Edited by: Akiva Cohen, Children’s Hospital of Philadelphia and Perelman School of Medicine - University of Pennsylvania, USA
Reviewed by: Dong Sun, Virginia Commonwealth University, USA; Itzhak Nissim, Children’s Hospital of Philadelphia and Perelman School of Medicine - University of Pennsylvania, USA
*Correspondence: Pramod K. Dash
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Methionine is an essential proteinogenic amino acid that is obtained from the diet. In addition to its requirement for protein biosynthesis, methionine is metabolized to generate metabolites that play key roles in a number of cellular functions. Metabolism of methionine via the transmethylation pathway generates S-adenosylmethionine (SAM) that serves as the principal methyl (−CH3) donor for DNA and histone methyltransferases (MTs) to regulate epigenetic changes in gene expression. SAM is also required for methylation of other cellular proteins that serve various functions and phosphatidylcholine synthesis that participate in cellular signaling. Under conditions of oxidative stress, homocysteine (which is derived from SAM) enters the transsulfuration pathway to generate glutathione, an important cytoprotective molecule against oxidative damage. As both experimental and clinical studies have shown that traumatic brain injury (TBI) alters DNA and histone methylation and causes oxidative stress, we examined if TBI alters the plasma levels of methionine and its metabolites in human patients. Blood samples were collected from healthy volunteers (HV;
It has been appreciated for more than 30 years that the resting metabolic expenditure of the severely injured brain is almost 40% higher than that of the non-injured brain, and is associated with a negative nitrogen balance (the difference between nitrogen uptake and nitrogen excretion), suggesting increased protein catabolism (Clifton et al.,
Methionine is an essential amino acid for protein synthesis and is often incorporated as the first amino acid. Metabolism of methionine occurs by two primary pathways: the transmethylation and the transsulfuration (Figures
In the present study, we measured the levels of methionine and several of its metabolites in plasma samples collected within the first 24 h of their injury from patients who experienced either a severe (GCS ≤ 8) or mild (GCS > 12) TBI (sTBI or mTBI). Plasma samples from healthy volunteers (HV) were used as controls. An acute time point for sample collection was chosen as experimental studies have shown that robust oxidative damage and cell death occurs during this period. Our results indicate that both mild and severe TBI cause significant reductions in plasma methionine levels. A decrease in the transmethylation product SAM was observed in sTBI patients, as were the plasma levels of choline, betaine, and dimethylglycine. The levels of the transsulfuration metabolite cysteine, as well as the gamma-glutamyl cycle metabolites (gamma-glutamyl amino acids and 5-oxoproline), were also found to be reduced after sTBI. Taken together, these results demonstrate that TBI decreases methionine and its key metabolites, which may alter the function of multiple organs, and suggest that supplementation of methionine metabolites may be beneficial for sTBI patients.
The University of Texas Health Science Center at Houston Committee for the Protection of Human Subjects approved the human subject protocol in accordance with the Declaration of Helsinki. In total, 60 subjects were recruited and provided written informed consented for participation in this study. All subjects were between 14–57 years of age and provided consent or proxy consent for their participation in this study. None of the subjects had drug dependency, or had active infections. Twenty sTBI (GCS ≤ 8), 20 mTBI (GCS ≥ 12), and 20 HV were recruited for this study. mTBI had no abnormalities on head computed tomography (CT) scans, but experienced one or more of the following: loss of consciousness, post-traumatic amnesia, altered mental status, neurologic deficits, or seizure. Demographic and clinical information on the study subjects is provided in Table
Group | Healthy volunteers | Mild TBI | Severe TBI |
---|---|---|---|
Number of subjects | 20 | 20 | 20 |
GCS (24 h of injury) | NA | 14.85 ± 0.37 | 3.65 ± 1.2 (intubated) |
Injury Severity Score | NA | 5.4 ± 2.4 | 27.5 ± 8.2 |
Age (years) | 25.2 ± 6.7 | 36.1 ± 13.3 | 25.8 ± 9.8 |
Female/Male | 4/16 | 6/14 | 4/16 |
Hispanic ethnicity | 6 | 3 | 4 |
Race | |||
White | 16 | 18 | 16 |
African American | 3 | 2 | 3 |
Asian | 1 | 0 | 1 |
