高效液相色谱法检测早期胃癌中胃液固有荧光的诊断价值

高效液相色谱法检测早期胃癌中胃液固有荧光的诊断价值,研究背景,1995年以来, 北京大学第三医院消化疾病研究中心对胃液荧光光谱在胃癌诊断中的应用进行了一系列临床及人群前瞻性双盲研究, 结果显示该方法诊断胃癌的敏感性为81.5%, 特异性为71.7%, 但对于早期胃癌的诊断仍不十分理想。,研究目的,研究高效液相色谱法检测早期胃癌中胃液固有荧光光谱的诊断价值。,研究方法,选择2004-08/2008-03在北京大学第三医院胃镜检查患者的胃液,采用MonoQ离子交换高效液相色谱分析方法检测胃液固有荧光,比较早期胃癌与胃良性疾病间的荧光峰差异并进行统计分析。,研究结果,胃液在MonoQ离子交换高效液相色谱条件下于保留时间30-32min之间规律出现一个强度最大的荧光峰,早期胃癌组均值明显高于胃良性病变组(P0.001)。,图 1 M o n o Q HPLC胃液固有荧光. A : 胃良性病变; B: 早期胃癌.,良恶性病变胃液荧光光谱峰值的比较,研究结果,以荧光强度0.12V为判别界值,诊断早期胃癌的敏感度是91.2%、特异度是87.8%。,结论,高效液相色谱法检测胃液固有荧光用于诊断早期胃癌具有较高的敏感性和特异性。,张月宁, 金新莲, 孟灵梅等. 高效液相色谱法检测早期胃癌中胃液固有荧光的诊断价值. 世界华人消化杂志. 2010. (21): 2262-2265.,High levels of aromatic amino acids in gastric juice during the early stages of gastric cancer progression,BACKGROUND,Early-stage gastric cancer is mostly asymptomatic and can easily be missed easily by conventional gastroscopy. Currently, there are no useful biomarkers for the early detection of gastric cancer, and their identification of biomarkers is urgently needed.,研究背景,早期胃癌通常没有症状，常规胃镜检查很容易漏诊。目前，无有效的早期胃癌检测生物标志物，迫切需要识别及鉴定有用的生物标志物。,METHODS,Gastric juice was obtained from 185 subjects that were divided into three groups: non-neoplastic gastric disease (NGD), advanced gastric cancer （AGC）and early gastric cancer (EGC). The levels of aromatic amino acids in the gastric juice were quantitated using high-performance liquid chromatography (HPLC).,研究方法,185例胃液标本被分为三个组：非恶性胃病（NGD）组，进展期胃癌（AGC）组和早期胃癌（EGC）组。使用高效液相色谱法测定胃液中芳香族氨基酸的含量。,RESULTS,The median values (25th to 75th percentile) of tyrosine, phenylalanine and tryptophan in the gastric juice were 3.8 (1.7-7.5) g/ml, 5.3 (2.3-9.9) g/ml and 1.0 (0.4-2.8) g/ml in NGD; 19.4 (5.8-72.4) g/ml, 24.6 (11.5-73.7) g/ml and 8.3 (2.1-28.0) g/ml in EGC.,研究结果,非恶性胃病(NGD)组胃液中酪氨酸、苯丙氨酸和色氨酸含量的平均值（四分位数间距）分别为3.8 (1.7-7.5) g/ml, 5.3 (2.3-9.9) g/ml 和1.0 (0.4-2.8) g/ml。早期胃癌(EGC)组分别为19.4 (5.8-72.4) g/ml, 24.6 (11.5-73.7) g/ml 和8.3 (2.1-28.0) g/ml。,Figure 1. Fluorescence chromatograms of aromatic amino acids in gastric juice. (A) NGD, (B) AGC and (C) EGC group. Tyr, tyrosine; Phe,phenylalanine; Trp, tryptophan.,RESULTS,Higher levels of tyrosine, phenylalanine and tryptophan in the gastric juice were observed in individuals of EGC group compared those of the NGD group (NGD vs. EGC, P0.0001).,研究结果,与非恶性胃病组相比，早期胃癌组胃液中酪氨酸、苯丙氨酸和色氨酸的含量更高(P0.0001)。,Figure 2. Scatter plot showing the levels of tyrosine, phenylalanine, tryptophan and total protein in gastric juice from NGD, AGC and EGC groups. 图2（a）散点图显示良性、进展期和早期恶性病变组患者胃液中酪氨酸、苯丙氨酸、色氨酸和总蛋白值。,RESULTS,For the detection of EGC, the areas under the receiver operating characteristic curves (AUCs) of each biomarker were as follows: tyrosine, 0.790 95% confidence interval (CI), 0.703-0.877; phenylalanine, 0.831 (95% CI, 0.750-0.911); and tryptophan, 0.819 (95% CI, 0.739-0.900).,研究结果,各生物标记物检测早期胃癌的受试者工作特征曲线下面积(AUC)如下：酪氨酸,0.790(95%可信区间,0.703 - -0.877)；苯丙氨酸,0.831(95%可信区间,0.750 - -0.911)；色氨酸,0.819(95%可信区间,0.739 - -0.900)。,Figure 3. Predictive ability of gastric juice analysis for gastric cancer. ROC curves of tyrosine, phenylalanine, tryptophan and protein in gastric juice for detection of (A) AGC. (B) EGC. 