A Method For Metabolism Detection Of S-Adenosine Homocysteine And Its Correlation With Stroke

Ischemic stroke is a common cerebrovascular disease.Due to its characteristics of high morbidity,high mortality and high disability rate,ischemic stroke has been regarded as the first cause of disability in adults in China,and it brought global health economic burden and the economic loss of the family couldn’t be ignored.Since 1969,many studies reported in the literature have found that a high level of homocysteine(HCY)is an independent risk factor for stroke.More and more evidences showed that the level of S-adenosyl homocysteine(SAH),a precursor of Hcy in plasma,is likely to be a more sensitive indicator of cardiovascular disease than Hcy.Epidemiological studies have found that the risk of ischemic stroke in high-risk stroke populations is several times than who have non-high-risk populations,and the risk factors including smoking,drinking,physical exercise,etc which could be changed have more significance for the incidence,treatment and prognosis of stroke.S-adenosylmethionine(SAM)is the upstream product of SAH in the methionine cycle.The detection method of SAH and SAM content in the blood is of great significance for the research of related diseases and have been widely concerned.SAH and SAM in the blood are metabolized and reabsorbed in the kidneys.The content of the two compounds in the urine is also important for the research of related diseases.At present,there are few reports on the detection methods of SAH and SAM in urine,and there is still a lack of an accurate,rapid and simple detection method.Oxygen consumption of brain is the highest in all the human organs,short-term ischemia and hypoxia can cause irreversible necrosis of neuronal cells.This kind of hypoxic microenvironment participates in the regulation of cell proliferation,differentiation,apoptosis and other processes,affecting the outcome of stroke and other diseases.Up to now,as an more sensitiveindicator of cardiovascular disease than Hcy,the potential mechanism of SAH on stroke have been unclear.Therefore,this study first explored the causative factors of ischemic stroke and the correlation between Hcy,SAH and cerebrovascular injury through population studies,and clarified the feasibility of SAH as a sensitive screening index and treatment target for ischemic stroke.Based on the application of micromolecular ion pair reagent trifluoroacetic acid,after optimizing three solid phase extraction cartridges for purifying urine samples and describing the relevant mechanisms.An accurate,rapid and simple ion pair chromatography-solid phase extraction method for determination of SAH and SAM in urine was established.The method was successfully applied to analysis of actual sample,and provided technical support for the follow-up research involving SAH and SAM.Exploring the mechanism of SAH apoptosis in the pathogenesis of ischemic stroke by oxygen-glucose deprivation cell model to,it provides theoretical basis and new ideas for further clarifying the pathogenesis of ischemic stroke and developing clinical treatment.Part 1: Correlation between S-adenosyl homocysteine and its metabolites and ischemic strokeObjective: To explore the causative factors closely related to the pathogenesis of ischemic stroke,to clarify the correlation between Hcy and SAH as potential factors that cause brain microvascular endothelial damage,and to clarify SAH as a screening sensitivity index and therapeutic target for ischemic stroke Point of feasibility.Methods: From June 2018 to October 2019,240 healthy individuals,143high-risk individuals with stroke,and 189 stroke patients were recruited in the First Hospital of Hebei Medical University,Shijiazhuang City,Hebei Province as the research object.By carrying out questionnaire surveys and combining the corresponding physical examination and laboratory examination methods,the three groups of poor lifestyle,previous chronic medical history and family history,and common clinical indicators were obtained,and the SAM,Differences in composition and metabolism of SAH and other substances.Results: After gender and age matching and elimination of missing information among the study subjects,78 healthy people,80 high-risk stroke patients,and 88 stroke patients were finally included in the statistical analysis.The measurement results of BMI index and waist-to-hip ratio showed that the measured values of the patient group and the high-risk group were larger than those of the healthy group(P <0.05).The proportion of smokers in the patient group and high-risk group was much higher than that in the healthy group,and the number of smokers in the two groups exceeded twice the number of smokers in