Metabolic Responses and Profiling of Bioorganic Phosphates and Phosphate Metabolites in Traumatic Brain Injury

This chapter constitutes a review of the recent literature on metabolic response and profiling of bioorganic phosphates and phosphate metabolites in disease related to traumatic brain injury (TBI). In this report we emphasize the emerging role of advanced imaging techniques in both the translational research of TBI biology and in the development of new modalities for the diagnosis and therapy of TBI-related diseases. To date, several neuroimaging techniques have been used for assessing phosphate metabolites related to TBI. These techniques include 31P-MRI/MRS imaging, magnetic resonance imaging, and incorporation of phosphate derivative hydrogels, all of which are of particular interest in identifying TBI. These advanced neuroimaging techniques are currently under investigation in an attempt to optimize properties for therapeutics purposes. In addition, this chapter also discusses the role of endogenous and exogenous phosphates related to TBI. TBI imaging is a rapidly evolving field, and a number of the recommendations presented will be updated in the future to reflect the advances in medical knowledge.


Introduction
As progress of medical science/technology and imaging accelerates into the future, this work is intended as an important review regarding the related chemistry and biochemistry of traumatic brain injury (TBI). While some pertinent reviews have also appeared [1], we review what is known regarding phosphate chemistry. This is of critical importance for future researchers, and relates to brain-related injury, especially TBI. We sought to cover phosphates and phosphorylation in this context. From a database search, a list of keywords (phosphate, phosphonate, phosphorylation, traumatic brain injury, and probe, imaging, or sensor) and approximately 35 references have been acquired (ISI Web of Science, accessed in 2017). . Instrumental techniques are also critically important and certain physical techniques are introduced and described as well (Figures 1 and 2). Reviews of biological phosphate imaging have emerged in the literature [36][37][38][39] and a combination of clinical and research aspects are presented. In addition, critically important phosphate species, probes, proteins, and related medicinal molecules are illustrated (Figures 3-6).

TBI introduction
According to the US Centers for Disease Control and Prevention (CDC), every year in the United States approximately 1.7 million individuals receive an injury classified as a TBI), and 52,000 of these cases led to death [40]. TBI can be defined as alterations in brain functions and brain metabolism due to head collision with a stationary or moving object, or striking of a physical subject or coupling of an external mechanical force (e.g., g-force, blast shockwave) with the head [41][42][43][44]. Research has revealed that TBI can be associated with a variety of outcomes, from mild shock upon a single impact, to developing chronic traumatic encephalopathy (CTE) at a later time, a neurodegenerative disorder linked to repetitive brain injuries [45]. Each damaging event may lead to a specific clinical condition, which requires specific observation and care to prevent long-term neurological damage.  Related head injuries can involve different motions of the event that ultimately impose a stretching force on neurons, commonly resulting in the dangerous formation of edema in the tissue, which increases tissue volume. Brain edema is influenced by complex molecular and cellular changes in blood-brain barrier (BBB) function, as well as cell volume regulation. These changes may also develop into pathological pathways. Edema resulting from the original sustained injury has a devastating impact on morbidity and mortality. These downstream effects of TBI increase intracranial pressure, impair cerebral perfusion and oxygenation, and contribute to additional ischemic injuries [46]. Therefore, these changes may also develop into pathological pathways. Other issues related to cerebral hypoperfusion range from loss of consciousness to devastating neuronal damage. The reason for these symptoms is the lack of high-energy phosphate compounds and high-energy metabolic demand caused by disruption of the continuous oxygen supply in the blood to the brain.
In general, there are three major types of traumatic brain edema. The first is vasogenic due to disruption of the BBB, which results in extracellular water accumulation. The second is cytotoxic/cellular due to sustained intracellular water collection. The third is called osmotic brain edema, which happens because of osmotic imbalances between blood and tissue. Rarely after TBI do we encounter a "hydrocephalic edema/interstitial" brain edema related to an obstruction of cerebrospinal fluid outflow [47]. Various detailed case studies have emerged that continue to raise the alarm and grab the attention of researchers to understand the effects of TBI. For many repeated types of injuries to the head, in certain individuals CTE has similarities to age-related neurodegenerative diseases [1]. Model systems such as rats [4,6,12,13,15,17,28,29,32,33] and mice [5,9,11,12,18,20,25] have been employed to better understand the mechanisms of TBI.
Phosphorus is a very important element in the body and is responsible for approximately 1.1% of total body mass. In the body, almost all of the phosphorus is combined with oxygen, forming phosphate. Phosphate acts as a body's electrolytes, carrying an electric charge in body fluids such as blood. The majority of phosphate in the body (85%) comes from bone [48]. The rest is stored as high-energy phosphate or in its free form, where it acts as a substrate for adenosine triphosphate (ATP) production. Even though phosphate metabolism in trauma has not been well studied, there are some interesting reports on phosphate in TBI that involve hypophosphatemia. In 2010 Lindsey et al studied 25 patients with TBI and found out that these individuals had a lower serum phosphorus concentration than those without TBI, suggesting ongoing phosphate loss in the TBI patients [49].
To date, conventional computed tomography (CT) is the main technique for the evaluation of TBI for patients' diagnoses. However, CT and magnetic resonance imaging (MRI) still cannot be used to predict neurocognitive functional deficits at any stage of TBI, because they do not image the functional pathology for the neurocognitive outcome [50]. Therefore, other techniques such as 31 P-magnetic resonance imaging/spectroscopy ( 31 P-MRI/MRS) and positron emission tomography (PET) are used as alternatives to provide insight into the metabolic changes that arise from TBI and to reveal the damage that contributes to short-and longterm impairment. In this chapter, a review of several relevant contributions of neuroimaging towards an improved understanding of TBI is presented, using both PET and 31 P-MRI/MRS.

