Amino Acids in Cerebrospinal Fluid of Patients with Aneurysmal Subarachnoid Haemorrhage: An Observational Study

Background The authors are aware of only one article investigating amino acid concentrations in cerebrospinal fluid (CSF) in patients with ruptured intracranial aneurysms, and this was published 31 years ago. Since then, both management of subarachnoid haemorrhage (SAH) and amino acid assay techniques have seen radical alterations, yet the pathophysiology of SAH remains unclear. Objective To analyse the pattern of concentrations of amino acids and related compounds in patients with different outcomes following aneurysmal SAH. Methods 49 CSF samples were collected from 23 patients on days 0–3, 5, and 10 post-SAH. Concentrations of 33 amino acids and related compounds were assayed by liquid chromatography tandem mass spectrometry in patients with good [Glasgow Outcome Scale (GOS) 1–3] and poor (GOS 4–5) outcome. Results Of the 33 compounds assayed, only hydroxyproline and 3-aminoisobutyric acid appeared not to increase significantly following SAH. In poor outcome patients, we found significantly higher concentrations of aspartic acid (p = 0.038), glutamic acid (p = 0.038), and seven other compounds on days 0–3 post-SAH; glutamic acid (p = 0.041) on day 5 post-SAH, and 2-aminoadipic acid (p = 0.033) on day 10 post-SAH. The most significant correlation with GOS at 3 months was found for aminoadipic acid on day 10 post-SAH (cc = −0.81). Conclusion Aneurysmal rupture leads to a generalised increase of amino acids and related compounds in CSF. The patterns differ between good and poor outcome cases. Increased excitatory amino acids are strongly indicative of poor outcome.

9.1, respectively, and the 28 day fatality rates 39 and 44% (1,2). Cumulative morbidity and mortality following SAH remain high, despite the considerable efforts of neuroclinicians worldwide (3,4). Traditionally, rebleeding and cerebral vasospasm have been regarded as the main causes of poor outcome in these cases (5). Although cerebral vasospasm has been extensively studied, and subjected to numerous drug trials during the past several decades, the outcome appears not to have been improved by its reversal (2,6,7). Several recent studies indicate that early brain injury (EBI), which occurs during the 24-72 h following aneurysmal rupture, makes a significant contribution to patient outcome, and may be responsible for the detrimental effects seen in patients after SAH (3,(8)(9)(10). EBI was first reported by Kusaka et al. (11). Since then, the knowledge of mechanisms involved in EBI significantly progressed, but it still warrants further investigation (12)(13)(14)(15)(16)(17)(18)(19)(20). During EBI, the central nervous system (CNS) suffers from "primary" insults (involving acute changes of intracranial pressure, cerebral perfusion pressure, and cerebral blood flow with vascular constriction and obstruction of the microcirculation), and "secondary" ischaemic processes (including anaerobic cellular respiration, energy depletion, impaired protein synthesis, excitotoxicity, free radical attack, neuronal stress, and DNA damage, leading to apoptosis and necrosis) (16). Many of these processes may potentially be initiated, mediated, or terminated by amino acids and related compounds. Von Holst and Hagenfeldt in 1985 appear to be the only group to have demonstrated increased levels of amino acids in cerebrospinal fluid (CSF) after SAH, and proposed mechanisms leading to it (21). Since then, no new studies analysing CSF amino acids involved in SAH has been published. Our present aim is to look into the role of amino acids and related compounds potentially involved in the process of brain injury following SAH. Determination of amino acid levels in CSF samples may provide a means of determining prognosis at an early stage and extending the knowledge about the pathophysiology of SAH.

MaTerials anD MeThODs ethics and consent
This is a prospective observational study conducted in a single medical centre in accordance with the Declaration of Helsinki. The local bioethics committee approved the study protocol, consenting protocol, and consent forms. Patients were assessed by two specialists (neurosurgeon and anaesthesiologist) as to their ability to give informed consent. Depending on this assessment, either the patient or next of kin gave consent for entry to the study and use a blinded medical data for analysis and further publication.

