Establishment And Clinical Application Of A Methodological Approach For The Detection Of Serum Unbound Bilirubin In Neonates By Peroxidase

BackgroundNeonatal jaundice(NNJ)is a common clinical problem in the early neonatal period,and in severe cases,bilirubin encephalopathy(BE)may develop,causing permanent neurological damage and even death.Serum unbound bilirubin(UB)is a well-recognized bilirubin neurotoxic factor,and dangerous concentrations of UB can cross the blood-brain barrier to invade specific anatomical regions of the central nervous system(including the pallidum,subthalamic nucleus,etc.)inducing neuronal apoptosis and necrosis.A increasing number of clinical studies have confirmed the highly predictive value of UB for the development of bilirubin-induced neurologic damage,and in particular its strong correlation with bilirubin-induced auditory impairments.Serum UB can be quantified by the peroxidase(POD)method,which is based on the combination of enzymatic reaction kinetics and spectrophotometry.Little research on UB detection has been reported in China.In order to further optimize the management of neonatal jaundice,there is an urgent need to carry out research and practice on the detection of serum UB concentrations for neonates.Objective1.To explore the methodology of POD for the detection of serum UB concentrations in neonates2.To evaluate the preliminary clinical application value of POD method in the management of neonatal jaundice.Methods1.By exploring the effects of different concentrations of horseradish peroxidase(HRP)and different concentrations of bilirubin standard solution conditions on the standardization of the primary rate constant Kp,and the effects of different-20℃freezing period on the activity of HRP,the changes of UB concentrations of bilirubin standard serum with different bilirubin to albumin molar ratios(BAMR)and different Pondus Hydrogenii(pH),and the gradual transition to the detection of UB concentrations in neonatal serum samples,the precision and stability of the POD method for the detection of serum UB were verified.2.A total of 60 jaundiced neonates with gestational age≥35 weeks admitted to the department of neonatology of Shenzhen Maternal and Child Healthcare Hospital between September 2021 and December 2022 were selected.The serum UB concentrations before receiving phototherapy at the time of admission were measured by POD method and the correlation between the clinical data and serum UB concentrations of the enrolled cases was analyzed.According to the results of brainstem auditory evoked potentials(BAEP),the enrolled cases were divided into normal and abnormal groups,and between-group differences were analyzed.Subsequently,logistic regression model of UB and abnormal BAEP were established,and ROC curves were plotted to assess the diagnostic value of UB for bilirubin-induced auditory impairment.Results1.Methodological section1.1 There were no significant effects of different bilirubin standard solution concentrations(1,2,3 μmol/L)and different HRP concentrations(0.019,0.038,0.075μg/mL)on the primary rate constant Kp at pH 7.4,reaction temperature 37.0℃ and protection from light.1.2 The UB concentrations of bilirubin standard serum increased significantly with increasing BAMR(range 0.1-2.0)at pH 7.4,reaction temperature 37.0℃ and protected from light.1.3 Fixed BAMR bilirubin standard serum showed no significant difference in UB concentrations in the pH range(7.0-7.6)at a reaction temperature of 37.0℃ and under light-protected conditions,while a corresponding significant increase and decrease in UB occurred at pH=6.8 and pH=8.0.1.4 There was no significant decrease in the UB concentrations of neonatal serum samples and HRP activity frozen at-20℃ for a week and 3 weeks,respectively.1.5 Good precision was achieved by the POD method for the detection of the UB concentrations in neonatal serum samples and bilirubin standard serum with different BAMRs,with an average intra-batch coefficient of variation of less than 10%and an average inter-batch coefficient of variation of less than 15%.2.Clinical study section2.1 Neonatal serum UB concentrations were significantly and positively correlated with total serum bilirubin(TSB)and also with BAMR.As the DB/TSB ratio increased,the slope increased and the coefficient of determination decreased.2.2 There were no significant differences in serum UB levels between near term infants and term infants,or between males and females,or between the different pH groups.2.3 The concentrations of TSB,BAMR,and UB were significantly higher in the abnormal BAEP group(n=6)than in the normal BAEP group(n=54).The logistic regression showed that UB,TSB,and BAMR were all significantly associated with abnormal BAEP outcomes(P<0.05).Among them,UB had the best predictive value for bilirubin-induced auditory impairment,with the area under the ROC curve of UB(0.961,P<0.001),which was greater than the AUC of the other two.At the optimal cut-off point UB≥24.1 nmol/L(or 1.4 μg/dL),the sensitivity was 83.3%and the specificity was 94.4%.Conclusions1.In this study,a method for the detection of serum UB by POD was established and applied to the detection of clinical serum samples from jaundiced neonates with gestational age≥3 5 weeks,which has the advantages of simple operation,rapid quantification,economic feasibility,as well as good precision,and is suitable for clinical application.2.The serum UB levels can be used as an indicator for early diagnosis,which have a predictive value for bilirubin-induced auditory impairment.Therefore,the determination of serum UB has some significance for the management of neonatal jaundice.