Our 1H NMR-based metabolomic approach demonstrated serum metabolic differences between patients in coma and individuals with diagnosis of brain death. The results obtained showed favorable separation and model parameters in cross-validated PLS-DA and OPLS-DA models with high predictability in both relative integral and whole spectra data for the studied sample. The discrimination potential between groups was also confirmed for specific metabolites with high ROC AUCs. The findings not only establish the potential of metabolomics in neurocritical care but may also provide an understanding of the pathogenic mechanisms underlying brain death. This is the first scientific publication in the literature relating to metabolomic studies of brain death.
In recent years, a few research projects aimed at metabolomic assessment of clinical conditions associated with central nervous system pathologies have been conducted; the goal in most cases was to identify diagnostic biomarkers for stroke or traumatic brain injury. The use of multivariate statistical analysis has made it possible to demonstrate significant separation between patients with cerebral pathology and healthy individuals in most of the studies , , –, –. For example, in the case of stroke the group of potential biomarkers includes, among others: lactate , , , , pyruvate , , , glycolate , formate , glutamine , methanol , acetate , , cysteine , folic acid , S-adenosyl homocysteine , oxidized glutathione , tyrosine, , , tryptophan , serine–, isoleucine , , valine , , , , glycine , leucine , , betaine , , carnitine ,  and ketone bodies (acetone, acetoacetate and β-hydroxybutyrate)  as blood biomarkers; citrate , hippurate , and glycine  as urine biomarkers; and finally acetic acid , 3-hydroxyisovaleric acid , 3-hydroxybutyric acid , choline , glycine , pyruvic acid , l-lactic acid , acetone  and branched chain amino acids (valine, leucine, isoleucine)  as cerebrospinal fluid biomarkers. Notably, in selected research projects, the adopted targeted metabolomic analysis proved to be helpful in differentiating cerebral infarction (CI) patients and those with intracerebral hemorrhage (ICH) , . Of course, the discriminatory power of the above-listed metabolites varied; however, statistical significance was maintained in each case. Similarly, favorable results have been obtained when using metabolomics in the analysis of states of traumatic brain injury (TBI), although far fewer research projects have been conducted here. The performed studies have shown that proline , phosphoric acid , β-hydroxybutyric acid , galactose , creatinine , valine , linoleic acid , arachidonic acid , medium-chain fatty acids (decanoic and octanoic acids)  and sugar derivatives including 2,3-bisphosphoglyceric acid  in the blood; as well as propylene glycol , lactate , , glutamine , creatine  and glutamate  in the cerebrospinal fluid should be considered potential markers of acute TBI and could even serve as death predictors . As stated before, there are no reports in the literature on metabolomics in brain-dead patients; however, a few research projects using magnetic resonance spectroscopy and magnetic resonance imaging have been performed to study in vivo metabolic changes in brain tissue. The conclusions from these studies have been that high levels of lactate, choline and lipids and decreased levels of n-acetyl aspartate are prognostic factors of brain death –. It has also been demonstrated that magnetic resonance spectra in these individuals are dominated by intense inorganic phosphate signals and are characterized by a complete absence of adenosine triphosphate (ATP) and phosphocreatine at the same time , .
Our results demonstrate that there are metabolites that can be considered potential biomarkers of brain death. The metabolomic serum analysis comparing brain-dead individuals to patients in coma revealed statistically significant increases in the concentrations of methanol, acetone, acetate and 3-methyl-2-oxovalerate and simultaneous statistically significant decreases in the concentrations of isoleucine, betaine, methylhistidine, glycine and valine. There were also significant changes in the concentrations of other metabolites that played significant roles in discrimination, although we were unable to identify them unambiguously (Table 3). All resonance signals underwent the primary identification procedure using statistical total correlation spectroscopy (STOCSY) and a two-dimensional NMR spectroscopic approach (analysis accuracy level 2 based on Sumer et al. . The unidentified resonance signals were determined by their multiplicity patterns and chemical shifts. To precisely identify them, advanced methods of serum purification should be carried out, and then mass spectrometry (MS) analysis should be performed. We plan to accomplish these steps in the next stage of research.
It is not possible to compare the results of our research with the analyses of other authors, as no studies on metabolomics in brain death have been conducted so far. Moreover, we did not make comparisons with healthy people but with comatose patients, which prevents the results of our research from being compared to analyses known from the literature of other brain pathologies, where healthy individuals were always the reference points. As a result, a lower concentration of an individual metabolite in our comparisons does not exclude its being higher than in healthy persons and vice versa. In the case of brain death, it should also be noted that this condition affects not only brain tissue but also the functioning of the entire organism in a more extensive way than does any other brain pathology.
