–88S.

Raper NR, Cronin FJ, and Exler J. (1992). Omega-3 fatty acid content of the US food supply. J Am Coll Nutr 11, 304–308.

Tur JA, Bibiloni MM, Sureda A, and Pons A. (2012). Dietary sources of omega 3 fatty acids: Public health risks and benefits. Br J Nutr 107, S23–52.

Simopoulos, A.P. (2011). Importance of the omega-6/omega-3 bal- ance in health and disease: evolutionary aspects of diet. World Rev. Nutr. Diet 102, 10–21.

Singh M. (2005). Essential fatty acids, DHA and human brain. Indian J Pediatr 72, 239– 242.

Dyall, S.C., and Michael-Titus, A.T. (2008). Neurological benefits of omega-3 fatty acids. Neuromolecular Med. 10, 219–235.

Salem, N., Jr., Litman, B., Kim, H.Y., and Gawrisch, K. (2001). Mechanisms of action of docosahexaenoic acid in the nervous sys-

Page 24 of 34

tem. Lipids 36, 945–959.

Cao, D., Xue, R., Xu, J., and Liu, Z. (2005). Effects of docosahex- aenoic acid on the survival and neurite outgrowth of rat cortical neurons in primary cultures. J. Nutr. Biochem. 16, 538–546.

Robson, L.G., Dyall, S., Sidloff, D., and Michael-Titus, A.T. (2010). Omega-3 polyunsaturated fatty acids increase the neurite outgrowth of rat sensory neurones throughout development and in aged animals.
Neurobiol. Aging 31, 678–687

Mills JD, Bailes, JE, Sedney CL, Hutchins H, and Sears B. (2011). Omega-3 fatty acid supplementation and reduction of traumatic axonal injury in a rodent head injury model. J. Neurosurgery 114, 77-84.

Mills JD, Hadley K, and Bailes JE. (2011). Dietary supplementation with the omega-3 fatty acid docosahexaenoic acid in traumatic brain injury. Neurosurgery 68, 474-481; discussion 481.

Homayoun, P., Parkins, N.E., Soblosky, J., Carey, M.E., Rodriguez de Turco, E.B., and Bazan, N.G. (2000). Cortical impact injury in rats promotes a rapid and sustained increase in polyunsaturated free fatty acids and diacylglycerols. Neurochem. Res. 25, 269–276.

Horrocks, L.A., and Farooqui, A.A. (2004). Docosahexaenoic acid in the diet: its importance in maintenance and restoration of neural membrane function. Prostaglandins Leukot. Essent. Fatty Acids 70, 361–372.

Ying Z, Feng C, Agrawal R, Zhuang Y, Gomez-Pinilla F. (2012). Dietary omega-3 deficiency from gestation increases spinal cord vulnerability to traumatic brain injury- induced damage. PlosOne volume 7, issue 12, e52998.

Wu Z, Ying Z, Gomez-Pinilla F. (2013). Exercise facilitates the action of dietary DHA on functional recovery after brain trauma. Neuroscience 248 (2013) 655-663.

Agrawal R, Tyagi E, Vergnes L, Reue K, Gomez-Pinilla F. Coupling energy homeostasis with a mechanism to support plasticity in brain trauma. Biochim Biophys Acta. 2014 Apr; 1842(4):535-46. doi: 10.1016/j.bbadis.2013.12.004. Epub 2013 Dec 15. PMID: 24345766

Stillwell W, and Wassall SR. (2003). Docosahexaenoic acid: Mem- brane properties of a unique fatty acid. Chem Phys Lipids 126, 1–27.

Stillwell W, Shaikh SR, Zerouga M, Siddiqui R, and Wassall SR. (2005). Docosahexaenoic acid affects cell signaling by altering lipid rafts. Reprod Nutr Dev 45, 559–579.

Page 25 of 34

Valentine RC, and Valentine DL. (2004). Omega-3 fatty acids in cellular membranes: A unified concept. Prog Lipid Res 43, 383–402.

Hering H, Lin CC, and Sheng M. (2003). Lipid rafts in the mainte- nance of synapses, dendritic spines, and surface AMPA receptor
stability. J Neurosci 23, 3262–3271.

Innis SM. (2008). Dietary omega 3 fatty acids and the developing brain. Brain Res 1237, 35–43.

