Management of neonatal hyperbilirubinemia: interpretation of the clinical management guidelines drafted by the American Academy of Pediatrics and other countries / 中华实用儿科临床杂志

Neonatal hyperbilirubinaemia, clinically presenting as jaundice, is a ubiquitous and commonly a benign metabolic condition in newborn infants.It is a leading cause of hospitalization of neonates in the first week of life.Serum bilirubin has been considered as the most potent superoxide with the peroxyl radical scavenger activity.However, uncontrolled hyperbilirubinaemia or rapidly rising bilirubin can reach a neurotoxic concentration, potentially leading to central nervous system sequelae.Thus, the health status of jaundiced newborn infants is dependent on striking an appropriate balance between the protective effects of serum bilirubin and the risk of bilirubin neurotoxicity.In order to standardize the management of neonatal hyperbilirubinemia (jaundice), many countries have developed clinical practice management guidelines.This review sorted out and briefly interpreted the main contents of clinical management guidelines for neonatal hyperbilirubinemia drafted by the American Academy of Pediatrics and other countries, aiming to provide references of clinical diagnosis and treatment practice to domestic pediatrician.

Determination of tromethamine residues in human coagulation factor Ⅷ by high performance liquid chromatography method / 中国输血杂志

【Objective】 To develop and verify high performance liquid chromatography (HPLC) detection for tromethamine (Tris) residues in human coagulation factor Ⅷ. 【Methods】 Alanine was used as internal standard, and AQC for pre-column derivation. Inertsil® ODS-SP was adopted, and acetic acid-sodium acetate buffer and acetonitrile were used for gradient elution at the flow rate of 1 mL/min and column temperature of 37℃. The 25 μL sample was loaded and determined by UV-detector with detection wavelength at 248 nm. This method was then verified. 【Results】 Glycine, sodium citrate and calcium chloride showed no interference with the detection of tromethamine (Tris) residues. The recovery rate of spike samples was within 90.0%~100.8%. The RSD in repeatability test were 2.5%, 0.7% and 1.2%, respectively, and in intermediate precision test 0.8%. The tromethamine (Tris) at concentrates of (0.5~4.0)μg/mL showed good linear relationship to the peak area to internal standard, of which the regression equation was Y=0.012 9X-0.000 782, R=0.999 97.The quantitative detection limit was 0.04μg/mL. 【Conclusion】 The HPLC for determination of tromethamine (Tris) residues in human coagulation factor Ⅷ was successfully developed, which showed good linearity as well as high specificity, precision and accuracy.

Establishment of kinetic detection method for PKA content / 中国输血杂志

【Objective】 To develop and verify a kinetic method for the determination of prekallikrein activator (PKA) content. 【Methods】 The optimal reaction conditions were determined by comparing the factors of pH and ionic strength of different sample dilution buffers, incubation time of each procedure, and incubation temperature. The accuracy, specificity, precision, linearity, stability and durability of the method were validated. 【Results】 The sample was diluted with 0.05 mol/L Tris-HCl buffer (pH8.5, containing 0.15 mol/L NaCl) and incubated by prekallikrein (PK) at 37℃ for 20 min. After that, the substrate S-2302 was added. Within 10 min before the measurement, the absorbance change rate reached △A405/min. The validation results indicated that the linear range of the method was (0.5~4.0)IU/mL, while the recovery of calibration standard was 96.9%~103.7% with the R2 value more than 0.99. The specificity test showed that human serum albumin, excipients of intravenous human immunoglobulin (pH4), low pH and protein content had no significant effect on the detection of PKA, The recovery rates of standard sample solution in the specificity experiment were 98.0% (0.9% sodium chloride solution), 95.3% (0.46% sodium caprylate solution), 96.7% (10% maltose solution, pH4.0), 94.0%(20%BSA), and 94.0%(5%BSA, pH4.0), respectively. The accuracy and precision of the method can meet requirements in the range between 0.5 and 4.0 IU/mL. The inter-batch recovery rate of quality control samples were between 96.4%~109.5% with the coefficients of variation(CV) between 0.2%~6.9%, while the intra-batch recovery rate were between 101.5%~102.9% with the CV between 2.6%~5.9%. The linearity, accuracy and precision of the assay can meet the requirements when PK and S-2302 were placed at room temperature for less than 6 hours, with the recovery rate of quality control samples between 94.9%~109.9%. The end-point method and kinetic method were used to determine the PKA in 20 batches of human serum albumin, and the consistency showed that there was no significant difference between the two methods(P>0.05). 【Conclusion】 A kinetic method for determination of PKA content with good linearity, specificity, accuracy, precision, stability and durability has been established. Compared with the method in ChP, the new method is more convenient, accurate and rapid to determine the content of PKA in human albumin and human immunoglobulin (pH4) for intravenous injection.

