Amino acid metabolism and prognostic markers in ischemic stroke patients

Ischemic stroke continues to be a serious public health problem worldwide, we compared the amino acid proles of convalescent patients with ischemic stroke with healthy subjects by High Performance Liquid Chromatography (HPLC), 48 convalescent patients with ischemic stroke and 48 healthy controls were recruited, nding that the levels of Phosphoserine(P-Ser), Glutamic Acid(Glu), Isoleucine(Ile), Leucine(Leu), Phenylalanine(Phe), Arginine(Arg), Proline(Pro) in stroke patients increased signicantly, and the levels of Taurine(Tau), Cystine(Cys), Methionine(Met), b-Alanine(b-Ala), Histidine(His) lowered in patients compared to controls (P<0.05). Furthermore, it was particularly important to nd indicators that could judge the prognosis of patients, the Tau/Glu level could be used as a potential prognosis marker and considering convalescent stroke patients have a good prognosis when the ratio > 0.79 in our study.


Introduction
Stroke is a complex neurological syndrome, which is one of the most important causes of disability and the second most common cause of death worldwide(H. . Most stroke patients will have different degrees of neurological, sensory and other dysfunctions after treatment, which seriously affects patients' daily life and work; Although the treatment of patients with stroke has improved greatly in recent years, it still has a high recurrence rate (Tu et al., 2017); Therefore, it is particularly important to investigate the metabolism and nd some indicators that can evaluate the prognosis of stroke patients for the optimized care and allocation of healthcare resources.
Metabolomics, which focuses on the pro ling of small metabolites in bio uids, cells and tissues, aiming to reveal the metabolic state of biological systems; metabolites are the substrates and products of metabolism that drive essential functions, such as energy production and storage, signal transduction and apoptosis, which can regulate or affect the environment in which they are produced (Johnson, Ivanisevic, & Siuzdak, 2016; Johnson, Patterson, Idle, & Gonzalez, 2012). Increase or decrease of the amino acid in the brain tissue have a toxic effect on the brain tissue, and it has been recognized as one of the mechanisms of stroke injury (Gomi, Karp, & Greenberg, 2000; Ramasamy, Dutta, Kannan, & Chandramouleeswaran, 2021). When a stroke occurs, brain tissue edema, in ammation or neuronal damage can also cause signi cant changes in serum amino acid metabolite group. Therefore, the levels of free amino acids in serum could partially re ect the status of metabolic disorders, which may help to distinguish stroke patients with healthy subjects and evaluate the prognosis of stroke patients (Xie & Li, 2015).
In this study, we compared the amino acid pro les of convalescent patients with ischemic stroke with healthy subjects by HPLC, aiming to nd the metabolic differences between two groups. At the same time, we monitored the trend of amino acid changes continuously and tried to seek some prognosis biomarkers for ischemic stroke patients.

Materials And Methods
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Study design and methods
Convalescent patients with ischemic stroke (after 14 days of the onset of ischemic stroke diagnostic) were recruited from Huguosi Hospital of TMC a liated to Beijing University of Chinese Medicine. The study was approved by ethics committee, and informed consent was obtained from all individual participants included in the study. Stroke severity was assessed on admission using the National Institutes of Health Stroke Scale (NIHSS) by a neurologist, and the NIHSS scores of all patients were uctuated between 5-15. Barthel index (BI) was used to assess the ability of daily living of these patients when they were discharged, and the patients were divided into a good prognosis group (BI>60) and a poor prognosis group (BI≤60). Healthy subjects (individuals without history of ischemic or hemorrhagic stroke) were included at the same time, which were considered as the control group. Those subjects had no statistical signi cance on basic characteristics, such as age sex other illnesses and so on, and the information of all subjects was listed in Table 1. Blood samples were collected according to entry criteria, and all the samples analysis were performed by HPLC using Amino Acid Analyzer (Hitachi L8900). vortexed for 10 s and reacted for 10 min to remove proteins, then centrifuged 10000rpm for 15min at 4 ℃ before experiment, and then the supernatant was used to analyze the amino acids through Amino Acid Analyzer.