Turner N, Else PL, and Hulbert AJ. (2003). Docosahexaenoic acid (DHA) content of membranes determines molecular activity of the sodium pump: Implications for disease states and metabolism. Nat- urwissenschaften 90, 521–523.

Innis SM. (2007). Dietary (n-3) fatty acids and brain development. J Nutr 137, 855–859.

Lane MA, and Bailey SJ. (2005). Role of retinoid signalling in the adult brain. Prog Neurobiol 75, 275–293.

Ahmad A, Moriguchi T, and Salem N. (2002). Decrease in neuron size in docosahexaenoic acid-deficient brain. Pediatr Neurol 26, 210– 218.

Aid S, Vancassel S, Poumes-Ballihaut C, Chalon S, Guesnet P, and Lavialle M. (2003). Effect of a diet-induced n-3 PUFA depletion on cholinergic parameters in the rat hippocampus. J Lipid Res 44, 1545– 1551.

Chalon S. (2006). Omega-3 fatty acids and monoamine neurotrans- mission. Prostaglandins Leukot Essent Fatty Acids 75, 259–269.

Innis SM, Gilley J, and Werker J. (2001). Are human milk long-chain polyunsaturated fatty acids related to visual and neural development in breast-fed term infants? J Pediatr 139, 532–538.

WainwrightRE,Bulman-FlemingMB,Lev́esqueS,MutsaersL,andMcCutcheonD. (1998). A saturated-fat diet during development alters dendritic growth in mouse brain. Nutr Neurosci 1, 49–58

Figueroa JD, Cordero K, Lian MS, De Leon M. Dietary omega-3 polyunsaturated fatty acids improve the neurolipidome and restore the DHA status while promoting functional recovery after experimental spinal cord injury. J Neurotrauma. 2013 May 15;30(10): 853-68. doi: 10.1089/neu.2013.2718.

Figueroa JD, Cordero K, Serrano-Illan M, Almeyda A, Baldeosingh K, Almaguel FG, De Leon M. Metabolomics uncovers dietary omega-3 fatty acid-dervied metabolites implicated in anti-nociceptive response after experimental spinal cord injury.

Page 26 of 34

Neuroscience 2013;255-1-18. doi: 10.1016/j.neuroscience.2013.09.012. Epub 2013 Sep 14

Hong S, Gronert K, Devchand PR, Moussignac RL, Serhan CN (2003) Novel docosatrienes and 17S-resolvins generated from docosahexaenoic acid in murine brain, human blood, and glial cells autacoids in anti-inflammation. J Biol Chem 278: 14677– 14687.

Mukherjee PK, Marcheselli VL, Serhan CN, Bazan NG (2004) Neuroprotectin D1: a docosahexaenoic acid-derived docosatriene protects human retinal pigment epithelial cells from oxidative stress. Proc Natl Acad Sci USA 101: 8491–8496

Schober ME, Requena DF, Abdullah OM, Casper TC, Beachy J, Malleske D, Pauly JR. (2016). Journal of Neurotrauma 33:390-402.

Kraft PR, Bailey EL, Lekic T, Rolland WB, Altay O, Tang J, Wardlaw JM, Zhang JH, Sudlow CL. (2012). Etiology of stroke and choice of models. Int. J. Stroke 7, 398-406.

FDA. Substances affirmed as generally recognized as safe: Menhaden oil. (1997). Final Rule: Federal Registry 30751–30757.

Hibbeln JR: Fish consumption and major depression. Lancet 1998; 351(9110): 1213

Hallahan B, Hiebbeln JR, Davis JM, Garland MR: Omega-3 fatty acid supplementation in patients with recurrent self-harm. Single-centre double-blind randomised controlled trial. Br. J Psychiatry 2007; 190: 118-22.

Nowrangi MA, Kortte KB, Rao VA. A perspectives approach to suicide after traumatic brain injury: case and review. Psychosomatics. 2014 Sep-Oct;55(5):430-7. doi: 10.1016/ j.psym.2013.11.006. Epub 2013 Nov 28. Review. PMID: 25223637

Lewis MD, Bailes J, (2011) Neuroprotection for the warrior: dietary supplementation with omega-3 fatty acids. Mil Med 2011 Oct; 176(10):1120-7.

Academy of Nutrition and Dietetics Evidence Analysis Library (2014). “Dietary Fatty Acids for Healthy Adults”. Position paper.

Davis BC, Kris-Etherton PM. Achieving optimal essential fatty acid status in vegetarians: Current knowledge and practical implications. Am J Clin Nutr. 2003;78(3 suppl):640S-646S.

