评价生物素营养的主要技术综述(2),营养学论文.docx

评价生物素营养的主要技术综述(2),营养学论文针对以上方式方法存在的各种缺陷，2018年日本学者HAYAKAWA等19对色谱柱的处理进行改良，利用胰蛋白酶处理的抗生物素蛋白固定色谱柱Bioptic AV-1,33 mm 4. 6 mm i. d. 特异性分离纯化生物素，然后用衍生剂ADAM将其衍生化为荧光 性 的biotin-ADAM酯，再 通 过 激发 波 长365 nm、发射波长412 nm下测定biotin-ADAM酯的荧光度确定生物素含量。结果显示血浆生物素回收实验回收率高达98%,血浆中游离生物素回收率为70. 7 10. 9%CV为15. 4%。该方式方法分析速度快、可靠、敏感、准确性高，同时不受其他营养素、抗生物素物质的干扰。 3. 5. 2 GC / MS法1989年MOCK等20使用GC / MS法测定成年人尿液中3HIA,同时实验处理经过中引入了3HIA内标，测得尿中3HIA的含量为112 38 mol/24 h肌酐。实验发现尿液中有机酸浓度和酰基肉碱底物水平的变化与体内生物素水平有明显的相关性，提示可作为评价生物素 营 养 状 况 的 有 效 指 标。 GC /MS法 测 定3HIA,样品制备经过复杂、耗时长； 技术要求高；同时3HIA的标记物在微量的水中就会水解，要求对样品及时分 析，使 得GC /MS的 推 广 使用受限。 3. 5. 3 UPLC-MS / MS法THOMAS等21针对上述缺点进行改良，采用UPLC-MS /MS测定8个健康成 年 人 正 常 尿 样3HIA含 量，其 平 均 值 为8. 5 3. 2mmol/mol肌酐。与GC /MS的结果相比后，两 种 方 法 测 定 数 据 之 间 的 相 关 性 为0. 97. BOGUSIEWICZ等22使 用 该 方 法 测 定10个生物素缺陷成年人尿液中酰肉碱底物浓度的结果显示： 随着生鸡蛋蛋白摄入时间的延长，尿液中Pc MMc、3HIAc MGc、Ac Mc比值明显上升，分别代表了羧化酶PCC、MCC和ACC活性的下降。研究结果显示这3比照值能够较好地反映人体边缘性生物素缺乏状态。这3比照值中的一项或3项联合使用对于发现孕妇早期生物素缺乏具有重要的意义。UPLC-MS /MS能够精到准确地分析生物素相关代谢产物的含量，不需要对样品进行提取和衍生化处理22.尿液中3对肉碱底物比值的测定，不受样品处理时间的影响，同时不需要个体酰基肉毒碱内标或制作标准曲线21,23-25,检测能力高、花费少，合适大人群快速测定工作。 3. 5. 4 LC-MS / MS法 近年来HORVATH等23建立了LC-MS /MS法测定生物素缺陷成年人血浆中3HIA-肉碱的含量，而3HIA-肉碱的生成与生物素代谢途径中MCC活性下降有关26,而MCC的活性 排除MCC基因的缺陷 与体内生物素的水平密切相关。HORVATH等23使用该方式方法测定生物素缺陷者尿液中3HIA-肉碱的浓度，标准曲线回归系数为0. 9992,准确度为3. 79% 5. 8%RE.HORVATH等24同时对有无液固萃取或液液萃取经过SPE 结果做了比拟，结果发现二者之间存在强相关性r = 0. 996 ,能够将SPE经过省略，提高分析速率。 血浆中3HIA-肉碱较少遭到肾功能的影响，使用LC-MS /MS测定3HIA-肉碱，通过测定准确度、精到准确度、检测限和动态范围来校正多种潜在性可能影响或引起机体生物素缺乏的因素23.同时LC-MS /MS测定3HIA-肉碱内标误差要比PCC的误差小，在各种液体基质中相比照较稳定，储存条件分布范围： 室温 - 80 27.LC-MS /MS法测定 尿 液 中3HIA-肉 碱 的 浓 度 要 比 测 定 血 清3HIA-肉碱更方便，由于尿样的处理相对简单、花费低，没有SPE经过； 兼具血清3HIA-肉碱测定的优点，便于在临床上推广使用25. 4瞻望 当前关于生物素营养状况评价方式方法的研究较多，研究趋势主要集中在下面几个方面： 1 确定有效的生物标志物，能够反响生物素的代谢及生物作用水平； 2 相关指标在人体内的正常范围值； 3 各指标不同生物作用水平之间的关联性；4 基质较复杂样品如血浆、尿液中生物素及相关代谢产物含量的测定技术尚不成熟，需要建立敏感、高效和快速的色谱技术。因而，关于生物素营养状况评价方式方法还有待深切进入研究，为生物素代谢评价及生物学应用方面提供根据。 以下为参考文献 1KUROISHI T,ENDO Y,MURAMOTO K,et al.Biotin deficiency up-regulates TNF-alpha productionin murine macrophagesJ. 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