Statistical analysis
The measured amino acid values were normally distributed, thus, differences in these parameters between two groups were assessed using the independent-samples t test; certain parameter change along time in one group was assessed using one-way analysis of variance (ANOVA); comparison of count parameters were assessed using Chi-square test. Furthermore, receiver operating characteristic (ROC) curve was used to evaluate the diagnostic e cacy of distinguishing healthy controls and ischemic stroke patients, and the results were reported as area under the curve (AUC). All statistical analysis was performed with SPSS 25.0 (SPSS, USA) and Graphpad Prism 8(GraphPad Software, USA). Statistical signi cance was de ned as P < 0.05.

Results
3.1 Different amino acid pro les between stroke patients and control subjects 23 amino acids were identi ed in our study, 12 were found signi cantly different between the ischemic stroke patients and the healthy controls ( Table 2). The average levels of phosphoserine, glutamic acid, isoleucine, leucine, phenylalanine, arginine, proline in the ischemic stroke patients were increased compared to controls, and the levels of taurine, cystine, methionine, b-alanine, histidine were decreased (P<0.05). These results showed that there were obvious metabolic differences between two groups. To evaluate the abilities using amino acid to distinguish stroke patients and healthy controls, the results of ROC curve analyses comparing elevated and lowered amino acids levels between two groups were shown in Figure 1 and

Exploration of prognostic biomarkers
The metabolic differences between convalescent patients with ischemic stroke and healthy subjects had been studied. At the same time, we monitored the trend of amino acid changes continuously and tried to seek some prognosis biomarkers for stroke patients. According to past studies, two typical amino acids (Tau, Glu) were selected and their ratio was considered as prognostic markers because that Tau can resist neurotoxicity of Glu. Tau/Glu was signi cant lower in stroke group in the rst week of admission than control group as shown in Figure 3. Daily serum samples were obtained for 5 weeks after admission in the stroke group, and the histogram (mean) of Tau/Glu in 5 weeks was shown in Figure 4, which indicated the value of Tau/Glu increased gradually with the prognosis getting better. Due to the neuroprotective effect of Tau resists neurotoxicity of Glu, showing that Tau/Glu may be related to the prognosis and could be used as the markers for monitoring prognosis. According to the BI when discharged, the patients were divided into a good prognosis group (BI>60) and a poor prognosis group (BI≤60), and the histogram (mean) of Tau/Glu in two sub-groups were shown in Figure 5a and 5b. The value of Tau/Glu in good prognosis group elevated signi cantly in the 5th week comparing to 1st week, however, the value of Tau/Glu in poor prognosis group didn't have signi cant difference in the 5 weeks. Independent-samples t test was used to compare Tau/Glu level in two sub-groups respectively in 5 weeks from admission, and there was signi cant difference in the 5th week in good and poor prognosis group showing in Figure 6. The ROC curve analyses comparing Tau/Glu between two sub-groups was shown in Figure 7, the AUC was 0.964, and the cut-off value was 0.79. Therefore, Tau/Glu level can be used as a prognosis marker and considering stroke patients have a good prognosis when the ratio > 0.79.

