CHROMATOGRAPHIC EVIDENCE FOR PYRRALINE FORMATION DURING PROTEIN GLYCATION IN-VITRO AND IN-VIVO
CHROMATOGRAPHIC EVIDENCE FOR PYRRALINE FORMATION DURING PROTEIN GLYCATION IN-VITRO AND IN-VIVO
复制标题
DOI：
10.1016/0167-4838(94)00209-y
复制
发表时间：
1995-02-22
期刊：
影响因子：
--
通讯作者：
MONNIER, VM
中科院分区：
文献类型：
--
作者：
PORTEROOTIN, M;NAGARAJ, RH;MONNIER, VM
吡拉林（β-2-（甲酰基-5-羟甲基-吡咯-1-基）-L-正亮氨酸）是在葡萄糖与蛋白质上赖氨酸残基的ε-氨基之间的非酶促反应中由3-脱氧葡糖醛酮形成的高级美拉德反应产物。虽然它的存在，以及在体外孵育的蛋白质与糖已被证明通过免疫化学方法使用多克隆和单克隆抗体，其在蛋白质中的形成最近受到质疑，通过类似的方法。为了澄清这个问题，我们研究了蛋白质中吡咯啉的形成，碱性水解和定量的高效液相色谱C-18反相柱。在与100 mM葡萄糖或50 mM 3-脱氧葡萄糖酮孵育的血清白蛋白中，注意到吡咯啉形成的时间和糖浓度依赖性增加。从3-脱氧葡萄糖醛酮形成吡咯啉在微酸性pH下迅速，证实了其通过该美拉德反应中间体的合成途径。通过这种方法，在人皮肤胶原蛋白库中也检测到低水平的吡咯啉（< 10 pmol/mg蛋白质），但没有明显的年龄效应。使用略微不同的方法，在酶消化和高效液相色谱分析后，在人血浆蛋白中检测到吡咯啉样物质。与对照相比，来自糖尿病患者的血浆显示出吡咯啉样物质的显著增加。糖尿病和正常个体中的水平分别为21.6 +/- 9.56和12.8 +/- 5.6 pmol/mg蛋白质（P = 0.005），从而反映了糖尿病血浆中吡咯啉的直接前体3-脱氧葡萄糖酮的水平升高。
Pyrraline (epsilon-2-(formyl-5-hydroxymethyl-pyrrol-1-yl)-L-norleucine) is an advanced Maillard reaction product formed from 3-deoxyglucosone in the non-enzymatic reaction between glucose and the epsilon-amino group of lysine residues on proteins. Although its presence in vivo as well as in in vitro incubations of proteins with sugars has been documented by immunochemical methods using polyclonal and monoclonal antibodies, its formation in proteins has recently been questioned by similar methodology. To clarify this issue, we investigated pyrraline formation in proteins following alcaline hydrolysis and quantitation by high-performance liquid chromatography on a C-18 reverse-phase column. Time- and sugar concentration-dependent increase in pyrraline formation was noted in serum albumin incubated with either 100 mM glucose or 50 mM 3-deoxyglucosone. Formation of pyrraline from 3-deoxyglucosone was rapid at slightly acidic pH, confirming its synthetic pathway through this Maillard reaction intermediate. Low levels of pyrraline(< 10 pmol/mg protein) were also detected in a pool of human skin collagen by this method, but no age effect was apparent. Using a slightly different approach, pyrraline-like material was detected in human plasma proteins following enzyme digestion and analysis by high performance liquid chromatography. Plasma from diabetic patients showed a significant increase in pyrraline-like material compared to controls. The levels in diabetic and normal individuals were 21.6 +/- 9.56 and 12.8 +/- 5.6 pmol per mg protein, respectively (P = 0.005), reflecting thereby the elevated levels of the immediate precursor of pyrraline, 3-deoxyglucosone, in diabetic plasma.