建立HPLC法检测血清尿刊酸并探讨其对儿童白血病的意义

Establishing a HPLC method to detect urocanic acid in serum and investigate its significance for childhood leukemia

目的建立利用高效液相色谱法(HPLC)检测血清尿刊酸(UCA)的方法,探讨血清UCA浓度对儿童急性白血病的临床意义。方法建立HPLC测定血清UCA浓度的方法,设定与优化HPLC色谱条件并进行验证。收集确诊为急性初诊白血病和非肿瘤血液疾病患者各90例的血清,检测UCA浓度,比较两组间的差异,并探究UCA浓度在白血病中的临床意义。结果成功优化并建立HPLC检测血清UCA方法,流动相乙腈:20 mmol/L KH2PO4,p H3.7为5∶95(V/V),流速为1.2 m L/min,检测波长264 nm的HPLC色谱条件下,反式尿刊酸(trans-UCA)和顺式尿刊酸(cis-UCA)浓度在0.125 g/L^5 g/L范围内与洗脱峰面积呈良好线性关系(R2分别为0.999 6和0.999 9)。批间测量和批内测量的相对标准偏差(RSD%)<5%。与对照组相比,急性白血病患儿血清中cis-UCA和trans-UCA浓度明显增高(P<0.001);与cis-UCA相比,trans-UCA对急性淋巴细胞白血病(ALL)的危险度分级诊断更有价值。结论 HPLC方法可以测定血清UCA浓度,儿童急性白血病血清中的UCA浓度可为诊断与预后提供线索,具有重要临床意义。

Objective To establish a method for detection of serum urocanic acid （UCA） by high performance liq- uid chromatography （HPLC） , and explore the clinical significance of serum UCA concentration for children acute leukemia. Methods The chromatographic conditions of HPLC were set up and optimized, and the linearity of standard curve, precision, accuracy and stability were validated.Then the serum from ninety acute leukemia children and ninety non-tumor blood disease children was collected, the concentration of serum UCA was detected with HPLC ,and the differences of two groups were compared to study the clinical significance of UCA in children acute leukemia.Results The HPLC method for detecting serum UCA was successfully established and optimized. The standard curves of trans-UCA and cis-UCA both showed good linearities （R2 = 0. 999 6 and 0. 999 9） at the condi- tion of the mobile phase of acetonitrile-20 mmol/L KHzPO4, pH 3.7 （5 ： 95, V/V）, flow rate of 1.2 mL/min, de- tection wavelength of 264 nm in HPLC. The relative standard deviation RSD% of intra-assay and inter-assay were lower than 5%. Compared with non-tumor blood disease, the serum concentration of cis urocanic acid （cis-UCA） and trans urocanic acid （trans-UCA） of children with acute leukemia were significantly increased （P〈0. 001 ）. Compared with cis-UCA, trans-UCA was more valuable for risk classification of acute lymphoblastic leukemia （ALL）. Conclusions HPLC is a good technology to titrate of UCA in serum. The concentration of serum UCA in children with acute leukemia may provide the clues for diagnosis and prognosis, with important clinical significance.