Guida E, Codini M, Palmerini C A, Fini C, Lucarelli C, Floridi A
Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Università di Perugia, Italy.
J Chromatogr. 1990 May 16;507:51-7. doi: 10.1016/s0021-9673(01)84180-7.
The development and the validation of a general strategy for the simple and accurate analysis of desmosines (isodesmosine and desmosine) in tissues coupled with the determination of collagen (as hydroxyproline) is described. The method is based on simplified sample (i.e., lung) pretreatment which involves, in a PTFE screw-capped Pyrex tube, homogenization, collagen extraction with hot 5% trichloroacetic acid and hydrolysis of the elastin-containing residue with 6 M hydrochloric acid, followed by cellulose minicolumn purification of desmosines from the hydrolysates, dansyl chloride pre-column derivatization of the purified desmosines and reversed-phase high-performance liquid chromatographic (HPLC) analysis of the dansyl derivatives using a Spherisorb ODS-2 column, an on-column enrichment sample device and a linear gradient of organic modifier (acetonitrile) in phosphate buffer. The simple sample pretreatment, the optimized chromatographic conditions and the short HPLC analysis time (less than 15 min) allow the accurate and rapid determination of desmosine and isodesmosine, thus permitting the determination of elastin in several kinds of tissues with a minimum of sample manipulation.
本文描述了一种通用策略的开发与验证，该策略用于简单、准确地分析组织中的锁链素（异锁链素和锁链素），并同时测定胶原蛋白（以羟脯氨酸计）。该方法基于简化的样品（即肺）预处理，具体步骤为：在聚四氟乙烯螺旋盖的派热克斯玻璃管中进行匀浆，用热的5%三氯乙酸提取胶原蛋白，并用6M盐酸水解含弹性蛋白的残渣，然后通过纤维素微型柱从水解产物中纯化锁链素，用丹磺酰氯对纯化后的锁链素进行柱前衍生化，并使用Spherisorb ODS - 2柱、柱上富集样品装置以及磷酸盐缓冲液中有机改性剂（乙腈）的线性梯度进行反相高效液相色谱（HPLC）分析丹磺酰衍生物。简单的样品预处理、优化的色谱条件以及较短的HPLC分析时间（少于15分钟）使得能够准确、快速地测定锁链素和异锁链素，从而只需最少的样品处理即可测定多种组织中的弹性蛋白。