Population, inclusion and exclusion criteria
132 patients with SAH [confirmed by non-contrast computed tomography (CT)] were referred to our department during study recruitment period (from May 2015 to October 2016). Inclusion criteria were as follows: (1) aneurysmal SAH treated endovascularly <24 h post rupture, (2) external ventricular drainage (EVD) placed <48 h post rupture. The aim was early prevention of rebleeding while managing acute hydrocephalus (HCP), but avoiding the trauma associated with open surgery. Exclusion criteria for the study were as follows: (1) history of CNS disease (meningitis, stroke), (2) active CNS infection, (3) active systemic disease (diabetes mellitus, rheumatoid arthritis, malignancy, cirrhosis, renal failure), (4) age below 18, and (5) pregnancy. Conditions with potential impact on CSF homeostasis as well as subpopulations with distinct SAH features were not enrolled. Control CSF was obtained during spinal anaesthesia from age-and sex-matched patients with a negative history of CNS diseases.

Management, Definitions, and end Points
On admission, the clinical status was assessed using GCS and specific SAH grading scales [Hunt and Hess (HH), World Federation of Neurosurgical Societies (WFNS)]. An initial head CT scan was used to confirm SAH and assess the presence of HCP. EVD was placed secondary to endovascular treatment in patients with GCS score below 15 and: (1) relative bicaudate index >1, (2) focal dilation of ventricular system due to obstruction, or (3) thick intraventricular blood clot. A second CT scan was performed within 24 h of aneurysmal occlusion and EVD placement to assess any procedure related brain injury. Patients received a continuous infusion of nimodipine for at least 10 days, hypotension was avoided using vasopressors, and euvolemia was maintained. Induced hypertension (20-30% above baseline levels) was used to treat patients diagnosed with delayed cerebral ischaemia (DCI), based on the appearance of a new focal deficit, or a drop of at least two points on the GCS lasting at least 2 h after the exclusion of systematic causes EVD infection screening involved CSF cell count at least twice per patient, and CSF culture at least once on day 10 post-SAH. The primary end point was the treatment outcome assessed at 3 months using the Glasgow Outcome Scale (GOS) (22). Patients were divided into two groups according to GOS. Good outcome consisted of those with no disability, moderate disability, and severe disability (GOS grades 5, 4, and 3); poor outcome were those with persistent vegetative state or death (GOS grades 2 and 1). DCI related infarction was defined as a new cerebral infarction identified on a head CT scan within 6 weeks of rupture and not present on the immediate posttreatment scan [as proposed by Vergouwen et al. (23)]. have been described in our previous report (24). The sample preparation comprised the following steps: protein precipitation by 10% sulphosalicyclic acid, dilution with borate buffer, amino acids labelling with aTRAQ™ reagent, and addition of internal standard mixture. Labelling efficiency was confirmed using norleucine (contained in sulphosalicylic acid solution) and norvaline (contained in borate buffer). LC-MS/MS assays were carried out on a high-performance liquid chromatography instrument 1260 Infinity (Agilent Technologies, Santa Clara, CA, USA) coupled with 4000 QTRAP triple quadrupole mass spectrometer (Sciex, Framingham, MA, USA). Chromatographic separation of amino acids was performed using a Sciex C18 column (4.6 mm × 150 mm, 5 µm) and a flow rate of 800 µL/ min in a gradient elution mode. Solvent A was water and solvent B was methanol, both with 0.1% formic acid and 0.01% heptafluorobutyric acid. The injection volume was 2 µL. The mass spectrometer was equipped with an electrospray ionisation source and operated in positive ionisation mode. For detection and quantification of amino acids, a highly selective schedule multiple reaction monitoring mode was used. Data acquisition and processing were performed with Analyst 1.5 software (Sciex, Framingham, MA, USA). The applied methodology is suitable for simultaneous determination of a wide range of 33 free amino acids, both proteinogenic and non-proteionogenic, with high sensitivity and specificity in time below 20 min (24)(25)(26). Due to simplification used in this article, o-phosphoethanolamine and ethylamine are counted to amino acids, yet in fact they are amino acids derivatives.