Degradation kinetics of larotaxel and identification of its degradation products in alkaline condition / 药物分析学报

Larotaxel, a new taxane compound prepared by partial synthesis from 10-deacetyl baccatin Ⅲ, is active against tumors. In this research, a selective LC–MS method was developed and validated for the study of degradation kinetics of larotaxel, which was carried out in aqueous solutions with different pH (1.5, 3.0, 5.0, 6.5, 7.4, 9.0, 10 and 11.0) and temperature (0, 25, 37 and 45 °C). The linear range was 0.5–25μg/mL, the intra-and inter-day precisions were less than 7.0%, and accuracy ranged from 97.4–104.5% for each analyte. The observed rate obtained by measuring the remaining intact larotaxel was shown to follow first-order kinetics. The activation energies for degradation were 126.7 and 87.01 kJ/mol at pH 1.5 and 11, respectively. Although larotaxel was stable in pH 5, 6.5 and 7.4 buffers at 37 °C for 24 h during our study, increasing or decreasing the pH of the solutions would decrease its stabilities. Moreover, three main degradation products in alkaline condition were separated by HPLC and identified by Q–TOF–MS. The three degradation products were confirmed as 10-deacetyl larotaxel, 7, 8-cyclopropyl baccatin Ⅲ and 10-deacetyl-7, 8-cyclopropyl baccatin Ⅲ.

Effects of intravenous infusion of bilirubin on the expression of myeloid differentiation factor 88 and in-terleukin-1 receptor associated kinase-4 in splenocytes with neonatal rats / 中国小儿急救医学

Objective To explore the effects of bilirubin on myeloid differentiation factor 88 (MyD88)and interleukin-1 receptor associated kinase-4(IRAK-4).Methods Seven-day-old Sprague Daw-ley rats (clean grade),male or female,weighing 12.0 to 15.0 g,were randomly assigned to 6 groups.There were normal saline group(Ⅰ),lipopolysaccharide(LPS)control group (LPS,Ⅱ),15 mg /kg bilirubin con-trol (free-LPS)group (Ⅲ),15 mg /kg group (Ⅳa),30 mg /kg group (Ⅳb)and 50 mg /kg group (Ⅳc), and then subsequently divided into 2 h,5 h and 24 h subgroups in each groups.Some of the 200 newborn rats died amid the experiment.Finally a total of 144 were involved in the analysis of results,and 8 rats in each subgroups.Newborn Sprague Dawley rats were administered at various doses of bilirubin (15 mg /kg, 30 mg /kg and 50 mg /kg respectively)intravenously;1 h after injection,the rats were administered LPS intrap-eritoneally at a dose of 1 mg /kg;MyD88 and IRAK-4 were detected by immunohistochemistry at 2 h,5 h and 24 h after the injection of bilirubin.Results (1 )LPS could stimulate the expression of MyD88 and IRAK-4 in spleen cells (qMyD88 2 h =49.89,qMyD88 5 h =139.54,qIRAK-4 2 h =7.93,qIRAK-4 5 h =24.30,qIRAK-4 24 h =6.97 ,P 0.05).Effects of medium and high concentration of bilirubin on LPS stim-ulation MyD88 were inhibitory(qⅣb 2 h =42.87,qⅣc 2 h =51.38,qⅣb 5 h =103.61 ,qⅣc 5 h =1 15.44,qⅣb 24 h =1.18,qⅣc 24 h =1 1.66,P <0.01 ).(4)Effects of low,medium and high concentration of bilirubin on LPS stimulation IRAK-4 were inhibitory(qⅣa 2 h =9.52,qⅣb 2 h =14.39,qⅣc 2 h =25.55,qⅣa 5 h =38.83,qⅣb 5 h =54.62,qⅣc 5 h =60.51 ,qⅣa 24 h =2.41 ,qⅣb 24 h =1.47,qⅣc 24 h =7.61 ,P <0.01 ).(5)The inhibition of biliru-bin to MyD88 and IRAK-4 was observed at 2 h,strengthened at 5 h,disappeared at 24 h in low-mid concen-trations of bilirubin(P <0.01 )while still visible at 24 h in high concentration of bilirubin.(6)There was neg-atively correlation between the expression level of MyD88,IRAK-4 and bilirubin concentration(rsMyD88 2 h =-0.86, rsMyD88 5 h =-0.92,rsMyD88 24 h =-0.53,rsIRAK-4 2 h =-0.82,rsIRAK-4 5 h =-0.86,rsIRAK-4 24 h =-0.57,P <0.01).(7) Under the effect of bilirubin and LPS,there were positively correlation between the expression levels of MyD88 and IRAK-4 of spleen cells(r2 h =0.77,r5 h =0.9,r24 h =0.67,P <0.01).Conclusion Bilirubin could inhibit the expression of MyD88 and IRAK-4.As the concentration of bilirubin increasing,its inhibition is more obvious and prolonged.The mechanism that bilirubin affects immune function of newborn rat may be related to regulation of expression of MyD88 and IRAK-4 at toll-like receptor 4 signal pathway.

