LC-MS/MS Method for the Determination of the contents of KDN, Neu5Ac and Neu5Gc in Different Tumor Tissues
- 环境生态－学位论文 
唾液酸(Sialic acid)是一类带羧基的九碳糖化合物酰基衍生物的通称。它存在于细菌、鱼类、哺乳动物等多种生物体内，参与并调节许多重要的生命活动如细胞识别、生物膜流动和细胞内吞作用等。现已知的唾液酸成员近50 多种，包括N-乙酰基神经氨酸(N-acetylneuraminic acid，Neu5Ac)、N-羟乙酰基神经氨酸(N- glycoulylneura minic acid，Neu5Gc) 和脱氨神经氨酸(2-keto-3-deoxy-D-glycero- D-galacto-nononic acid，KDN)三种核心单体，其余的唾液酸均由这三种单体衍生而来。1986年，Inoue等人在虹鳟鱼卵中首次分离出KDN。KDN普遍存在于哺乳动物、低等脊椎动物和细菌中，在低等脊椎动物和细菌中的表达量很高，而在哺乳动物中的表达量却很低。 近几年，有研究报道，在卵巢肿瘤组织中游离KDN的含量明显比正常卵巢中的要高，并且游离KDN的含量会随着肿瘤恶性程度的增高呈正相关增长，KDN在卵巢癌组织中高表达暗示了KDN很可能是肿瘤发生时的“早期预警信号”或是恶性程度的预测指标，其在肿瘤中的表达及其与肿瘤的相关性研究逐步增多。以往相关研究主要采用荧光HPLC检测手段来分析组织中唾液酸的含量，而本文采用更为快捷、精确及敏感的LC-MS/MS的分析方法测定肿瘤组织中KDN，Neu5Ac和Neu5Gc的含量，主要研究包括： 1. 确定了HPLC-MS/MS法分析组织中唾液酸的色谱及检测器条件:分析物采用C18柱分离，以乙腈-水(含0.1%的醋酸铵)为流动相进行梯度洗脱，负离子质谱检测，多反应监测模式(MRM)定性定量测定。 2. 用建立的分析方法对60例喉癌、42例乳腺癌、8例卵巢癌、34例肺癌、6例子宫癌、6例宫颈癌和7例肝癌患者癌组织及淋巴结组织中的KDN， Neu5Ac和Neu5Gc的含量进行测定，分析它们在这些组织中表达的特点和差异，探讨KDN，Neu5Ac和Neu5Gc的含量与肿瘤组织各种临床指标的关系，及其在肿瘤进程中的作用，以及作为喉癌、乳腺癌、肺癌、卵巢癌、子宫癌、宫颈癌及肝癌临床诊断标志物的可能性。 在上述质谱联用条件下，KDN，Neu5Ac和Neu5Gc可以达到基线分离，KDN，Neu5Ac和Neu5Gc分别在0.005-10 mol/L、0.05-100mol/L、0.05-2 mol/L浓度范围内线性良好，相关系数(r2)均大于0.992，方法的检出限均在0.002-0.003mol/L范围内，日内日间的相对标准偏差(RSD)均小于6%，加标回收率为93.6%-102.4%。符合组织样品处理得真实度和精密度。 在人类癌组织及淋巴结组织中Neu5Ac是含量最高的单唾液酸，其次是KDN，Neu5Gc的含量很低。我们发现组织中Neu5Ac主要以结合形式存在， KDN则主要以游离形式存在，而Neu5Gc在喉癌中主要以结合形式存在，在肺癌、子宫癌、宫颈癌及肝癌则主要以游离形式存在。 不同肿瘤中，KDN，Neu5Ac和Neu5Gc的含量有差别。同时，癌组织中KDN， Neu5Ac和Neu5Gc的含量远比淋巴结组织中的多。 在喉癌中，癌组织中KDN含量与淋巴结中KDN含量具有相关性，P＜0.01。中低分化癌组织和淋巴结中KDN和Neu5Gc的含量要远远高于高分化癌组织和淋巴结中KDN的含量，因此我们推测KDN的表达与喉癌的恶性潜能呈正相关，是判断喉癌恶性程度的肿瘤标志物。 在乳腺癌与肺癌中，癌组织及淋巴结组织中KDN和Neu5Ac的含量与淋巴结是否转移有关，因此KDN和Neu5Ac可能是检测淋巴结是否转移的临床诊断指标。这些新发现强调了KDN在今后研究中的重要性。 在子宫癌、宫颈癌及肝癌组织中，KDN，Neu5Ac和Neu5Gc均有表达。我们发现在卵巢癌及子宫癌中存在随着肿瘤分化程度的增高，KDN表达水平下降的趋势；而在宫颈癌及肝癌中存在随着肿瘤分化程度的增高，KDN表达水平上升的趋势，但由于各组织样本量太少，无法进行统计学分析，有待于进一步研究探讨。Sialic acid (Sia) is a family of nine-carbon sugar compounds with a carboxylic acyl derivatives. It exists in bacteria, fish, mammals and other living organisms, participates in and regulates many important life events, such as cell recognition, membrane flow, endocytosis and so on. Sialic acid is usually located in the outermost layer of the sugar part of the cell membrane and the key positions of secreted glycoconjugates(glycolipids, glycoprotein and lipopolysaccharide). Sia is a important material foundation for variety of the structure and founction of glycoconjugates. Sialic acid has been known nearly 50 members, including N-acetylneuraminic acid(Neu5Ac), N-glycoulylneuraminic acid(Neu5Gc) and 2-keto-3-deoxy-D-glycero- D-galacto-nononic acid (KDN) as its core monomer. The rest of the sialic acids are derived from these three monomers. In 1986, Inoue et al discovered the occurrence of KDN in rainbow trout eggs for the first time. KDN is ubiquitous in mammals, lower vertebrates and bacteria. Its expression is very high in lower vertebrates and bacteria, but its expression is very low in mammals. In recent years, the studies have reported an increased level of free KDN in human ovarian tumors and in red blood cells during the early stages of hematopoiesis. This finding suggested that KDN may be an oncofetal antigen in ovarian cancer/or a marker for detection of recurrence of disease. To follow up this unexpected finding, we sought to determine if the elevated levels of free KDN was unique to ovarian cancer, or occurred in other human cancers. Previous studies