MRI techniques for TBI that involve phosphates
In terms of MRI for TBI, various techniques have been employed (Figures 1 and 2). For example, T 2 -weighted MRI has been used [5]. Interestingly, in 1990 Heiss et al. used PET of [ 18 F]fluorodeoxyglucose (FDG)coupled with 31 P-MRS to diagnose tumors in the brain. The study suggested that both methods can examine different aspects of tumors in the brain and can be used as a tool for further classification of brain tumors or diseases related to the brain such as TBI [51]. A further study in 2002 by Greenman et al. used a method called threedimensional rapid acquisition with relaxation enhancement (RARE) pulse sequence for direct measurement of phosphocreatine (PCr) images of the human myocardium. The aim of this study was to assess the metabolic state of myocardial tissue in several disease states and determine the efficacy of therapeutic mediation [52]. Then, in 2005, Greenman et al. published a work related to 31 P-MRS to evaluate the metatarsal head region of the foot in neuropathic diabetic patients. The study concluded that a very uniform net magnetization can be achieved and the use of double-tuned birdcage radiofrequency coils can improve the quality of MRI/MRS examinations [50]. A study in 2018 conducted by Chen et al. using in vivo 31 P-MRS magnetization transfer (MT) suggested that MRS provides a direct measure of neuronal activity at the metabolic level by investigating the change in cerebral ATP metabolic rates in healthy adults upon repeated stimulation [45]. 31 P-MRS has also proven effective in detecting a selective saturation sequence for ATP and phospholipids. Thus, the 31 P-MRS-MT technique at 3 T is a good candidate for neurological and neuropsychiatric disorders because of the noninvasive nature of NMR studies. Additionally, 31 P-MRS was reported and discussed in 2004 by Cernak et al. [6] The technique involving metals such as manganese is also applicable: manganese-enhanced MRI was used in a 2011 study by Tang et al. [29] Additionally, ex vivo diffusion tensor imaging was implemented in a study in 2012 by Jin and coworkers [5].
1 H, 31 P, and 13 C in vivo MRS are complementary techniques that allow noninvasive measurement of different aspects of brain metabolism that may contribute to the clinical management of patients with acute TBI [53]. 13 C-MRS measures the breakdown of intake of 13 C-labeled sugar (e.g., glucose) via glycolysis and the tricarboxylic acid cycle. Even though not many 13 C-MRS studies have been conducted, the development of in vivo hyperpolarized techniques shows a potential to detect TBI. On the other hand, 31 P-MRS allows measurement of high-energy phosphates (ATP and PCr) produced by oxidative phosphorylation and creatine kinase in mitochondria [54]. Changes in these metabolites have been noted in several patients and animal studies (further study might reveal the role of the high-energy phosphates). 1 H-MRS is the most commonly used MRS technique for studying brain metabolism following TBI. It has the potential to measure various metabolites: some are associated such as lactate, Glu and Gln, which can also be measured by 13 C-MRS. Creatine and N-acetylaspartic acid are associated with the ATP and PCr, which can also be measured with 31 P-MRS. Thus, the ratios of high-energy phosphates are thought to represent a balance in the brain. In addition, the chemical shift difference between inorganic phosphate and PCr enables calculation of intracellular pH. 13 C-MRS detects the 13 C isotope of carbon in brain metabolites [55].