图3 使用酪氨酸(Tyr),苯丙氨酸(Phe),色氨酸(Trp)和总蛋白绘制的受试者工作特征（ROC）曲线用于检测进展期和早期胃癌。,RESULTS,The sensitivity and specificity of phenylalanine were 75.5% and 81.4%, respectively, for detection of EGC. A multiple logistic regression analysis showed that high levels of aromatic amino acids in the gastric juice were associated with gastric cancer (adjusted coefficients ranged from 1.801 to 4.414, P0.001).,研究结果,苯丙氨酸用于早期胃癌检测的敏感性和特异性分别为75.5%和81.4%。多元逻辑回归分析表明，胃液中芳香族氨基酸的含量较高与胃癌相关(系数调整范围从1.801到4.414, P 0.001)。,DISCUSSIONS,The enhanced production of aromatic amino acids in EGC corresponds to the results for a previous study and is consistent with early reports in other malignant diseases (blood, lung, breast, bladder, etc.). We speculate that the following reasons may account for this phenomenon.,讨论,早期胃癌患者芳香族氨基酸的产生增多与我们之前研究的结果一致，这种现象在早期报告的其他恶性疾病(血液,肺,乳房,膀胱,等等)中也存在。 我们推测，以下原因可以解释这一现象：,DISCUSSIONS,First, tumour cells require sufficient aromatic amino acids to accumulate in the cancer foci to meet the sharply increased needs for protein synthesis to support rapid growth.A high expression of amino acids transporters (such as L-type amino acid transporter 1) in various tumours has been reported in many studies.,讨论,首先,肿瘤细胞需要在癌症病灶周围积累足够的芳香族氨基酸，为满足急剧增加的蛋白质合成的需求，来支持快速生长。氨基酸转运蛋白(如I型氨基酸转运蛋白)在许多的肿瘤中的高表达已有报道。,DISCUSSIONS,Amino acid transporters may enhance the transport of amino acids and accumulate aromatic amino acids near cancer foci.The infiltration of excessive aromatic amino acids may significantly increase in gastric juice in EGC compared to NGD.,讨论,氨基酸转运蛋白可能会加强氨基酸的转运和芳香族氨基酸在癌灶周围的积聚。芳香族氨基酸的过度浸润可能会使其在早期胃癌患者胃液中的含量显著多于良性病变者。,DISCUSSIONS,Secondly, aberrant metabolism in cancer cells would precede the visible changes in stomach carcinogenesis.It is known that the abnormalities of amino acid transport and metabolism in a cancerous region occur frequently in the early stage of gastric cancer.,讨论,其次,肿瘤细胞的代谢异常会先于胃癌形成过程中可见的形态学变化。众所周知,癌变部位氨基酸转运和代谢异常经常发生在胃癌的早期阶段。,DISCUSSIONS,Gastric juice, which is generated by the gastric epithelium, contains a substantial amount of information about the metabolic status of the epithelium.Therefore, the information obtained from analysing the gastric juice may be useful in separating biomarkers for the early detection of gastric cancer.,讨论,胃上皮细胞产生的胃液中含有大量的反应上皮细胞的代谢状态的信息。因此，从分析胃液所获得的信息可能有助于分离出胃癌的早期检测的生物标志物。,DISCUSSIONS,Finally, matrix metalloproteinases (MMPs), which are enzymes that degrade the extracellular matrix and basement membrane, play roles in the early phase of tumourigenesis.The over-expression and activity of MMPs accelerates the degradation of extracellular matrix and basement membrane.,DISCUSSIONS,The degradation of matrix generates aromatic amino acids in the gastric juice surrounding tumour cells.Additionally, blood aromatic amino acids could easily penetrate the impaired basement membrane and epithelia of carcinoma foci and enhance amino acid levels in gastric juice.,讨论,最后，金属基质蛋白酶(MMPs)能降解细胞外基质和基底膜，在肿瘤形成的早期阶段扮演主要角色。金属基质蛋白酶的过表达和活性增加会加速细胞外基质和基底膜的降解。,讨论,胃液中肿瘤细胞周围细胞外基质的降解会生成芳香族氨基酸。此外,血液中的芳香族氨基酸可以轻易地穿透受损的基底膜和癌灶的上皮细胞,使胃液氨基酸的水平增高。,DISCUSSIONS,These causes c