Hegele Jörg, Buetler Timo, Delatour Thierry
Nestlé Research Centre, Nestec Ltd., Vers-chez-les-Blanc, CH-1000 Lausanne 26, Switzerland.
Anal Chim Acta. 2008 Jun 9;617(1-2):85-96. doi: 10.1016/j.aca.2007.12.027. Epub 2008 Jan 3.
Free and protein-bound forms of early and advanced glycation-induced lysine (Lys) modifications were quantified in dairy products by LC-MS/MS using a stable isotope dilution assay. The glycation profiles for N(epsilon)-fructoselysine (FL), N(epsilon)-carboxymethyllysine (CML) and pyrraline (Pyr) were monitored in raw and processed cow milk to investigate whether free glycation products could serve as fast and simple markers to assess the extent of protein glycation in dairy products. In all milk samples, the fraction of free glycation adducts was predominantly composed of advanced modifications, e.g. 8.34+/-3.81 nmol CML per micromol of free Lys (Lys(free)) and 81.5+/-87.8 nmol Pyr micromol(-1) Lys(free)(-1) vs. 3.72+/-1.29 nmol FL micromol(-1) Lys(free)(-1). In contrast, the protein-bound early glycation product FL considerably outweighed the content of CML and Pyr in milk proteins of raw and processed cow milk, whereas severely heat treated milk products, e.g. condensed milk, contained a higher amount of protein-bound advanced glycation adducts. Typical values recorded for milk samples processed under mild conditions were 0.47+/-0.08 nmol FL micromol(-1) of protein-bound Lys (Lys(p-b)), 0.04+/-0.03 nmol CML micromol(-1) Lys(p-b)(-1) and 0.06+/-0.02 nmol Pyr micromol(-1)Lys(p-b)(-1). It was particularly noticeable, however, that mild heat treatment of raw milk, i.e. pasteurization and UHT treatment, did not significantly increase the amount of both free and protein-bound Lys modifications. In conclusion, the profiles of free and protein-bound glycation-induced Lys modifications were found to be different and a screening of free glycation adducts does, therefore, not allow for a conclusion about the protein glycation status of dairy products.
采用稳定同位素稀释分析法，通过液相色谱-串联质谱法（LC-MS/MS）对乳制品中早期和晚期糖基化诱导的赖氨酸（Lys）修饰的游离形式和蛋白质结合形式进行了定量分析。监测了原料奶和加工牛奶中N-ε-果糖赖氨酸（FL）、N-ε-羧甲基赖氨酸（CML）和吡咯赖氨酸（Pyr）的糖基化谱，以研究游离糖基化产物是否可作为快速简便的指标来评估乳制品中蛋白质糖基化的程度。在所有牛奶样品中，游离糖基化加合物的部分主要由晚期修饰组成，例如每微摩尔游离赖氨酸（Lys（游离））中含有8.34±3.81纳摩尔CML和81.5±87.8纳摩尔Pyr/微摩尔Lys（游离）-1，而每微摩尔Lys（游离）-1中含有3.72±1.29纳摩尔FL。相比之下，在原料奶和加工牛奶的乳蛋白中，与蛋白质结合的早期糖基化产物FL的含量大大超过CML和Pyr的含量，而经过严重热处理的乳制品，如炼乳，含有较高含量的与蛋白质结合的晚期糖基化加合物。在温和条件下加工的牛奶样品的典型值为每微摩尔与蛋白质结合的赖氨酸（Lys（p-b））中含有0.47±0.08纳摩尔FL、0.04±0.03纳摩尔CML/微摩尔Lys（p-b）-1和0.06±0.02纳摩尔Pyr/微摩尔Lys（p-b）-1。然而，特别值得注意的是，原料奶的温和热处理，即巴氏杀菌和超高温瞬时灭菌处理，并未显著增加游离和与蛋白质结合的Lys修饰的量。总之，发现游离和与蛋白质结合的糖基化诱导的Lys修饰谱不同，因此，对游离糖基化加合物的筛查不能得出关于乳制品蛋白质糖基化状态的结论。