Seventeen of the 20 HV did not eat after midnight, the night before sample collection (at least 12 h before sample collection). The mTBI subjects had a last recorded meal that was 7.5 ± 4.4 h prior to sample collection. sTBI samples were collected 15.07 ± 5.0 h after the time of their injury. Blood samples were obtained within the first 24 h after injury and were coded to protect anonymity. Samples were collected in potassium EDTA tubes (Becton Dickinson, Franklin Lakes, NJ, USA), placed on ice, and processed within an hour of draw. Plasma was isolated by centrifugation at 4°C as described by the vendor. Aliquots were prepared and frozen at −80°C until needed. Plasma was processed by Metabolon, Inc. (Durham, NC, USA) using a proprietary series of extractions designed to increase the sensitivity of small molecule detection. Samples were placed briefly on a TurboVap® (Caliper Technologies Corp., Hopkinton, MA, USA) to remove any organic solvent. Each sample was then frozen and dried under vacuum. The samples were analyzed by liquid chromatography-mass spectrometry (LC-MS) or gas chromatography-mass spectrometry (GC-MS) depending on the analyte being interrogated. Methionine and its metabolites were identified by comparison to purified standards. A selection of quality control compounds was added to every sample. Relative levels of each metabolite were quantified using Metabolon’s proprietary peak integration software. The integrated peak values from all subjects were averaged and used for normalizing the values for each individual subject.
SAM was measured using a competitive ELISA as described by the vendor (BioVendor, Asheville, NC, USA). A standard curve for calculating the abundance of SAM was generated by serial dilution of a purified standard. The range of the standards was based on the vendors’ instructions. Standards and plasma samples (50 μl) were added to a 96-well plate containing immobilized antibodies specific to SAM. Biotinylated SAM was immediately added to the well, after which the plate was incubated at 37°C for 1 h. After extensive washing, a streptavidin-horseradish peroxidase conjugate was added and incubated for 30 min. The plate was washed, and developed using tetramethylbenzidine (TMB). The reaction was terminated by the addition of 2N sulfuric acid. The optical density was measured using a microplate reader at 450 nm. Concentration of SAM in the plasma was calculated using a 4-parameter logistic curve.
Data was initially evaluated using a Shapiro-Wilk normality test, followed by a one-way analysis of variance (ANOVA) across the four subject groups. Any data found to not have a normal distribution was analyzed using a Kruskal-Wallis ANOVA on ranks. Groups with differences were identified using a Dunn’s pairwise comparison as the
Methionine is metabolized by two primary metabolic cycles: the transmethylation pathway to generate SAM and homocysteine (Figure
The transmethylation cycle involves the metabolism of methionine to generate SAM, S-adenosylhomocysteine (SAH), and homocysteine (Figure
A number of methyltransferases use SAM as a methyl donor for DNA, protein and lipid methylation and convert SAM to SAH (Cantoni,
Transsulfuration of homocysteine is essentially an irreversible reaction that generates cystathionine (via cystathionine β-synthase), which is further metabolized by cystathionine gamma-ligase to produce cysteine and alpha-ketobutyrate. Alpha-ketobutyrate is then reduced by α-hydroxybutyrate dehydrogenase to generate 2-hydroxybutyrate (Figure
Glutamate cysteine ligase (GCL) ligates cysteine with glutamate to generate gamma-glutamylcysteine that is then combined with glycine by glutathione synthase (GS) to generate glutathione (Figure
A decrease in cysteine and glycine would be expected to result in a decrease in the levels of glutathione. Unfortunately, plasma glutathione could not be detected by our mass spectrometry analysis. In addition to its role in the reduction of oxidative stress, glutathione is used to facilitate the transport of amino acids into cells via the gamma-glutamyl cycle (Orlowski and Meister,
The essential amino acid methionine not only acts as a building block for protein synthesis, but also serves as the substrate for the synthesis of key molecules such as SAM and glutathione. As such, changes in the levels of methionine metabolites could impact a number of biological processes such as epigenetic regulation of gene expression and cytoprotection. Our measurements of methionine and its metabolites in plasma samples from sTBI, mTBI, and HV revealed four key findings: (1) the relative plasma levels of methionine and SAM are significantly reduced in sTBI patients; (2) the levels of cysteine and glycine, the precursors for the synthesis of glutathione, are also reduced in sTBI patients; (3) in contrast to that observed in sTBI patients, the plasma levels of cysteine were significantly elevated in mTBI; and (4) the relative levels of several gamma-glutamyl amino acids and 5-oxoproline are significantly reduced in the plasma of sTBI patients. These findings are summarized in Figure
