Detection And Analysis Of Nitric Oxide In Children With Autism Spectrum Disorder

PurposeAutism spectrum disorder（ASD）is a neurodevelopmental disorder,which causes great economic pressure on families and society,and it is projected to account for 1%of the national GDP by 2030.Currently,it still lacks effective diagnostic biomarkers in clinic for ASD.Nitric oxide（NO）,a kind of free radical,has both neuroprotective and neurotoxic effects.It was found that the total concentrations of NO stable metabolites nitrite and nitrate（NOx）in the blood samples of ASD children was higher,but not clear in urine and saliva.In order to explore novel biomarkers for non-invasive detection,this study accurately detected and analyzed NO levels in urine and saliva of ASD children,the following research methods were mainly used.Methods（1）The accuracy,precision,detection limit,quantitative limit and stability of Nitric Oxide Analyzer Chemiluminescence（NOA）were assessed.The pretreatment of nitrite（NO₂^-）and nitrate（NO₃^-）in urine were optimized by comparing several known pretreatment techniques in parallel.The optimal storage temperature and storage time were clarified by observing the relation between the stability of NO₂^-and NO₃^-in urine and the time and temperature.Subsquently,the levels of NO₂^-and NO₃^-in urine collected from 140 ASD children,140 ND children and 102 healthy adults were then accutatly detected by NOA.（2）The change of p H values of urine at different storage temperatures and time points were measured by p H meter.（3）The pretreatment of saliva was optimized by parallel comparison of several known pretreatment techniques.By observing the effect of spitting sampling,cotton swab sampling and optimized pretreatment（sodium hydroxide and centrifugation）on the stability of NO₂^-in saliva,the sampling and pretreatment methods were clarified.Then the concentration of NO₂^-in saliva collected from 140 ASD children,140 ND children and 102 healthy adults was then accurately detected by NOA.Results（1）The detection limits of NO₂^-and NO₃^-were 0.03μM and 0.02μM respectively and the quantitative limits of NO₂^-and NO₃^-were 0.11μM and 0.07μM respectively,the relative standard deviation was less than 5%,the recovery was in range of 97.7%and 107.5%when analyzing NO₂^-and NO₃^-standard solutions by NOA.The results show that the NOA method is accurate and reliable.（2）It’s found that all kinds of chemical substances or filtration operations lead to instability of the sample results by comparing various pretreatment methods of urine.Therefore,the urine samples were injected directly into the NOA reactor without treatment for detection.In addition,although the urine samples could be stored stably at-20℃for 3 hours,it still needs to be tested promptly.（3）It was found that NO₂^-、NO₃^-in urine did not fluctuate significantly during testing of adult samples over 13 hours a day.Furthermore,it was found that exercise decreased the levels of NO₂^-and NO₃^-in urine by observing urine samples before and after exercise within 4 days.（4）The levels of NO₂^-in urine were 1.0710±0.1849μM in ASD children,0.3706±0.0377μM in ND children,0.4965±0.0418μM in healthy adults.The levels of NO₃^-in urine were 2.9280±0.1898 m M in children with ASD,2.350±0.1122 m M in ND children,3.5800±0.2587 mmol/L in healthy adults.The levels of NO₂^-and NO₃^-in urine of ASD children were evidently higher than ND children,p values were 0.0003,0.0094 separately.The levels of urinary NO₂^-and NO₃^-of healthy adults were evidently higher than ND children,p values were<0.0001,0.0002 separately.（5）Gender did not cause differences in NO₂^-and NO₃^-levels in urine.（6）It was feasible to collect saliva by cotton swab for ASD children,ND children and healthy adults.Saliva treated with sodium hydroxide and PBS could be stored stably at-20℃for at least 24 hours,and cotton swab did not affect the detection of NO₂^-in saliva.（7）The salivary NO₂^-levels were 6.3410±0.5308μM in ASD children,1.5770±0.1188μM in ND children,1.2560±0.0741μM in healthy adults.Compared to ND children,NO₂^-levels in saliva were significantly higher in ASD children（p<0.0001）and significantly lower in healthy adults（p=0.0069）.（8）Gender did not cause differences in NO₂^-levels in saliva.ConclusionThe levels of NO₂^-and NO₃^-in urine and saliva in ASD children are significantly higher than ND children,indicating that NO may be a potential diagnosis of ASD oxidative stress biomarker.