the healthy group(P <0.05).The health group has the largest number of people who have physical exercise habits,and the distribution of this indicator among the three groups has a statistically significant difference(P <0.05).There were no significant statistical differences between the three groups in marital status and whether they were drinking alcohol.The systolic and diastolic blood pressure measurement results of the three groups of study subjects showed that the patient group> high-risk group> healthy group(P<0.05).The results of high-density lipoprotein cholesterol(HDL-C)showed that the healthy group> high-risk group> patient group(P <0.05).The triglyceride level in the high-risk group was higher than that in the patient group and the healthy group(P <0.05).The blood glucose level of the patient group was slightly lower than that of the high-risk group and the healthy group(P <0.05).The measurement results of high-sensitivity C-reactive protein showed that the patient group> high-risk group> healthy group(P<0.05).The mean value of uric acid in the high-risk group and the patient group was significantly higher than that in the healthy group(P <0.05).There were no significant statistical differences between the total cholesterol and low-density lipoprotein cholesterol(LDL-C)between the three groups.The number of patients with history of hypertension was the largest in the patient group,followed by the high-risk group,and those with no history of hypertension in the healthy group(P <0.05).The proportion of those without a history of diabetes in the healthy group and those with a history of diabetes in the high-risk group was slightly higher than that in the case group(P <0.05).Among the individuals with previous TIA episodes,17 were in the patient group and 14 were in the high-risk group.There were no subjects with previous TIA episodes in the healthy group(P <0.05).The number of patients with a history of stroke in the patient group was much higher than that in the high-risk group and the healthy group(P <0.05).There were more individuals with a family history of stroke in the high-risk group than in the patient group and the healthy group(P <0.05).The plasma Hcy content in the patient group was significantly higher than that in the high-risk group and the healthy group,and there was a statistically significant difference in the Hcy content between the three groups(P <0.05).The SAM content in the healthy group was the highest,followed by the high-risk group,and the lowest in the patient group(P <0.05).There was no statistically significant difference in plasma SAH content among the three groups(P> 0.05).The results of the SAM / SAH ratio showed that the patient group <high-risk group <healthy group,and there was a significant statistical difference between the patient group and the healthy group(P <0.05).Conclusion: The harm of Hcy to the onset of stroke has always existed.The relationship between SAH and ischemic stroke is complex and inseparable.Further investigation and research are still needed to demonstrate the exact relationship between the two.At the same time,the screening and intervention of people at high risk for stroke will have an important impact on reducing the incidence of stroke and improving the prognosis.Part 2 Establishment and Application of Micromolecule Ion Pair Chromatography for Detection of S-adenosylmethionine and S-adenosylhomocysteine in Human Urine and Study of the Mechanism of Solid Phase ExtractionObjective: To establish a new Ion pair chromatography method for simultaneously determining the concentration of SAM and SAH in human urine and study the mechanism of solid phase extraction.Method: The effects of three type of solid phase extraction columns including Elut PBA,Bond Elut PRS and Bond Elut CBA on extracting SAH and SAM in urine samples were investigated,and the related extraction mechanisms were studied.Samples were added with 30% perchloric acid to precipitate proteins,and the supernatant were purified by the optimal solid phase extraction columns.The eluent was filtered through a 0.25 μm filter membrane and collected in a sample vial for later use.The SAH and SAM are separated by ion pair chromatography which trifluoroacetic acid were as the ion pair reagent and quantified by a diode array detector.Results: The optimized solid phase extraction columns was determined as Bond Elut PBA,and the relevant extraction mechanism was clarified.Good linearity values were obtained in the 0.5-60 μmol/L and 0.1-10 μmol/L ranges for SAM and SAH,respectively.The linear equations were of SAH and SAM were y = 9.4968 x + 0.8157(R2=0.9999)and y = 4.6492x-0.0718(R2=0.9998)respectively.The limits of detection were 0.006 and 0.01 μmol/L