Molecules of importance
There are various small molecules used either as diagnostic agents or potential therapies in the context of phosphate TBI studies. We can also consider small molecule probes and those coupled with the use of pharmaceuticals (Figures 3-6).

PET imaging
PET is an important clinically used instrumental technique that requires an administration of artificial diagnostic agents (Figure 1). The artificial agents used involve one disintegrating atom such as the 18 F or 11 C isotope. The isotope is generated and then covalently attached (by a simple chemical reaction and protocol) to a small molecule prior to nuclear medical examination [13,21].
PET imaging is well known for its sensitivity for small molecular changes (nanogram scale) compared to milligram or microgram for MRI or CT. PET also is able to provide important information on brain metabolism. As a result, PET imaging is used to measure a change in the glucose metabolism after TBI. The magnitude and duration have been correlated with worse behavioral and cognitive outcomes [56]. These results regarding cerebral glucose utilization were obtained using deoxyglucose (DG) labeled with 14 C and autoradiography [57]. DG was chosen because DG is phosphorylated but not further metabolized, becoming trapped in the cell with a slow clearance rate. For noninvasive imaging, a positron-emitting isotope such as 18 F can be incorporated within DG, resulting in the production of [ 18 F]FDG; this then accumulates in brain tissue in proportion to glucose uptake and the level of phosphorylation and is quantifiable using the technique of PET imaging [58].
For more information on nuclear chemistry and the mechanism of positron/electron capture as well as the preparative chemistry, please see other sources.

Phosphate species
There is a range of phosphate species used in biology. In some ways, the phosphates are central to the discussion, but in other ways they are peripheral to the thrusts of literature reports.
The phosphates under discussion are shown in Figure 3 and listed below.