Burdge G. Alpha-linolenic acid metabolism in men and women: Nutritional and biological implications. Curr Opin Clin Nutr Metab Care. 2004;7(2):137-144

Page 27 of 34

Harris, W.S., Kris-Etherton, P.M., and Harris, K.A. (2008). Intakes of long-chain omega-3 fatty acid associated with reduced risk for death from coronary heart disease in healthy adults. Curr. Atheroscler. Rep. 10, 503–509.

Lien, E.L. (2009). Toxicology and safety of DHA. Prostaglandins Leukot. Essent. Fatty Acids 81, 125–132.

Maresta, A., Balduccelli, M., Varani, E., Marzilli, M., Galli, C., Heiman, F., Lavezzari, M., Stragliotto, E., and De Caterina, R.;ESPRIT Investigators. et al. (2002). Prevention of post-coronary angioplasty restenosis by omega-3 fatty acids: main results of the Esapent for Prevention of Restenosis Italian Study (ESPRIT). Am. Heart. J. 143, E5.

Filion, K.B., El Khoury, F., Bielinski, M., Schiller, I., Dendukuri, N., and Brophy, J.M. (2010). Omega-3 fatty acids in high-risk cardio- vascular patients: a meta-analysis of randomized controlled trials. BMC Cardiovasc. Disord. 10, 24.

Marik, P.E., and Varon, J. (2009). Omega-3 dietary supplements and the risk of cardiovascular events: a systematic review. Clin. Cardiol. 32, 365–372.

Woodman, R.J., Mori, T.A., Burke, V., Puddey, I.B., Barden, A., Watts, G.F., and Beilin, L.J. (2003). Effects of purified eicosa- pentaenoic acid and docosahexaenoic acid on platelet, fibrinolytic and vascular function in hypertensive type 2 diabetic patients. Atherosclerosis 166, 85–93.

Kris-Etherton, P.M., Harris, W.S., and Appel, L.J. (2003). Omega-3 fatty acids and cardiovascular disease: new recommendations from the American Heart Association. Arterioscler. Thromb. Vasc. Biol. 23, 151–152.

Bays, H.E. (2007). Safety considerations with omega-3 fatty acid therapy. Am. J. Cardiol. 99, 35C–43C.

Calo, L., Bianconi, L., Colivicchi, F., Lamberti, F., Loricchio, M.L., de Ruvo, E., Meo, A., Pandozi, C., Staibano, M., and Santini, M. (2005). N-3 Fatty acids for the prevention of atrial fibrillation after coronary artery bypass surgery: a randomized, controlled trial. J. Am. Coll. Cardiol. 45, 1723–1728.

Covington, M.B. (2004). Omega-3 fatty acids. Am. Fam. Physician 70, 133–140.

Bailes JE, Patel V, (2014). The potential for DHA to mitigate mild traumatic brain injury. Mil Med 2014 Nov;179(11 Suppl):112-6. doi: 10.7205/MILMED-D-14-00139.

Painter TJ, Rickerds J, Alban RF. (2015). Immune enhancing nutrition in traumatic brain injury – A preliminary study. International Journal of Surgery 21, 70-74.

McClave SA, Martindale RG, Vanek VW, et al., Guidelines for the Provision and Assessment of Nutrition Support Therapy in the Adult Critically Ill Patient: Society of

Page 28 of 34

Critical Care Medicine (SCCM) and American Society for Parenteral and Enteral Nutrition (A.S.P.E.N.). JPEN J Parenteral Enteral Nutr. May-Jun;2009 33(3):277-316. PubMed ID: 19398613

McClave SA, Taylor B, Martindale RG, et al. Guidelines for the Provision and Assessment of Nutrition Support Therapy in the Adult Critically Ill Patient: Society of Critical Care Medicine (SCCM) and American Society for Parenteral and Enteral Nutrition (A.S.P.E.N.) J Parenteral Enteral Nutr. Feb 2016 159-211.

Colombo, J., Carlson, S.E., Cheatham, C.L., Shaddy, D.J., Kerling, E.H., Thodosoff, J.M., Gustafson, K.M., and Brez, C. (2013). Long- term effects of LCPUFA supplementation on childhood cognitive outcomes. Am. J. Clin. Nutr. 98, 403–412.