for SAM and SAH,respectively,and the limits of quantitation were 0.02 and 0.04 μmol/L for SAM and SAH,respectively.The intra-and interday precisions for SAM and SAH determined were less than 1.38,and the recovery rates of SAM and SAH in urine were between 83.65 and 98.41%.Conclusion: Finally,a accurate,rapid,economical and simple method for the determination of SAM and SAH in human urine by micromolecule ion pair chromatography combined with solid phase extraction was established.The method was successfully applied for the determination of SAM and SAH in actual urine samples,and the results are generally consistent with previously published reference.Part 3: Molecular mechanism of apoptosis of S-adenosyl homocysteine in the pathogenesis of ischemic strokeObjective: To explore the specific molecular mechanism of S-adenosyl homocysteine-induced apoptosis in b.End3 cells.Method: Using b.End3 cells to build an oxygen and glucose deprivation(OGD)model,and add 3-deazaadenosine(3-DZA)on this basis,so that SAH content in b.End3 cells were accumulated.By detecting the total lactate dehydrogenase(LDH)in the cells and CCK-8 colorimetry to determine the cell survival rate,the damage of b.End3 cells was analyzed.Annexin V-PI double staining flow cytometry was used to determine the apoptosis rate of different intervention groups.The expression levels of apoptosis-related proteins and signaling pathway-related proteins in each treatment group were measured by Western Blot immunoblotting.Results: According to the growth state of the cells at different seeding densities,it was determined to use a seeding density of 9 * 10 3 / 100μl for subsequent studies.According to the results of different oxygen-glucose deprivation(OGD)time leading to cell damage and cell survival rate,25 hours of OGD was determined as the most suitable duration of intervention.The results of LDH measurement showed that the LDH release of the 3-DZA group,OGD group,and 3-DZA + OGD group were significantly increased compared with the Con group,and the LDH release amount of each treatment group increased sequentially.There were significant statistical differences in the amount of release(P <0.05).The cell survival rate measurement results showed that the cell survival rates of the 3-DZA group,OGD group,and3-DZA + OGD group all decreased significantly,with the 3-DZA + OGD group having the lowest cell survival rate.In the results of the apoptosis rate measured by Annexin V-PI double staining flow cytometry,the total apoptosis rate of the 3-DZA + OGD group was significantly higher than that of the other treatment groups,and there was a total apoptosis rate among the treatment groups.Significant statistical difference(P <0.05).Western blot results showed that the relative expression levels of Bcl-2 / Bax protein in the 3-DZA group,OGD group,and 3-DZA + OGD group were significantly reduced compared with the Con group.The relative expression of the pro-apoptotic protein Cleaved-caspase-3 / Caspase-3 protein showed an increasing trend in the 3-DZA group,OGD group and 3-DZA + OGD group.The trend of relative expression of JNK signaling pathway protein p-JNK / JNK is consistent with that of Cleaved-caspase-3 / Caspase-3 protein.There were significant statistical differences in protein expression between the groups in Western blot results(P <0.05).After adding blocking agent SP600125,the intracellular LDH release in the 3-DZA + OGD + IN group was less than that in the 3-DZA+ OGD group,and the cell survival rate of the former was significantly higher than that of the latter(P <0.05).The results of flow cytometry measurement of the apoptosis rate of the three groups showed that the total apoptosis rate of the cells in the 3-DZA + OGD + IN group was significantly lower than that in the 3-DZA + OGD group(P <0.05).The relative expression of Bcl-2 / Bax protein in cells of 3-DZA + OGD + IN group was significantly higher than that of 3-DZA + OGD group.The relative expression levels of the pro-apoptotic protein Cleared-caspase-3 / Caspase-3 protein and the signaling pathway protein p-JNK / JNK are consistent,both of which are expressed in the 3-DZA + OGD + IN group,which is lower than in the 3-DZA + OGD group(P <0.05).Conclusion: SAH can cause b.End3 cell damage and lead to increasement in the apoptosis rate of b.End3 cells and increasement in the expression of related pro-apoptotic proteins and decrease in the expression of inhibitory apoptotic proteins.Western blot detection of the pathway determined the specific regulatory signaling pathway of SAH-induced apoptosis--JNK pathway.Based on the recovery experiments,it was further clarified that SAH can induce apoptosis in b.End3 cells through the activation of JNK signaling pathway.