Study On The Stability Of Cyanide Metabolites In Biological Samples

Objective:1.Study on the stability of cyanide metabolite SCN^-and 2-aminothiazoline-4-carboxylic acid?ATCA?in biological samples;2.To investigate the reference value range of SCN^-and ATCA in smokers?18-27years old?.Method:1.Decomposition dynamics modelSixteen healthy rabbits were randomly divided into two groups.In the first group,2LD₅₀ potassium cyanide was administered orally to the stomach,and the blood of the rabbits was dissected and fully mixed immediately after the disappearance of respiration,heartbeat and reflexes of the rabbits.In the second group,the same amount of normal saline was infused through the mouth,and the blood of the rabbits was taken and fully mixed after death.The blood of the two groups was divided into four parts on average,which were stored at 20?,4?,-20?and 20?with 1%NaF.Samples were taken at 0d,7d,15d,30d,50d,80d,120d,150d to detect the contents of SCN^-and ATCA in each group.2.Sample collection of smokers in normal population100 volunteers?male 94,female 6?,aged 18-27.All participants had different levels of smoking habits?see the table below for details?,without basic diseases.All volunteers filled in the volunteer information form and signed the informed consent after fully understanding the purpose of the experiment.All the samples involved in this experiment have passed the ethical review.In the morning,25 mL of blood was collected from the elbow vein in an empty stomach.The blood was stored in the blood vessels without additives and those with heparin sodium.After 30 minutes of standing,all the blood vessels were centrifuged in a centrifuge at 5000 rpm for 5 minutes to obtain serum,plasma and whole blood from different blood vessels.Take the supernatant respectively and store it in the frozen storage tube,put it in the refrigerator at-80?for inspection.3.Extraction and detectionDetection of thiocyanate?SCN^-?:the biological samples to be tested are extracted by liquid-liquid method,and then detected by ion chromatography.The retention time of standard addition experiment is used for qualitative analysis,and the standard curve method?external standard method?is used for quantitative analysis.Detection of 2-aminothiazoline-4-carboxylic acid?ATCA?:the biological sample to be tested is added with internal standard ATCA-¹³C,¹⁵N₂ is extracted by liquid-liquid,ATCA is detected by LC-MS/MS method,the proportion is determined by retention time,quantitative ion and qualitative ion,and the standard curve?internal standard method?is quantitative.4.Data analysisSPSS 20.0 was used for ANOVA of single sample repeated measurement data;CAS3.2.8 pharmacokinetic software;GraphPad Prism 7 software.Results:1.StabilityThe results of the stability study of CN^-showed that the P<0.05 of each detection time point and corresponding different storage conditions showed that the SCN^-concentration measured at different times and the interaction of SCN^-concentration under different storage conditions were statistically significant,that is,the SCN^-concentration changed with time,and different storage conditions had different effects on SCN^-.By statistical software analysis,under different storage conditions,P<0.001,that is to say,the concentration of SCN^-was not equal under different storage conditions.At the first three time points?0d,3d,7d?,the change trend of the four storage conditions was similar,and there was a small decline trend.After 7d,the measured value under20?storage conditions increased at first,then decreased gradually,and the overall SCN^-concentration was higher than that of the other three groups In addition,under the condition of 4?,20?+1%NaF,the concentration of SCN^-increased slightly,while under the condition of-20?,the concentration of SCN^-decreased.The overall trend is that the SCN^-concentration is the highest at 20?,the next time at 20?+1%NaF,and then at 4?,the lowest at-20?.The results of ATCA stability study showed that under different storage conditions,P?0.001,that is to say,the concentration of ATCA under different storage conditions was not equal.As a whole,ATCA concentration increased at 20?,4?and 20?+1%NaF.At the same time point,the change trend of ATCA concentration in 20?storage was similar to that in 4?,and there was no significant difference?P>0.05?.At the same time point,ATCA concentration in 20?was higher than that in-20?under20?+1%NaF,the difference was statistically significant?P<0.05?.2.Reference value range of cyanide metabolites in smoking populationThere is no statistical difference between genders,which can be analyzed as a whole.The data showed that the SCN^-content in the serum of 100 smoking volunteers was 738.47±654.77ng/mL,ranging from 20.09ng/mL to 3685.99ng/mL.The content of SCN^-in plasma was 715.68±542.99ng/mL,ranging from 86.59ng/mL to 2054.91ng/mL.The content of SCN^-in whole blood was 463.99±435.51ng/mL,ranging from41.25ng/mL to 1979.46ng/mL.The statistical results showed that there was no significant difference between the SCN^-content in serum and that in plasma,but both of them were higher than that in whole blood.The serum ATCA content of 100 smoking volunteers was 126.07±81.12ng/mL,ranging from 12.57.6ng/mL to 396.10ng/mL.The content of ATCA in plasma was 150.55±89.07ng/mL,ranging from 41.18ng/mL to379.00ng/mL.The content of ATCA in the whole blood was 57.33±42.61ng/mL,ranging from 11.11ng/mL to 284.03ng/mL.The results showed that there was no significant difference between ATCA content in serum and that in plasma,but both of them were higher than that in whole blood.3.The change of ATCA concentration in the blood of cyanide poisoning patientsThe concentration of the cyanide metabolite ATCA in the blood of 4 cases of cyanide poisoning also increased first and then decreased in a certain range of preservation time,and the increase was related to the preservation temperature.The higher the temperature,the faster the increase,the greater the increase.This may be caused by the transformation of ITCA into ATCA.After the peak value is reached due to the complete transformation,the trend of decrease due to decomposition will appear.ATCA was stable at-20?.Conclusion:1.The results showed that SCN^-and ATCA of cyanide metabolites in preserved blood increased first and then decreased,which was the most stable when stored at-20?,which was consistent with the change of metabolite concentration in the blood of cyanide poisoning cases,suggesting that ATCA could be used as a biomarker of cyanide prebiotic.Relevant test materials should be stored at-20?to avoid the false negative result of cyanide metabolite detection caused by decomposition after death;2.The upper limit of normal values of SCN^-and ATCA in whole blood,plasma and serum of healthy smokers in Taiyuan was 1170.86ng/mL,1596.07ng/mL,1800.88ng/mL and 126.42ng/mL,294.91ng/mL and 257.55ng/mL,respectively.The SCN^-content was significantly higher than that of healthy nonsmokers,and there was no significant difference between the ATCA content and that of healthy nonsmokers.3.The results of ATCA in 4 cases of cyanide poisoning were higher than the upper limit of normal value of ATCA in healthy people of Taiyuan City,which suggested that ATCA could be used as a marker of cyanide poisoning death.