Among the identified metabolites that allowed us to discriminate individuals in the state of brain death from comatose patients, methanol featured the greatest statistical significance. Methanol is a naturally occurring compound in normal, healthy individuals and is formed through anaerobic fermentation by intestinal bacteria – and metabolic processes involving S-adenosylmethionine, highly localized in the pituitary gland , . It is widely recognized as a putative causative agent of neurodegenerative diseases , –; however, it has been found to be decreased in stroke patients . The increase in methanol levels in the brain death state may be related to intestinal paralysis commonly occurring in this condition, followed by increased bacterial translocation, as well as due to insufficiency of metabolic clearance mechanisms that physiologically maintain low levels of methanol in the organism . Ketone bodies such as acetone, acetoacetate and beta-hydroxybutyrate are the source of energy for the brain when glucose is less available ,  and could provide up to 60% of the human brain’s metabolic needs . The effect of this is the increase in acetone concentration observed not only in the plasma of patients with ischemic stroke  but also in their cerebrospinal fluid . The administration of ketones has even been shown to provide neuroprotection after brain hypoxia/ischemia or traumatic brain injury , . Elevated acetone in individuals with brain death compared to patients in coma is a result of the obvious extreme energy deficiencies of the brain tissue in such cases. The same also applies to acetate, which in our research turned out to be another metabolite at increased concentrations. Under normal resting conditions, acetate may contribute up to 10–15% of the basal energy demands of brain astrocytes –, so its upregulation may be important for meeting bioenergetic demands in a critical state. On the other hand, a significant reduction in acetate uptake and utilization by astrocytes in cerebral ischemia , , as well as its formation by N-acetylaspartate hydrolysis during brain infarction , have been described, possibly contributing to acetate elevation in the state of brain death. Our observations indicating the discriminatory potential of acetone as a biomarker of brain death are indirectly confirmed by the results of autopsy studies on animals, which have shown its significant postmortem increase in brain tissue , ; however, it is also elevated in the ischemic core and penumbra after cerebral infarction . The last metabolite identified to be significantly elevated in brain-dead individuals compared to comatose patients in our study was 3-methyl-2-oxovalerate. This abnormal metabolite arising mainly from the incomplete breakdown of isoleucine is a well-known neurotoxin that plays a critical role in neurological damage , .
Isoleucine and valine, the concentrations of which decrease significantly in the state of brain death compared to coma, are branched chain amino acids (BCAAs) that participate in the synthesis of brain cell signaling molecules such as serotonin, dopamine and norepinephrine and influence brain function by the production of energy via the citric acid cycle (CAC) , . Reduction in levels of isoleucine , ,  and valine , ,  is commonly observed in ischemic stroke; moreover, it is described as associated with infarction severity and poor neurological outcome . It has also been observed in several critical illnesses such as traumatic brain injury, sepsis, burn injury and heart diseases , , , , . It is presumed that BCAA depletion could arise from their utilization as amine neurotransmitters or CAC intermediates for brain function recovery processes , and it is also a possible cause of their even more profound reduction in the state of brain death. A similar situation is related to the observation of changes in glycine. This simplest amino acid is one of the major inhibitory neurotransmitters in the central nervous system , and its release might be inhibited as a consequence of neural excitation during brain ischemia and injury . High levels of glycine protect neuronal cells during brain ischemia , , whereas a decrease in glycine has a detrimental effect on ischemic neuronal damage , . The downregulation of glycine in serum is commonly presented in the literature of stroke , , ; however, there is also one report describing the opposite observation . Betaine is another metabolite whose significant decrease in concentration differentiates the state of brain death from coma in our observations and, as in the previous case, the literature describes its changes in other brain pathologies in different ways (an increase  as well as a decrease  in serum of patients in stroke). Betaine is hypothesized to influence pathways of inhibitory neurotransmitter production and recycling , but most of all, it is a protective substance that helps to reduce ,  high levels of homocysteine - a well-documented promoter of ischemic injury to endothelial cells in brain vessels, an oxidative stress inductor, and a compound that influences the pro-thrombotic system . Betaine-dependent protective mechanisms seem to be devastated in cerebral destruction processes . The explanation of the drop in the concentration of the last compound with discriminatory potential that we identified, methylhistidine, is not as obvious as in the cases of the previously described metabolites. Approximately 75% − 90% of this histidine derivative is formed in skeletal muscles , –; therefore, its measurement (especially in urine and less frequently in plasma) is a commonly used biomarker for skeletal muscle protein breakdown , , , . Considering the above, it is not surprising that the urine excretion of methylhistidine in sepsis , thermal injuries  and traumatic brain injury  has been found to be increased, indicating the upregulation of skeletal muscle catabolism in critical states of the organism. On the other hand, it has also been shown that an elevation of the urinary concentration of methylhistidine can occur, with an accompanying paradoxical decrease in plasma, as demonstrated in patients with Alzheimer's disease . We believe that the reduction in methylhistidine concentration in our study could indicate the relative downregulation of skeletal muscle catabolism in individuals with brain death in comparison to coma patients, remembering that in both of these states, increased catabolic processes are of course unquestionably present.
Medicine does not have any laboratory tests that are able to confirm brain death, so this condition is diagnosed only on the basis of clinical examination, optionally complemented by instrumental methods, which are not always easily accessible. From this point of view, the results of our study demonstrating the potential of 1H NMR-based metabolic serum fingerprinting with multivariate metabolomic data analysis are particularly valuable. Further studies in this field should not only be regarded as constituting a great scientific challenge but also as a necessity for modern medicine, especially intensive care and transplantation medicine.