基于同位素稀释质谱技术的多酚类物质生物学标志物的测定研究

Cardiovascular disease (CVD) is one of the most serious disease threatening human health. Regulating dietary patterns and pursuing healthy life styles are the most simple, economical, safe and effective pathway to reduction of morbidity of CVD. Some epidemiology researches demonstrate that increasing the intake of foods rich in polyphenols, such as fruits and vegetables, can reduce the onset risk of cardiovascular diseases, but other researches do not acknowledge that. These researches always use the dietary survey to evaluate the intake of polyphenols, which are not objective enough and may lead to some biases. Therefore, it is urgent to carry out the study on the determination of biomarkers reflection to the intake of polyphenols, and then apply it to relative researches. Based on our early work, this current project aims to develop a specific, sensitive and precise isotope dilution mass spectrometry to detect some potential biomarkers of polyphenols and develop a Folin-Ciocalteu colorimetric technique to assess total phenols content in biological samples. With our developed methods, the levels of these biomarkers and total phenols in serum and urine of healthy and CVD high-risk subjects will be determined and compared with the intake levels assessed by dietary survey and the serum concentrations of β-carotene and vitamin C, and finally their association with other risk factors of CVD will be analyzed. This project will help us find an effective way to conduct assessment of nutritional status and to monitor the effect on the intervention of life styles, and it will also offer us some new scientific evidence for support of the prevention of polyphenols intake.

心血管病(CVD)是危害人类健康最严重的疾病之一，而调整膳食结构、推行健康生活方式是CVD防治最为简便、经济、安全和有效的途径。流行病学研究发现，增加富含多酚类物质食物如水果蔬菜等的摄入，能够降低CVD 的发病风险，但还有一些结果不支持，这些研究多通过膳食调查的方法对其摄入进行评价，不够客观且容易引入偏倚。故需要建立准确可靠的反映其摄入的生物学标志物的测定方法，以应用于相关研究的开展。本项目拟在前期工作基础上，建立特异灵敏精密的同位素稀释质谱法测定多酚类物质生物学标志物以及Folin-Ciocalteu 比色法常规测定总酚的方法。测定健康人群和CVD高危人群体内生物学标志物和总酚的水平，与膳食调查法估计的摄入水平及血清β-胡萝卜素和维生素C水平进行比较，并探讨它们与CVD危险因素的关系。本课题将为营养评估和生活方式干预效果的监测提供有效技术手段，为多酚摄入对CVD 的防治提供新的科学依据。

大量流行病学研究发现，增加富含多酚类物质食物如水果蔬菜等的摄入，能够降低心血管疾病（CVD）的发病风险，但目前尚缺乏评价其摄入的客观准确的方法。故需要建立准确可靠的反映其摄入的生物学标志物的测定方法，以应用于相关研究的开展。本项目遵照课题申请书中的内容及年度执行计划从以下三个方面展开工作：. 1）建立了固相萃取Folin-Ciocalteu比色法测定尿总酚、同位素稀释-液相色谱串联质谱（ID-LC/MS/MS）测定尿液白藜芦醇及其代谢物、LC/MS/MS测定尿液22种多酚类物质生物学标志物的方法，上述方法易于操作、精密、可靠，适合临床应用。. 2）健康志愿者水果干预试验发现多酚类生物标志物对膳食多酚摄入响应良好，验证了多酚类生物学标志物与多酚摄入的剂量反应关系；. 3）用本项目建立的研究方法测定了健康志愿者尿总酚以及22种多酚生物学标志物水平、分布及其与CVD危险因素的关系。通过与膳食调查法估计的摄入水平比较，尿总酚以及多酚类生物标志物浓度能够反映水果、蔬菜的摄入，经常食用水果组尿总酚显著高于很少吃水果组，食用蔬菜多于肉类组尿总酚、山奈酚、高香草酸以及3,4-双羟基苯乙酸的含量显著高于肉食为主组，经常食用柑橘类水果组，柚皮苷和槲皮素的浓度显著高于食用其他水果组；平时常吃苹果组，高香草酸的含量显著高于食用其他水果组。此外，大部分多酚类生物学标志物均与反映蔬菜水果摄入的血清β-胡萝卜素和维生素C正相关；尿总酚、大部分羟基苯甲酸类、黄酮醇类以及肠道菌群代谢物类的多酚标志物与CVD危险因素呈显著负相关，而与CVD保护因素呈显著正相关。. 总之，本研究首次揭示了健康人群正常饮食的多酚类物质生物学标志物与日常膳食习惯及CVD危险因素关系，方法将在多酚生物学标志物研究、多酚类物质的健康促进研究以及CVD危险分析中发挥重要作用。