mainly used fluorescence HPLC detection methods to analyze the content of sialic acid in tissues. And in this thesis we use a more efficient, accurate and sensitive method called LC-MS/MS to determine the content of KDN, Neu5Ac and Neu5Gc in tumor tissues. The main studies include: 1. The chromatography and detector conditions of HPLC-MS/MS to analyze the sialic acid in tissues: use C18 separate, acetonitrile-water(containing 0.1% ammonium acetate) as mobile phase for gradient elution, negative ion mass spectrometry, multiple reaction monitoring mode (MRM) qualitative and quantitative determination. 2. Use this analytical method to analyze the content of KDN, Neu5Ac and Neu5Gc in tumor and lymph node tissues of 60 cases throat cancer, 42 cases breast cancer, 8 cases ovarian cancer, 34 cases lung cancer, 6 cases uterine cancer, 6 cases cervical cancer and 7 cases liver cancer, analyze the characteristics and differences of them in these tissues. Investigate the relationship between the content of KDN, Neu5Ac and Neu5Gc and various clinical indicators, as well as the clinical diagnosis possibilities of throat cancer, breast cancer, lung cancer, ovarian cancer, cervical cancer, uterine cancer and liver cancer. The linear range of KDN was 0.005-10 mol/L, Neu5Ac was 0.05-100mol/L and Neu5Gc was 0.05-2 mol/L. Correlation coefficient(r2) are more than 0.992. The lowest quantification was between 0.002 to 0.003mol/L. The method recovery is 93.6%-102.4%. The intra-day and inter-day RSD less than 6%. Meet the validity and precision of tissue samples treatment. Neu5Ac is the highest content of sialic acid in human tumor and lymph node tissues, followed by KDN, the content of Neu5Gc is very low. And we also found Neu5Ac mainly existed as the bound form in tissues, KDN mainly existed as the free form, but Neu5Gc mainly existed as the bound form in throat tumor and mainly as free form in lung tumor, uterine tumor, cervical tumor and liver tumor. The contents of KDN, Neu5Ac and Neu5Gc are different in different tumors. Meanwhile, the contents of KDN, Neu5Ac and Neu5Gc in tumor are much higher than that of lymph node tissues. In throat cancer, correlation is significant between KDN in tumors and matched lymph nodes of cancer patients, P＜0.01. The level of KDN and Neu5Gc in tumor and lymph node which is poorly- moderately- differentiated is much higher than the one which is well- differentiated. It suggesting that the content of KDN was positively correlated with the malignant potential of throat cancer and maybe KDN is a warning signal to determine the degree of maligancy of throat cancer. In breast and lung cancer, there is relationship between the content of KDN and Neu5Ac in tumor and lymph node tissues and the lymph node metastasis. Therefore, KDN and Neu5Ac maybe the clinical diagnostic criteria for detecting lymph node metastasis. These new findings highlight the importance of KDN research in the future. KDN, Neu5Ac and Neu5Gc were all expressed in uterine cancer, cervical cancer and liver cancer. We found the expression of KDN declined in ovarian and uterine tumor tissues which are with high degree of tumor differentiation. And the expression of KDN incresed in cervical and liver tumor tissues which are with high degree of tumor differentiation. But the amount of the tissue samples are too small for statistical analysis, so we need more further studies.