Fleith, M. and Clandinin, M.T. (2005). Dietary PUFA for preterm and term infants: review of clinical studies. Crit. Rev. Food Sci. Nutr. 45, 205–229.

Stonehouse, W. (2014). Does consumption of LC omega-3 PUFA enhance cognitive performance in healthy school-aged children and throughout adulthood? Evidence from clinical trials. Nutrients 6, 2730–2758.

Russell, K.L., Berman, N.E., Gregg, P.R., and Levant, B. (2014). Fish oil improves motor function, limits blood-brain barrier disruption, and reduces Mmp9 gene expression in a rat model of juvenile traumatic brain injury. Prostaglandins Leukotr. Essent. Fatty Acids 90, 5–11.

Nestle Health Science “Impact Peptide 1.5 Product Details” (2017). https://

http://www.nestlehealthscience.us/brands/impact/impact-peptide-1-5
Fresenius Kabi “SMOFLIPID Description” (2017). http://smoflipid.com/Smoflipid-PI.pdf

B-complex references:

Haar, C.V., Peterson, T.C., Martens, K.M., Hoane, M.R. (2015). Vitamins and nutrients as primary treatments in experimental brain injury: clinical implications for nutraceutical therapies. Brain Research 1640, 114-129. doi: 10.1016/j.brainres.2015.12.03

Hoane, M. R., Wolyniak, J. G., & Akstulewicz, S. L. (2005). Administration of riboflavin improves behavioral outcome and reduces edema formation and glial fibrillary acidic protein expression after traumatic brain injury. Journal of Neurotrauma, 22(10), 1112-22. doi:http://dx.doi.org.libproxy1.usc.edu/10.1089/neu.2005.22.1112

Hoane, M.R., Akstulewicz, S.L., Toppen, J. Treatment with Vitamin B3 Improves Functional Recovery and Reduces GFAP Expression following Traumatic Brain Injury in Rats. (2003) Journal of Neurotrauma. 20(1): 1189-1199

Page 29 of 34

Hoane MR, Kaplan SA, Ellis AL. The effects of nicotinamide on apoptosis and blood- brain barrier breakdown following traumatic brain injury. Brain Res. 2006 Dec 13;1125(1):185-93. Epub 2006 Nov 14. PMID: 17109832

Swan AA, Chandrashekar R, Beare J, Hoane MR. Preclinical efficacy testing in middle- aged rats: nicotinamide, a novel neuroprotectant, demonstrates diminished preclinical efficacy after controlled cortical impact. J Neurotrauma. 2011 Mar;28(3):431-40. doi: 10.1089/neu.2010.1519. Epub 2011 Jan 9. PMID: 21083416

Rahmani A, Hatefi M, Dastjerdi MM, Zare M, Imani A, Shirazi D. Correlation Between Serum Homocysteine Levels and Outcome of Patients with Severe Traumatic Brain Injury. World Neurosurg. 2016 Mar;87:507-15. doi: 10.1016/j.wneu.2015.09.016. Epub 2015 Sep 18. PMID: 26386458

Schaeffer MC. Excess dietary vitamin B-6 alters startle behavior of rats. J Nutr. 1993 Aug;123(8):1444-52. PMID: 8336216

Naim, M.Y., Friess, S., Smith, C., Ralston, J., Ryall, K., Helfaer, M.A. & Margulies, S.S. (2011). Folic acid enhances early functional recovery in a piglet model of pediatric head injury. Developmental Neuroscience, 32(5-6), 466-79. doi:http:// dx.doi.org.libproxy2.usc.edu/10.1159/000322448

Paul RT, McDonnell AP, Kelly CB. Folic acid: neurochemistry, metabolism and relationship to depression. Hum Psychopharmacol. 2004 Oct;19(7):477-88. Review. PMID: 15378677

Lizer MH, Bogdan RL, Kidd RS. Comparison of the frequency of the methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism in depressed versus nondepressed patients. J Psychiatr Pract. 2011 Nov;17(6):404-9. doi: 10.1097/01.pra.0000407963.26981.a6

Bhatia P, Singh N. Homocysteine excess: delineating the possible mechanism of neurotoxicity and depression. Fundam Clin Pharmacol. 2015 Dec;29(6):522-8. doi: 10.1111/fcp.12145. Epub 2015 Sep 17. PMID 26376956

Nazki FH, Sameer AS, Ganaie BA. Folate: metabolism, genes, polymorphisms and the associated diseases. Gene. 2014 Jan 1;533(1):11-20. doi: 10.1016/j.gene.2013.09.063. Epub 2013 Oct 1. PMID: 24091066

Delport D, Schoeman R, van der Merwe N, van der Merwe L, Fisher LR, Geiger D, Kotze MJ. Significance of dietary folate intake, homocysteine levels and MTHFR 677 C>T genotyping in South African patients diagnosed with depression: test development for clinical application. Metab Brain Dis. 2014 Jun;29(2):377-84. doi: 10.1007/ s11011-014-9506-7. Epub 2014 Feb 18.