Effects of intravenous infusion of bilirubin on the expression of phospho-p38 mitogen-activated protein kinase and the apoptosis in splenocytes of neonatal rats / 中华实用儿科临床杂志

Objective To explore the effects of bilirubin on myeloid differentiation factor phospho-p38 mitogen-activated protein kinase (p-p38MAPK) and apoptosis in splenocytes of neonatal rats.Methods Seven-day-old Sprague Dawley rats (clean grade),male or female,weighting 12.0-15.0 g,were randomly assigned to 6 groups.There were blank control group (Ⅰ),lipopolysaccharide (LPS) control group (Ⅱ),15 mg/kg bilirubin control (free-LPS) group (Ⅲ),15 mg/kg group (Ⅳa),30 mg/kg group (Ⅳb) and 50 mg/kg group (Ⅳc),and then subsequently divided into 2 h,5 h and 24 h subgroups in each groups.Some of the 200 newborn rats died amid the experiment,tinally,a total of 144 cases were involved in the analysis of results,and 8 rats in each subgroups.Newborn Sprague Dawley rats were administered at various doses of bilirubin (15 mg/kg,30 mg/kg and 50 mg/kg,respectively) intravenously; 1 h after injection,the rats were administered LPS intraperitoneally at a dose of 1 mg/kg;p-p38MAPK were detected by immunohistochemistry;Apoptosis in splenocytes was detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling methods at 2 h 5 h and 24 h after the injection of bilirubin.Results 1.Expression of p-p38MAPK in each group:bilirubin in low-mid concentrations of range inhibited LPS-induced p38MAPK activation (qⅣa =20.93,10.37,respectively at 2 h,and 5 h,all P ＜ 0.01 ;qⅣ b =79.97,14.79,all P ＜ 0.01).The inhibition strengthened with increasing concentration of bilirubin.The effect was observed at 2 h,strengthened at 5 h,disappeared at 24 h.Bilirubin in the high concentrations of range stimulated the expression of p-p38MAPK (qⅣc =32.55,19.23,27.72,respectively at 2 h,5 h and 24 h,all P ＜0.01),observed at 5 h,reduced at 24 h.2.Effects of bilirubin on apoptosis in splenocytes:LPS could increased the apoptosis index (AI) of splenocytes(q =54.62,P ＜ 0.01);The AI of splenocytes had no significant change in low concentrations of range of bilirubin (q =43.92,P ＞ 0.05).Low-mid concentration of bilirubin with LPS reduced the AI of splenocytes (q Ⅳ a =4.48,P ＜ 0.01 ;q Ⅳ b =2.07,P ＜ 0.05),while high concentration of bilirubin with LPS increased the AI of splenocytes (q =5.08,P ＜ 0.01).Conclusions Bilirubin in low-mid concentrations of range could inhibit the expression of LPS-induced p38MAPK,while bilirubin in high concentrations of range stimulated the expression.As the concentration of bilirubin elevated,its inhibition was prolonged.Bilirubin in high concentrations of range bilirubin could induce apoptosis in splenocytes.The immune dysfunction in neonatal hyperbilirubinemia may have something to do with the regulation of phosphorylation of p38MAPK and activation of apoptotic pathways.