Phosphorylation
Phosphorylation of proteins (serine, threonine, and tyrosine), for example, is an essential theme in biology. It is a constantly monitored and investigated process in biological systems, and continues as a vital aspect in the study of neurodegenerative disease research because it relates to kinase and phosphatase activity (Figure 3). For example, tau protein has been central in Alzheimer's disease (AD) hypotheses for many years. Hyperphosphorylation is considered to be an important step in disease pathology [59].
Among many kinases proteins, mitogen-activated protein kinases (MAPKs), protein kinase B (also known as Akt), and glycogen synthase kinase (GSK) are the major kinases involved in cellular signaling, and as confirmed by the study from Joseph T. Neary, MAPKs, Akt, and GSK respond to trauma of the central nervous system (CNS). Therefore, it is very important to conduct further studies of these proteins to provide a better understanding of their role in the pathogenesis of many disorders, including traumatic injuries of the brain [58]. A study by Naoki Otani et al. showed that the extracellular signal-regulated kinase (ERK) pathway is triggered in lesions in regions of selective vulnerability after TBI and has a devastating effect on the hippocampus. The results show that pretreatment with U0126 (an ERK inhibitor) decreases neuronal cell loss after TBI [60]. Meanwhile, a study conducted by Noshita et al. also suggested that phosphorylation of Akt at serine-473 and DNA fragmentation after TBI in mice showed that phospho-Akt was decreased in the injured cortex 1 h after TBI and temporarily increased at 4 h in the perifocal damaged cortex. They concluded that the degree of Akt phosphorylation is dependent on the intensity of cellular damage after TBI [61].
Another study revealed that MAPKs are involved in pathophysiological TBI. Thus, regulating the MAPK pathway-mediated cerebral damage after acute injury could be a direction for the development of the novel therapeutic target in TBI [62][63][64]. Study of a simple chemical compound, sodium selenite, was performed. Sodium selenite was found to upregulate proteins that help to remove the phosphorylation group from its position on the amino acids in particular proteins. The specific enzyme is called PP2A/PR55 (protein phosphatase 2A regulatory subunit PR55). In a study from 2014 by Zhu et al., phosphorylation of various molecules was considered as a result of cerebral contusion (mouse model). The following molecules were studied: Akt, mTOR (mammalian target of rapamycin), and S6RP [35]. For example, the Thr308 and Ser473 sites of Akt are important phosphorylation sites for activating Akt. Thr308 becomes phosphorylated by PKD1 and other enzymes, including PDK2 phosphorylate Ser473. Activated Akt mediates several responses, including phosphorylating a range of intracellular proteins. mToR and S6RP are downstream targets of the PI3K/Akt pathway. Phosphorylation of a precursor stimulates activation of mTOR and S6RP [65][66][67]. Some phospholipids are ubiquitous and have been the subject of imaging regarding cell membrane dynamics.
A study from Alice E. Pasvogel et al. showed that following TBI, membrane integrity of neurons and neuroglia is compromised resulting in elevated phospholipid levels in the cerebrospinal fluid. The pattern of change and the concentration of each of the phospholipids were different for those who died and those who survived following TBI. In conclusion, the study found the increase concentration of lysophosphatidylcholine in those who died. These findings give a preliminary proof of greater disruption of central nervous system membrane phospholipids in patients who died after TBI [69,70].

Extracellular phosphates
In addition to the endogenous phosphate species that are produced in the biological system, there are also exogenous or xenobiological compounds that can be discussed. Chitosan combined with β-glycerophosphate disodium (β-GP) for use as a thermosensitive hydrogel was first reported by Chenite in 2000. This gel-forming biopolymer can be used for the development of therapeutic implants. Further study by Dong et al. from 2015 involved a hydrogel that consisted of derivatives of phosphate groups [10]. The result suggests that an injectable thermosensitive chitosan/gelatin/β-glycerol phosphate (C/G/GP) hydrogel could release the phenolic antioxidant ferulic acid (FA), which can inhibit the neurological oxidative stress and effectively protect the brain from further impairments. Another study from Ibrahim Jalloh et al. in 2015 also suggested that there was a shift in glucose metabolism from glycolysis to pentose phosphate pathways (PPPs) with decreasing brain tissue oxygen concentrations after TBI. This finding gives another perspective on the roles of PPPs and glycolysis after TBI, and whether they can be manipulated to enhance the potentially antioxidant role of PPPs and give better outcome to TBI patients [71].
In 2014, Brend L. Fiebich et al. suggested that prostaglandin E2 (PGE2), produced by the enzymatic activity of cyclooxygenases (COX) 1 and 2, is the common mediator for the inflammatory brain that leads to TBI. The group proposed a two-hit model for neuronal injury. First, an initial localized inflammation mediated by PGE2 was then followed by the release of ATP by injured cells (second hit). In this study, it was concluded that by inhibiting the P2 receptor in the second hit using P2 receptor-based antiinflammatory drugs (PBAIDs) the activity of specific ectonucleotidases and release of excessive ATP could be increased and is another approach to counter neuroinflammation [72,73].

