Simultaneously Measurement Of Neopterin And Biopterin In Urine And Serum By High Performance Liquid Chromatography

Objective:To?establish?a?method?to?detect neopterin (NP) and? biopterin(BP) in?urine and serum simultaneously.To detect the concentrations of NP and BP in clinical serum and urine samples by HPLC and to assist diagnosis of diseases.Methods: 1.Urine?and serum samples?were?first precipitated?with trichloro- acetic?acid, following centrifuged?at?12000 rpm? for?15?min　at 4oC ,then?added NaOH-phosphate buffer to neutralize the?supernatant, injection volume:30μL.2.NP and BP in urine and serum were detected by RP-HPLC－FD? , Conditions: mobile?phase?:methanol/water was 10/90(v/v); column: Hypersil BDS?C18 (i.d?4.6×200mm?5μm),Lichrospher C18 (i.d 4.6×250mm 5μm);flow?rate:0.5ml/min,?column temperature:20℃,detection wavelength: Excitation wavelength 360nm;Emission wavelenfth 440nm. 3. The concentrations of NP and BP in serum and urine were detected by RP-HPLC-FD and the concentrations of Cr in urine were determined by Automatic Biochemistry Analyzer(Olympus AU600)Results:?1. The detection limitation?of standards NP?and?BP?were?0.01μg/L and 0.1μg/L respectively.The?precisions(RSD)of?NP?andBP?in?urine?were?09%?and 3.39%,The?regression?equations?of?NP?and BP?in?urine were Y(NP)?=?1.1952x?+?9.1557?(r2?=?0.9997)and Y(BP) = 1.2276x +3.244(r2 = 0.9997)?.The?average?recoveries of NP?and BP in?urine were?101.3% and 98.6%,. In?serum the regression equations of NP and BP were Y(NP)=1.0255x-0.0077 (r2=0.9998);Y(BP)=0.7027x+0.0394 (r2=0.999),respectively. The precisions (RSD) of NP and BP in serum were 2.87% and 3.96% and the?average?recoveries of NP?and BP in?serum were?97.9% and 94.3% .2.The normal serum concentrations of NP and BP were 5.76±2.44ug/L and 1.81±0.57μg/L, respectively.In controls, the concentrations of NP and BP in adult (age>50)serum and the ratios of NP/Cr and BP/Cr in adult(age>50) urine were significantly higher than that in youngers'(age<50) (P<0.05).but there is no significant variance of the ratio of NP/BP between the adult and the youngers.None of these variations could be observed between man and woman(P>0.05). But The AMI's serum concentrations of NP and BP were 12.96±6.95μg/L and 3.36±1.73μg/L .The serum concentrations of NP and BP in AMI group increased significantly more than that in normal group (P<0.01). The concentrations of NP and BP in serum and ratios of NP/BP and NP/Cr and NP/BP in urine were higher for uremia patients (n＝12) than that for controls(n=30)(P<0.05),but there was no significant variance of the ratio of BP/Cr in urine between uremia group and controls.The concentrations of NP and BP in uremia patients' serum increased more significantly before dialysis than that after dialysis. The results indicates that the NP and BP in serum can be used as the markers for dysfunction of kidey.Conclusion: 1. A? simple, fast, accurate, ?sensitive, reliable and convenient ?method?for?the?detection?of NP?and?BP?in?urine?and serum was?established,?It?could?be?used?to detect ?NP?and?BP ?for?clinical?urine?and serum?samples?and?to provide?a?more?convenient?and accurate?tool for the diagnosis?of?patients.2. By measurment of NP and BP in urine and serum with AMI and uremia patients , the concentrations of NP not only was a warning- factor in advance for AMI,but also was a marker for uremia cell immune and to assess the effect of blood dialysis .