使用单柱高效液相色谱法直接检测人黄韧带水解产物中胶原蛋白和弹性蛋白的交联

Direct detection of crosslinks of collagen and elastin in the hydrolysates of human yellow ligament using single-column high performance liquid chromatography.

作者信息

Chen J R, Takahashi M, Kushida K, Suzuki M, Suzuki K, Horiuchi K, Nagano A

机构信息

Department of Orthopedic Surgery, Hamamatsu University School of Medicine, Hamamatsu, 431-3192, Japan.

出版信息

Anal Biochem. 2000 Feb 15;278(2):99-105. doi: 10.1006/abio.1999.4412.

DOI:10.1006/abio.1999.4412

PMID:10660450

Abstract

Collagen and elastin are recognized as two major connective tissue proteins of human yellow ligament. In both collagen and elastin there are many kinds of intra- or intermolecular crosslinks. Pyridinoline (Pyr) and deoxypyridinoline (Dpyr) are mature crosslinks which maintain the structure of the collagen fibril. Desmosine (Des) and isodesmosine (Isodes) represent the major crosslinking components of elastin. Pentosidine (Pen), which is a senescent crosslink and one of the advanced glycation end products, accumulates with age in tissue proteins including collagen. We developed a direct and one-injection HPLC method to measure Pyr, Dpyr, Des, Isodes, and Pen in the hydrolysate of human yellow ligament. This method used one column and two detectors. Recovery rates of Pyr, Dpyr, Pen, Des, and Isodes were 86.4-98.3, 83.6-96.8, 78.7-95.6, 83.6-97.9, and 85.6-99.3%, respectively (n = 8). The intraassay coefficients of variation for Pyr, Dpyr, Pen, Des, and Isodes were 3.7, 4.1, 5.4, 4.5, and 4.7%, respectively (n = 8), and the interassay coefficients of variation for Pyr, Dpyr, Pen, Des, and Isodes were 4.4, 5.1, 4.9, 4.6 and 4.1%, respectively. Linear regression analysis showed the linearity (r = 0.99, P = 0.0001) of calibration line for each Pyr, Dpyr, Pen, Des, and Isodes. Using this method, we investigated age-related changes in the crosslinks of collagen and elastin in human yellow ligament. There was a significant correlation between Pen and age, but no correlations with Pyr, Dpyr, Des, and Isodes. We believe that this method is useful for investigating the content of these crosslinks in both collagen and elastin under various conditions.

摘要

胶原蛋白和弹性蛋白被认为是人体黄韧带的两种主要结缔组织蛋白。在胶原蛋白和弹性蛋白中，都存在多种分子内或分子间交联。吡啶啉（Pyr）和脱氧吡啶啉（Dpyr）是维持胶原纤维结构的成熟交联。锁链素（Des）和异锁链素（Isodes）是弹性蛋白的主要交联成分。戊糖苷（Pen）是一种衰老交联，也是晚期糖基化终产物之一，会随着年龄增长在包括胶原蛋白在内的组织蛋白中积累。我们开发了一种直接的单注射高效液相色谱法来测定人黄韧带水解产物中的Pyr、Dpyr、Des、Isodes和Pen。该方法使用一根色谱柱和两个检测器。Pyr、Dpyr、Pen、Des和Isodes的回收率分别为86.4 - 98.3%、83.6 - 96.8%、78.7 - 95.6%、83.6 - 97.9%和85.6 - 99.3%（n = 8）。Pyr、Dpyr、Pen、Des和Isodes的批内变异系数分别为3.7%、4.1%、5.4%、4.5%和4.7%（n = 8），批间变异系数分别为4.4%、5.1%、4.9%、4.6%和4.1%。线性回归分析表明，每种Pyr、Dpyr、Pen、Des和Isodes校准曲线的线性度（r = 0.99，P = 0.0001）。使用该方法，我们研究了人黄韧带中胶原蛋白和弹性蛋白交联的年龄相关变化。Pen与年龄之间存在显著相关性，但与Pyr、Dpyr、Des和Isodes无相关性。我们认为该方法有助于在各种条件下研究胶原蛋白和弹性蛋白中这些交联的含量。