Discussion
Ischemic stroke continues to be a serious public health problem worldwide, with a poor prognosis once it occurs. Fast diagnosis and appropriate treatment are of utmost signi cance to improving the outcome in patients with acute ischemic stroke or convalescent patients with ischemic stroke. Therefore, it is particularly important to investigate the metabolism and nd some indicators that can evaluate the prognosis of stroke patients for the optimized care and treatment(Ormstad, Verkerk, & Sandvik, 2016; X. . Many studies have reported amino acid metabolism in acute ischemic stroke. In this study, we compared the amino acid pro les of convalescent patients with ischemic stroke with healthy subjects by HPLC, aiming to nd the metabolic differences and prognostic markers between two groups. 24 amino acids were identi ed in our study, the levels of P-Ser, Glu, Ile, Leu, Phe, Arg, Pro in stroke patients increased signi cantly, and the levels of Tau, Cys, Met, b-Ala, His lowered in patients compared to controls, which indicated that most amino acids had high diagnostic e ciency in convalescent patients with ischemic stroke and healthy controls. Ile and Leu were reduced in acute ischemic stroke(Kimberly, Wang, Pham, Furie, & Gerszten, 2013), but we observed that they were increased in convalescent patients in our study, which was consistent to reported research (Goulart et al., 2019). Glutamate, as a nerve excitatory amino acid, is an indicator of neural excitation and neurotoxin, and is found to be highly increased in stroke patients as studies demonstrated (Gomi et al., 2000; Meng et al., 2015). These amino acid metabolic differences between convalescent patients and healthy controls could be used as diagnostic markers in two groups. Given the complexity of neurobiological mechanisms of ischemic stroke, it might need a combination, rather than a single metabolite using as the diagnostic marker(D. Wang, Kong, Wu, Wang, & Lai, 2017). So, further studies are needed.
Although advances in medicine have greatly improved the treatment of patients with ischemic stroke in recent years, there is still a high recurrence rate. Therefore, it is particularly important to nd indicators that can judge the prognosis of patients. Amino acids are important nutrients in human body, the levels of free amino acids in serum could partially re ect the status of metabolic disorders, which may help to evaluate the status of patients (Xie & Li, 2015). The stroke patients exhibited signi cantly higher Glu and lower Tau and Tau/Glu than the healthy controls in our study. Glu is the main excitatory neurotransmitter in the central nervous system, impaired uptake or excessive release of Glu lead to the extracellular concentration rising sharply in ischemic stroke patients, and ultimately the excitotoxicity of Glu can induce neuronal death and brain damage ( weeks explore whether Glu, Tau or Tau/Glu can be used as prognosis biomarkers for stroke patients. According to our results, the value of Tau/Glu increased gradually with the prognosis getting better. The value of Tau/Glu in good prognosis group elevated signi cantly in the 5th week comparing to 1st week, however, the value of Tau/Glu in poor prognosis group didn't have signi cant difference in the 5 weeks, and there was signi cant difference in the 5th week in good and poor prognosis group. The ROC curve comparing Tau/Glu between two sub-groups was analyzed, the AUC was 0.964, and the cut-off value was 0.79. Therefore, Tau/Glu level can be used as a potential prognosis marker and considering convalescent stroke patients have a good prognosis when the ratio > 0.79. Further studies are also needed to investigate the role of Tau/Glu in more patient groups as well.

Conclusion
There were obvious amino acid metabolic differences between convalescent stroke patients and healthy controls, and most amino acids had high diagnostic e ciency in two groups. The Tau/Glu level can be used as a potential prognosis marker of convalescent stroke patients. However, a limitation of this study is that the number of participants is less, we need to include more subjects to verify the potential prognosis marker. Anymore, further studies are needed to seek more biomarkers for better treatment of stroke patients.

Disclosure statement
No potential con ict of interest was reported by the author(s).
Ethics approval and consent to participate: The study was approved by the ethics committee in march 2019, and informed consent was obtained from all individual participants included in the study.

Consent for publication:
All the authors listed have approved for the publication of the manuscript that is enclosed. Funding: This research did not receive any speci c grant from funding agencies in the public, commercial, or notfor-pro t sectors.

Data availability statement
The data presented in this study are available upon request. Histogram (mean) of Tau/Glu in the 5 weeks after admission Figure 5 Histogram (mean) of Tau/Glu in the 5 weeks after admission; a:Barthel index >60 b: Barthel index ≤60 Figure 6