Page 30 of 34

Mech AW, Farah A. Correlation of clinical response with homocysteine reduction during therapy with reduced B vitamins in patients with MDD who are positive for MTHFR C677T or A1298C polymorphism: a randomized, double-blind, placebo-controlled study. J Clin Psychiatry. 2016 May;77(5):668-71. doi: 10.4088/JCP.15m10166. PMID: 27035272 Free Article.

Achour O, Elmtaoua S, Zellama D, Omezzine A, Moussa A, Rejeb J, Boumaiza I, Bouacida L, Rejeb NB3, Achour A, Bouslama A. The C677T MTHFR genotypes influence the efficacy of B9 and B12 vitamins supplementation to lowering plasma total homocysteine in hemodialysis. J Nephrol. 2016 Oct;29(5):691-8. doi: 10.1007/ s40620-015-0235-8. Epub 2015 Nov 11. PMID: 26559681

Petchkrua W, Burns SP, Stiens SA, James JJ, Little JW. Prevalence of vitamin B12 deficiency in spinal cord injury. Archives of Physical Medicine and Rehabilitation. November 2003, Vol.84(11): 1675-1679, doi:10.1053/S0003-9993(03)00318-6. http:// http://www.sciencedirect.com.libproxy1.usc.edu/science/article/pii/S0003999303003186?via %3Dihub.

Walters JL, Buchholz AC, Martin Ginis KA. Evidence of dietary inadequacy in adults with chronic spinal cord injury. Spinal Cord (2009) 47, 318-322;doi:10.1038/sc.2008.134 published online 11 November 2008. http://www.nature.com/sc/journal/v47/n4/full/ sc2008134a.html

Amino Acid References:

Institute of Medicine, April 22, 2011. Nutrition and Traumatic Brain Injury: Improving Acute and Subacute Health Outcomes in Military Personnel.

Dash PK, Hergenroeder GW, Jeter CB, Choi HA, Kobori N, Moore AN. Traumatic Brain Injury Alters Methionine Metabolism: Implications for Pathophysiology. Front Syst Neurosci. 2016 Apr 29;10:36. doi: 10.3389/fnsys.2016.00036. eCollection 2016. PMID: 27199685

Durham WJ, Foreman JP, Randolph KM, Danesi CP, Spratt H, Masel BD, Summons JR, Singh CK, Morrison M, Robles C, Wolfram C, Kreber LA, Urban RJ, Sheffield-Moore M, Masel BE. Hypoaminoacidemia Characterizes Chronic Traumatic Brain Injury. J Neurotrauma. 2017 Jan 15;34(2):385-390. doi: 10.1089/neu.2015.4350. Epub 2016 Jun 16. PMID: 27178787

Amorini AM, Lazzarino G, Di Pietro V, Signoretti S, Lazzarino G, Belli A, Tavazzi B. Severity of experimental traumatic brain injury modulates changes in concentrations of cerebral free amino acids. J Cell Mol Med. 2017 Mar;21(3):530-542. doi: 10.1111/jcmm. 12998. Epub 2016 Oct 3. PMID: 27696676 Free PMC Article

Page 31 of 34

Aquilani R, Iadarola P, Boschi F, Pistarini C, Arcidiaco P, Contardi A. Reduced plasma levels of tyrosine, precursor of brain catecholamines, and of essential amino acids in patients with severe traumatic brain injury after rehabilitation. Arch Phys Med Rehabil. 2003 Sep;84(9):1258-65.

