Single-column high-performance liquid chromatographic fluorescence detection of immature, mature, and senescent cross-links of collagen
Single-column high-performance liquid chromatographic fluorescence detection of immature, mature, and senescent cross-links of collagen
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DOI：
10.1006/abio.1997.2350
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发表时间：
1997-11-01
影响因子：
2.9
通讯作者：
Ishioka, N
中科院分区：
文献类型：
--
作者：
Saito, M;Marumo, K;Ishioka, N
建立了一种不使用放射性同位素和初步分级步骤的可还原（未成熟）和不可还原（成熟和衰老）胶原交联的高效液相色谱-荧光检测方法。该方法使用含有 7% 乙醇的柠檬酸钠缓冲液的梯度洗脱程序。可还原交联（二羟基赖氨酸正亮氨酸、羟基赖氨酸正亮氨酸和赖氨酸正亮氨酸）和不可还原交联（组氨酸羟基赖氨酸正亮氨酸）通过柱后邻苯二甲醛衍生化检测，而其他不可还原交联（吡啶啉、脱氧吡啶啉和戊糖苷）通过自然荧光检测。邻苯二甲醛衍生物交联和天然荧光不可还原交联的含量的线性范围分别为20-600、5-500（吡啶啉、脱氧吡啶啉）和0.2-20pmol（戊糖素）。含有 1-2 mg 干胶原蛋白的组织足以进行可还原和不可还原交联的重复分析。该 HPLC 系统可分析相当于 0.25 毫克的水解胶原蛋白。使用该系统，还研究了人类结缔组织胶原蛋白交联与年龄相关的变化。 (C) 1997 年学术出版社。
A high-performance liquid chromatographic-fluorescence detection method of reducible (immature) and nonreducible (mature and senescent) cross-links of collagen was established without the use of a radioisotope and preliminary fractionation step. This method used a gradient elution procedure of sodium citrate buffer containing 7% ethanol. The reducible cross-links (dihydroxylysinonorleucine, hydroxylysinonorleucine, and lysinonorleucine) and nonreducible cross-link (histidinohydroxylysinonorleucine) were detected by O-phthalaldehyde derivatization with the postcolumn method, whereas other nonreducible cross-links (pyridinoline, deoxypyridinoline, and pentosidine) were detected by natural fluorescence. The linear ranges of contents of the O-phthalaldehyde derivative cross-links and the natural fluorescent nonreducible cross-links were 20-600, 5-500 (pyridinoline, deoxypyridinoline), and 0.2-20 pmol (pentosidine), respectively. Tissue containing 1-2 mg dry mt of collagen was adequate for duplicate analyses of the reducible and nonreducible cross-links. An equivalent of 0.25 mg of hydrolyzed collagen could be analyzed by this HPLC system. Using this system, age-related changes in the cross-links of collagen from human connective tissues were also investigated. (C) 1997 Academic Press.