MRI contrast agents
In TBI phosphate literature, MRI contrast agents have been previously described [31]. Structural information about the brain can be quantified using brain volume based on T 1 -weighted MRI. Even though the most common contrast agents are based on gadolinium, new pharmaceuticals (for example, gadobenate benate dimeglumine (Gd-BOPTA)) have been developed with higher T 1 and T 2 relaxivity to improve signal intensity enhancement and thereby improve lesion visualization [74]. Garcia-Martin et al. used a phosphonated Gd 3+ -based contrast agent to measure intravascular acidification in rat gliomas. To distinguish the differences in pH, [75,76] the contrast agent used undergoes changes in T 1 relaxivity over a broad range from pH 6 to 8 [77]. This application is an example of an alternative for TBI symptom detection.
Another study using manganese-enhanced MRI (MEMRI), in which the manganese ion acts as an MRI contrast agent, was used to study rats subjected to a controlled cortical impact. The results suggest that MEMRI detected early indications of excitotoxic injury and BBB disruption that preceded vasogenic edema in the hyperacute phase and offer a novel contrast that complements conventional MRI in the study of TBI [78,79]. In 2009 Chapon et al. revealed that MRI contrast agent can detect the inflammatory progression by radiolabeled peptide (IELLQAR) to target E-selectin, an important intercellular adhesion molecule involved in the leukocyte cycle [78].

Therapeutics tested
Small molecules are at the heart of medically treating people who have received TBI. (R,S)-2-Chloro-5-hydroxyphenylglycine (CHPG [5]) was studied. This compound has been studied by David J loane et al. in 2013 [80] and the result in mice model demonstrate that activation of mGluR5 using the selective agonist and CHPG, within 30 minutes after the moderatelevel TBI significantly improved sensorimotor and cognitive function recovery and reduced TBI-induced lesion volumes in the mice model. Next, edaverone (Figure 5) was used [11]; it was found to be effective in the mouse model under study. The theme of concussioninduced depression is elucidated in this paper [11].

Proteins and enzymes
There are also related proteins in these studies. Perhaps the most central protein in a discussion of neurodegeneration is the beta amyloid protein (Aβ)-one of the hallmarks of AD. It can be used as a baseline measurement. Studies by the Smith [81] and Sharp groups [82] showed that Aβ plaques may be found within TBI patients. The study also suggests that rapid Aβ plaque formation may result from the accumulation of amyloid precursor protein in damaged axons and a disturbed balance between Aβ genesis and catabolism during the process of TBI. In this study, the authors took an image of Aβ plaque burden in long-term survivors of TBI and made determinations to generate a correlation between traumatic axonal injury and Aβ concentration. By comparing the distribution of Aβ to AD, they found that Aβ-comprised plaque in the TBI survivors decreased in neocortical regions but increased in another brain region, the cerebellum. This then suggested that TBI may dispose one to an AD-like fate [25].
There are also phosphate-related reports involving studying the reduction of certain proteins after the onset of TBI. Such proteins include CREB and PSD95 [26]. Then there is carbamylated erythropoietin (EPO) [4]; EPO is a pleiotropic cytokine that identified its role in erythropoiesis (the process by which red blood cells are produced) [83]. EPO was recognized for its hematopoietic properties; however, many researchers around the globe were attracted by its function as a tissue protector. In 2004, a study from Leist et al. showed that the carbamylation of EPO formed a kind of nonhematopoietic derivative, cEpo. This reaction surprisingly eliminated its erythropoietic effects; however, it keeps its function in tissue protection [84]. These results led to another study conducted by Fiordaliso et al. from 2004, which suggested that the erythropoietic and tissue-protective effects of EPO were based on different receptors [85]. These discoveries have brought many researchers to design and synthesize EPO derivatives with tissue-protective effects only. To date, there are two major, developed, modified EPO molecules that have tissue-protective effects: cEpo and asialoerythroprotein (asialoEpo). Interestingly, the first modification of EPO through carbamylation was reported by Leist et al.; however, the method of producing cEpo was described in a patent by Warren Pharmaceuticals [86]. This newly reported research may shed new light on the development and application of cEpo, a prospective drug candidate for neuroprotection. There are studies that involve delayed mGluR5 activation and targeting of intermediate proteins [3]. One study found that activation of metabotropic glutamate receptor 5 (mGluR5) by CHPG decreases microglial activation and release of associated proinflammatory factors in vitro, which is mediated, in part, through inhibition of reduced NADPH oxidase. These results suggested that treatment with CHPG may significantly limit lesion progression in TBI through mGluR5 receptors [87].

