血浆L-瓜氨酸和L-精氨酸浓度分析方法建立及应用

目的 建立反相高效液相色谱(RP-HPLC)-紫外检测法同时测定血浆L-瓜氨酸和L-精氨酸浓度的方法并应用于临床。方法 采用三氯乙酸结合加热法除血浆蛋白,用异硫氰酸苯酯(PITC)进行柱前衍生化反应,梯度洗脱,柱温46℃,波长254 nm,进样20 μl,流速1.0 ml/min; 通过分析21例消化道晚期恶性肿瘤病人、21例非恶性肿瘤病人和39例健康人的空腹血浆L-瓜氨酸和L-精氨酸浓度,验证方法的可行性。结果 两种氨基酸的峰面积和浓度之间呈线性关系,线性范围从0～1 000 μmol/L,两者r=0.999 95。L-瓜氨酸和L-精氨酸的定量下限分别为0.240 μmol/L和0.448 μmol/L。日内和日间变异系数(CVs)分别小于3.4%和7.2%,平均回收率在95.2%～104.1%。血浆L-瓜氨酸/L-精氨酸的比值均值,消化系统肿瘤病人组高于年龄性别相匹配的健康对照组,差异有统计学意义(t检验,P=0.009); 两种氨基酸比值诊断消化系统恶性肿瘤的敏感度和特异度分别为81%和71.4%。结论 这一新方法可用于临床测定血浆L-瓜氨酸/和L-精氨酸浓度; 与浓度相比,血浆两种氨基酸浓度比值也许是评估消化系统恶性肿瘤更加敏感的指标。

Establishment and Clinical Application of the Analytical Method for Plasma L-citrulline and L-arginine Concentration

Objective To establish a method for simultaneously measuring L-citrulline and L-arginine concentration in plasma using reversed-phase high performance liquid chromatography(RP-HPLC)with ultraviolet detection,and the novel method is applied to the patients with digestive system tumor.Methods Plasma samples were deproteinized by trichloroacetic acid with heating method.Phenylisothiocyanate(PITC)was used as derivatization reagent and a gradient elution was adopted.The feasibility verification of method was carried out by detecting plasma L-citrulline and L-arginine acid concentration in 21 cases of advanced digestive tract malignant tumor patients,21 cases of non malignant tumor patients and 39 cases of healthy people.Results The linearity for L-citrulline and L-arginine ranged from 0 to at least 1 000 μmol/L.r=0.999 95 for both.The lower limitsof quantification for L-citrulline and L-arginine were 0.240 μmol/L and 0.448 μmol/L respectively.The intraday and interday coefficients of variation(CVs)were less than 3.4% and 7.2%,respectively.The average recovery ratewas from 95.2% to 104.1%.The ratio of plasma L-citrulline /L-arginine in patients with digestive cancar was significantly higher than that in the healthycontrol group,t-test,P=0.009.The sensitivity and specificity of plasma L-citrulline/L-arginine ratio in the malignant tumor of digestive systemwere 81% and 71.4% respectively.Conclusion This methodcan provide a reliable and efficient method for the clinical determination of the L-citrulline and L-arginine concentration in plasma.The ratio of plasma citrulline/arginine increased obviously in patients with digestive cancer,suggesting that this ratio is a more sensitive index than single concentration of L-citrulline or L-arginine when evaluating patients with digestive tumor.