statistical analysis
Statistical analysis was performed using STATISTICA 10 (Stat Soft Inc., Tulsa, OK, USA). Values for normally distributed numerical data have been expressed as mean and SDs; for ordinal or non-normally distributed numerical data as median and interquartile range, and for categorical data as counts and percentages. The normality of data distribution was assessed using the Shapiro-Wilk test. Amino acids levels are presented on figures as median and interquartile range since in nearly all cases they do not show normal distribution. Comparisons were made by using (1)  Rupture of an aneurysm led to a significant elevation of 27 of the 33 compounds in the CSF at days 0-3 post-SAH ( Table 2). On days 0-3 post-SAH, the six exceptions were ethylamine (p = 0.798), gamma-aminobutyric acid (p = 0.699), 3-amino-isobutyric acid (p = 0.668), hydroxyproline (p = 0.164), threonine (p = 0.079), and arginine (p = 0.062). On day 5 post-SAH, three compounds showed no significant difference-aspartic acid (p = 0.175), 3-amino-isobutyric acid (p = 0.563), and hydroxyproline (p = 0.658). On day 10 post-SAH, there were four-aspartic acid (p = 0.054), 3-amino-isobutyric acid (p = 0.806), hydroxyproline (p = 0.289), and ethylamine (p = 0.292). Thus, hydroxyproline and 3-amino-isobutyric acid are the only 2 of the 33 substances assayed which appear to show no increase following SAH.
In the course of the study, it was possible to undertake assays at all three time points in 10 of the subjects; 7 of these were in the good outcome group. In this group, the Friedman test with Conover-Iman post hoc, or ANOVA test with Fisher post hoc, revealed significant changes in the concentrations of 18 of the compounds tested ( Table 4). The number of poor outcome patients was too small to carry out such testing.
The levels of four of the compounds tested showed a significant correlation with GOS at 3 months. These were 2-amino-adipic acid on day