Conclusions and future outlook
There are various ways that the wide variety of phosphates that exist in biology are involved in health and disease; ions such as phosphates can be exploited in many prospective ways in the future and in particular they could be imaged in new ways. This review concerned phosphates and TBI reports in which the discussion or study involved molecular imaging. The reports were clinical and involved laboratory studies. Animal models were often used. A great deal of biochemistry was described; often, enzyme activities were monitored and these trends were published.
This fresh review was intended to help medicinal chemists make new connections. The major goals are intended to help achieve future innovation of potential treatment of TBI with chemical or biological agents. Administration within the "golden hour" for the best efficacy is an essential point to make. In terms of imaging there are new MRI techniques and experiments that are available as well. Some of the most important instrument manufacturers such as GE Healthcare (Milwaukee, WI), Bruker, Hitachi Medical Corporation, Phillips, and Toshiba Medical Corporation provide the current hardware for the task at hand [88][89][90][91][92]. However, biochemistry can allow for additional innovative imaging to be undertaken. Below are a few detailed aspects for future study with regard to TBI and phosphate research.

Future
More commonly, research in the future will prominently feature the effects on phosphate metabolism. With phosphate metabolism still in its infancy [93], a fuller treatment would involve a great deal of related research. Therefore, we have described some related papers that involve important points about phosphates.
• Much research effort involves the status of enzyme activity. The importance of accurately carrying out immunohistochemistry involving phosphorylated proteins can be underscored [16,47]. How well Western blots and other related assays are prepared and conducted by laboratory personnel and how they can be best carried out and executed are extremely important for the field.
• The theme of subcellular redistribution of phosphates can be made more pronounced [16]. Novel chemical probes that can "chase" the constituents between different cellular compartments can be designed and studied.
• The importance of the maintenance of vasculature and smooth muscle cells that help constitute the microvessels within neurological tissue can be further studied. How these structures are effected by TBI in, e.g., mouse models can be further determined [16].
• Overabundant Reactive Oxygen Species (ROS) concentration driven by Fenton reaction has major role in the transformation of many highly radical species such as ROS/RNS. These highly reactive species, can lead to many disturbances such as TBI. See references herein and elsewhere for an introduction to ROS and their analysis. MRI is a very common theme in research [1,15,28,29,31], as well as the closely related instrumental technique of NMR spectroscopy.
• How phosphates are interrelated (via brain injury) with the range of ROS is an important quest in basic science.
• More research about phosphates in gliosis needs to be researched. How can gliosis best be imaged and can it relate to the homeostasis of phosphates?
• What is the range of factors that delays mGluR5 activation and how do phosphates or phosphonates become involved?
• How can researchers parse between secondary and primary pathology at the chemical level regarding both experimental and clinical research of TBI phosphate activity?
• What divergent effects might arise from prior organophosphate/organophosphonate pesticide exposure (a history of exposure) in which phosphonates are located where phosphorylation usually takes place? How does this effect hinder or perhaps help in etiology? How can medicine take advantage of this artificial preloading?