PMID: 13680559

Aquilani R, Boselli M, Paola B, Pasini E, Iadarola P, Verri M, Viglio S, Condino A, Boschi F. Is stroke rehabilitation a metabolic problem? Brain Inj. 2014;28(2):161-73. doi: 10.3109/02699052.2013.860470. PMID: 24456056

Børsheim E, Bui QU, Wolfe RR. Plasma amino acid concentrations during late rehabilitation in patients with traumatic brain injury. Arch Phys Med Rehabil. 2007 Feb; 88(2):234-8. PMID: 17270522

Vuille-Dit-Bille RN, Ha-Huy R, Stover JF. Changes in plasma phenylalanine, isoleucine, leucine, and valine are associated with significant changes in intracranial pressure and jugular venous oxygen saturation in patients with severe traumatic brain injury. Amino Acids. 2012 Sep;43(3):1287-96. doi: 10.1007/s00726-011-1202-x. Epub 2011 Dec 22. PMID: 22189890

Jeter CB1, Hergenroeder GW, Ward NH 3rd, Moore AN, Dash PK. Human mild traumatic brain injury decreases circulating branched-chain amino acids and their metabolite levels. J Neurotrauma. 2013 Apr 15;30(8):671-9. doi: 10.1089/neu. 2012.2491. Epub 2013 Apr 6. PMID: 23560894

Winkler EA, Yue JK, Ferguson AR, Temkin NR, Stein MB, Barber J, Yuh EL, Sharma S, Satris GG, McAllister TW, Rosand J, Sorani MD, Lingsma HF, Tarapore PE, Burchard EG, Hu D, Eng C, Wang KK, Mukherjee P, Okonkwo DO, Diaz-Arrastia R, Manley GT; TRACK-TBI Investigators.COMT Val 158 Met polymorphism is associated with post- traumatic stress disorder and functional outcome following mild traumatic brain injury. J Clin Neurosci. 2017 Jan;35:109-116. doi: 10.1016/j.jocn.2016.09.017. Epub 2016 Oct 18.PMID: 27769642

Winkler EA, Yue JK, McAllister TW, Temkin NR, Oh SS, Burchard EG, Hu D, Ferguson AR, Lingsma HF, Burke JF, Sorani MD, Rosand J, Yuh EL, Barber J, Tarapore PE, Gardner RC, Sharma S, Satris GG, Eng C, Puccio AM, Wang KK, Mukherjee P, Valadka AB, Okonkwo DO, Diaz-Arrastia R, Manley GT; TRACK-TBI Investigators. COMT Val 158 Met polymorphism is associated with nonverbal cognition following mild traumatic brain injury. Neurogenetics. 2016 Jan;17(1):31-41. doi: 10.1007/s10048-015-0467-8. Epub 2015 Nov 17. PMID: 26576546

Cole JT, Mitala CM, Kundu S, Verma A, Elkind JA, Nissim I, Cohen AS. Dietary branched chain amino acids ameliorate injury-induced cognitive impairment. Proc Natl Acad Sci U S A. 2010 Jan 5;107(1):366-71. doi: 10.1073/pnas.0910280107. Epub 2009 Dec 7. Erratum in: Proc Natl Acad Sci U S A. 2010 Feb 2;107(5):2373. PMID: 19995960

Page 32 of 34

Guerriero RM, Giza CC, Rotenberg A. Glutamate and GABA imbalance following traumatic brain injury. Curr Neurol Neurosci Rep. 2015 May;15(5):27. doi: 10.1007/ s11910-015-0545-1. Review. PMID: 25796572

Shen Q, Hiebert JB, Hartwell J, Thimmesch AR, Pierce JD. Systematic Review of Traumatic Brain Injury and the Impact of Antioxidant Therapy on Clinical Outcomes. Worldviews Evid Based Nurs. 2016 Oct;13(5):380-389. doi: 10.1111/wvn.12167. Epub 2016 May 31. Review. PMID: 27243770

Dal Negro RW, Testa A, Aquilani R, Tognella S, Pasini E, Barbieri A, Boschi F. Essential amino acid supplementation in patients with severe COPD: a step towards home rehabilitation. Monaldi Arch Chest Dis. 2012 Jun;77(2):67-75. PMID: 23193843

Rondanelli M, Opizzi A, Antoniello N, Boschi F, Iadarola P, Pasini E, Aquilani R, Dioguardi FS. Effect of essential amino acid supplementation on quality of life, amino acid profile and strength in institutionalized elderly patients. Clin Nutr. 2011 Oct;30(5): 571-7. doi: 10.1016/j.clnu.2011.04.005. Epub 2011 Jun 1. PMID: 21636183

Aquilani R, Iadarola P, Contardi A, Boselli M, Verri M, Pastoris O, Boschi F, Arcidiaco P, Viglio S. Branched-chain amino acids enhance the cognitive recovery of patients with severe traumatic brain injury. Arch Phys Med Rehabil. 2005 Sep;86(9):1729-35.