As methionine is an essential amino acid, its decrease in both mild and severe TBI patients could have resulted from a reduction in dietary intake. However, the time from last meal was comparable for the HV and sTBI groups, with all groups having not eaten within 7 h of sample collection. Thus, the changes in methionine levels we observed in TBI patients may not be solely due to lack of food intake. Previous clinical studies have shown that sTBI causes a nitrogen imbalance in which nitrogen excretion exceeds nitrogen uptake (Clifton et al.,
Only a few clinical studies have examined the consequences of amino acid supplementation after TBI. Ott et al. (
SAM is a key methyl group donor for methylation of intracellular molecules including DNA, proteins and phospholipids (Grillo and Colombatto,
As the brain has a high metabolic activity and high lipid content, it is particularly vulnerable to oxidative damage. Glutathione is the major antioxidant molecule, and a decrease in its levels can exacerbate brain damage. Although intracellular glutathione levels are relatively high, circulating levels of glutathione are low, due to its extremely short half-life (ranging from seconds to minutes; Wendel and Cikryt,
Amino acids are thought to be transported into the cell via their conversion to gamma-glutamyl amino acids, a process dependent on glutathione (Orlowski and Meister,
In addition to changes in methionine metabolism in sTBI patients, we found a modest, but significant, decrease in plasma methionine levels in mTBI as compared to HV (Figure
A number of weakness need to be considered when interpreting our results. As methionine and its metabolites were measured in the plasma, the source of these molecules cannot be ascertained. Additional weakness of the present study include that the activity and levels of the enzymes for methionine metabolism were not measured. Alterations in their activity, in conjunction with decreased methionine levels, could have given rise to the changes we observed. If supplementation of protein/methionine restores the levels of SAM and other metabolites, this would suggest that the metabolic enzymes of methionine metabolism are not altered. However, this is yet to be examined. Another weakness of our study is that the consequence of SAM reduction on the methylation of cellular proteins, DNA and phospholipids has not been determined. A study by Yi et al. (
In summary, our results show that sTBI patients have low levels of circulating methionine, and that methionine metabolites generated via the transmethylation and transsulfuration cycle are significantly reduced. In addition, flux through the gamma-glutamyl cycle is decreased. As methionine and its metabolites are critical for a number of cellular functions and cytoprotection, decreases in their plasma levels may contribute to brain injury pathology. Alternatively, these biochemical changes may serve a protective function in the acute stage of injury, and their persistence may be detrimental. The systemic changes in methionine and metabolite levels we observed may not only influence the function and pathology of the injured brain, but may alter the function of other organs as well.
PKD, GWH, CBJ, HAC, NK and ANM all contributed to either the conception, design, acquisition, analysis, or interpretation of data for the manuscript. Further, PKD, GWH, CBJ, HAC, NK and ANM all contributed to either drafting the manuscript or revising it for intellectual content. PKD, GWH, CBJ, HAC, NK and ANM have all approved the version of the manuscript to be published and agree to be accountable for all aspects of the work in ensuring that questions of the accuracy or integrity of any part of the work are appropriately investigated and resolved.
This study was supported by Grants from NIH (NS087149), Mission Connect/TIRR Foundation, and the Gilson-Longenbaugh Foundation.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer IN and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.
We would like to thank the nurses at Memorial Hermann Hospital-Texas Medical Center and Elizabeth B. Jones, MD for assistance with recruiting patients. The authors would like to thank Norman H. Ward III, Travis Shields, and Jose Barrera for their help in sample collection.
analysis of variance
branched-chain amino acids
betaine homocysteine methyltransferase
computed tomography
Disability Rating Scale
gas chromatography-mass spectrometry
glutamate cysteine ligase
Glasgow coma scale
gamma-glutamyl transpeptidase
gamma-glutamyl cyclotransferase
Hamilton depression rating scale
healthy volunteers
liquid chromatography-mass spectrometry
L-methionine S-adenosyltransferase
mild TBI
reactive oxygen species
S-adenosylhomocysteine
S-adenosylmethionine
serotonin reuptake inhibitors
traumatic brain injury
tetramethylbenzidine.