DiscUssiOn
In this study, we have analysed the profiles of amino acids and related compounds in the CSF of patients following SAH. The most significant findings are as follows: (1) Increase in 31 of 33 compounds in the days following SAH, with 26 increasing in the first 3 days after rupture (2) Significant increase in 18 of these compounds between days 0-3 and days 5 and 10 (3) Higher levels of excitatory amino acids (EAAs) (glutamic acid, aspartic acid, and 2-amino-adipic acid) appear to predict a poor outcome.
As far as we are aware, it is 31 years since an article investigating amino acids in SAH has been published. In view of the paucity    With the introduction of microdialysis, interest in this subject has been renewed since it allows in vivo sampling of brain interstitial fluid (27). This method allows continuous monitoring of brain metabolism, but this is limited to the tissue around the probe (28). On the other hand, the commonly available CSF analysis gives a more general picture of conditions in the brain. Physiologically, the exchange between brain interstitial fluid and CSF is bidirectional (29). In mice, levels of amino acids in brain interstitial fluid were found to be approximately 5-10 times lower than in the CSF (30). Although the latest studies of amino acids in SAH have focussed on microdialysis, widespread use of this monitoring technique is limited by its high cost. By contrast, EVD is a relatively inexpensive procedure commonly performed in patients following SAH. In the clinical setting, CSF seems to be a more convenient source for examining biomarkers. In our patients, rupture of an aneurysm led to an increase in 31 of 33 amino acids and related compounds we assayed. In the study by von Holst et al., there was no increase in taurine levels following SAH. Because the concentration of taurine in whole blood is four to eight times greater than in blood plasma alone, von Holst et al. concluded that red blood cell (RBC) lysis did not contribute to the amino acid concentrations (31). We do not agree with this statement as in our series, the CSF taurine levels increased by a factor of 2. RBC lysis begins within 2-4 h of SAH and continues at least until the clot has cleared (32,33). In our opinion, this process contributes to the amino acid levels at every stage following SAH. Microdialysis studies indicate that an early increase of taurine in the brain interstitial fluid is a reliable marker of poor outcome (34,35). In the both articles, brain cells activated in the course of SAH were indicated as a potential source of taurine in the interstitial fluid. In our series, significantly higher taurine levels were observed on days 0-3 post-SAH in patients with a poor outcome. This observation suggests that taurine may have some value as a clinical marker and encourage further studies. In experimental settings, both detrimental and beneficial roles for taurine have  Among 33 assayed compounds, only 2 did not increase at any stage; these were hydroxyproline and 3-aminoisobutyric acid, neither of which is encoded in the eukaryotic genetic code.    Hydroxyproline is produced by hydroxylation of proline and incorporated into collagen protein (36). In patients with blood-CSF barrier dysfunction, hydroxyproline has a smaller biological variation in CSF when compared with other amino acids (37). Hydroxyproline is increased in the blood plasma in Alzheimer's disease, but shows no increase in Parkinson's disease (38,39).
High protein bound of hydroksyproline in human erythrocytes could be an explanation of its elevation absence in CSF following SAH (40). Even less data are available for 3-aminoisobutyric acid, although it is known that it is a product of thymine catabolism and plays a role in fatty acid metabolism (41). The alterations of 3-aminoisobutyric acid could be a potential marker for the monitoring of the blood-brain barrier condition in the future studies (42). Immediately after aneurysmal rupture, significantly higher levels of nine of the compounds investigated identified the patients with a poorer prognosis. Furthermore, none of the substances showed a significant decrease in CSF following SAH. We suspect that the initial increase is due to extravasated blood, and its extent related to the amount of blood. The main source is likely to be plasma, since the majority of amino acids (except glutamine and glutamic acid) have a CSF:plasma ratio of 0.1-0.2 (43). This is consistent with the fact that patients with a greater volume of subarachnoid blood have a poorer outcome (44).
In 18 of the compounds we investigated, there was a significant increase from days 0-3 post-SAH to days 5 and 10. There are several possible mechanisms for this delayed rise: (1) increased amino acid turnover as a response to injury. Zetterling et al. proposed this mechanism as an explanation for an increase in the concentrations of eight non-transmitter amino acids in brain interstitial fluid (45). (2) Cytokine-stimulated amino acid release. High-mobility group box 1 protein (HMGB1), which is a proinflammatory cytokine [found in CSF of SAH patients (46)], which induces the release of the glutamate analogue from gliosomes (glial resealed subcellular particles) in a concentration-dependent manner (47). Conversely, HMGB1 was found to accumulate in glutamate treated primary cortical culture media, and supernatants collected from these cultures were found to trigger microglial activation (48). (3) Disruption of CSF homeostasis affecting transport with both the bloodstream and the interstitial fluid. (4) Lysis of RBCs (mentioned above) subsequent to their continuous release from the clot during its clearance (33).
Glutamic acid, as an important EAA, is the most extensively studied amino acid in SAH. Levels of glutamic acid in the interstitial fluid increase within minutes of SAH, peak at 40 min, and remain elevated for days (49). The increase is most pronounced in patients with acute ischaemic neurological deficit (50). Increase in interstitial glutamic acid was identified as one of the earliest markers of impending ischaemia, typically increasing before the onset of clinical symptoms (27,50). Our results are in line with these observations. Glutamic acid, aspartic acid, and 2-aminoadipic acid levels increase on days 0-3 post-SAH, and are all significantly higher at some point in the poor outcomes group of patients. In fact, in this group, outcome was most accurately predicted by 2-aminoadipic acid levels in CSF on day 10 post-SAH. 2-Aminoadipic acid is a structural homologue of glutamic acid and a natural product of lysine metabolism in mammalian cells (51). Huck et al. described gliotoxic properties of this amino acid (52), while Kato et al. observed enhanced susceptibility of glial cell to oxidative stress after 2-aminoadipic acid administration (53).
In this prospective observational study, some limitations need to be considered. First, specific enrolment criteria (acute HCP and EVD insertion) interfere with typical SAH pathophysiology. They will aggravate the SAH-associated brain injury and may well alter amino acid concentrations. Consequently, our observations may only be applicable to this subpopulation of SAH patients. Second, the relatively small number of examined samples and enrolled patients limits the extent of our conclusions. Nevertheless, the statistical relationships in our study follow the pattern of large scale studies (e.g., high correlation between admission status and treatment outcome). Third, EVD infection and CNS microbial inflammation could potentially affect the results, but our protocols specifically aim to minimise such problems. We have assumed a relationship between CSF and interstitial fluid, but this may itself be corrupted by the pathology. Future studies should include more good-grade patients without severe complications (e.g., acute HCP) with CSF drawn by LP. cOnclUsiOn Aneurysmal subarachnoid haemorrhage leads to a generalised increase of amino acids and related compounds in CSF. The patterns of concentrations differ between good and poor outcome patients. Increased EAAs are strongly indicative of poor outcome.

eThics sTaTeMenT
This is a prospective observational study conducted in a single medical centre in accordance with the Declaration of Helsinki. The local bioethics committee approved the study protocol, consenting protocol, and consent forms.