Lim MM, Elkind J, Xiong G, Galante R, Zhu J, Zhang L, Lian J, Rodin J, Kuzma NN, Pack AI, Cohen AS. Dietary therapy mitigates persistent wake deficits caused by mild traumatic brain injury. Sci Transl Med. 2013 Dec 11;5(215):215ra173. doi: 10.1126/ scitranslmed.3007092. PMID: 24337480

Fernstrom JD. Branched-chain amino acids and brain function. J Nutr. 2005 Jun;135(6 Suppl):1539S-46S. PMID: 15930466

Sharma B, Lawrence DW, Hutchison MG. Branched Chain Amino Acids (BCAAs) and Traumatic Brain Injury: A Systematic Review. J Head Trauma Rehabil. 2017 Jan 5. doi: 10.1097/HTR.0000000000000280. [Epub ahead of print] PMID: 28060208

Elkind JA, Lim MM, Johnson BN, Palmer CP, Putnam BJ, Kirschen MP, Cohen AS. Efficacy, dosage, and duration of action of branched chain amino Acid therapy for traumatic brain injury. Front Neurol. 2015 Mar 30;6:73. doi: 10.3389/fneur.2015.00073. eCollection 2015. PMID: 25870584

Eakin K1, Baratz-Goldstein R2, Pick CG2, Zindel O2, Balaban CD3, Hoffer ME4, Lockwood M1, Miller J1, Hoffer BJ5. Efficacy of N-acetyl cysteine in traumatic brain injury. PLoS One. 2014 Apr 16;9(4):e90617. doi: 10.1371/journal.pone.0090617. eCollection 2014.

Page 33 of 34

Günther M, Davidsson J, Plantman S, Norgren S, Mathiesen T, Risling M. Neuroprotective effects of N-acetylcysteine amide on experimental focal penetrating brain injury in rats. J Clin Neurosci. 2015 Sep;22(9):1477-83. doi: 10.1016/j.jocn. 2015.03.025. Epub 2015 Jun 19. PMID: 26100161

Choi BY, Kim IY, Kim JH, Lee BE, Lee SH, Kho AR, Jung HJ, Sohn M, Song HK, Suh SW. Decreased cysteine uptake by EAAC1 gene deletion exacerbates neuronal oxidative stress and neuronal death after traumatic brain injury. Amino Acids. 2016 Jul; 48(7):1619-29. doi: 10.1007/s00726-016-2221-4. Epub 2016 Apr 4. PMID: 27040821

Wang Q, Fan W, Cai Y, Wu Q, Mo L, Huang Z, Huang H. Protective effects of taurine in traumatic brain injury via mitochondria and cerebral blood flow. Amino Acids. 2016 Sep; 48(9):2169-77. doi: 10.1007/s00726-016-2244-x. Epub 2016 May 7. PMID: 27156064

Huxtable RJ (1992) Physiological actions of taurine. Physiol Rev 72:101–163

Schaffer S, Takahashi K, Azuma J (2000) Role of osmoregulation in the actions of taurine. Amino Acids 19:527–546

Georgia B, Schuller L, Eunkyue P (2003) Taurine: new implications for an old amino acid. FEMS Microbiol Lett 226:195–202

El Idrissi A (2008) Taurine increases mitochondrial buffering of cal- cium: role in neuroprotection. Amino Acids 34:321–328

Curtis L, Epstein P (2014) Nutritional treatment for acute and chronic traumatic brain injury patients. J Neurosurg Sci 58:151–160

Sun M, Zhao Y, Gu Y, Xu C (2012) Anti-inflammatory mechanism of Taurine against ischemic stroke is related to down-regulation of PARP and NF-κB. Amino Acids 42:1735–1747

Rondanelli M, Aquilani R, Verri M, Boschi F, Pasini E, Perna S, Faliva A, Condino AM. Plasma kinetics of essential amino acids following their ingestion as free formula or as dietary protein components. Aging Clin Exp Res. 2017 Aug;29(4):801-805. doi: 10.1007/ s40520-016-0605-7. Epub 2016 Jul 12. PMID: 27406393

Thank you to all of the researchers cited in this article for their tedious work and dedication.

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