A novel method for histidinohydroxylysinonorleucine measurement and its clinical aaplication

组氨酸羟基赖氨酸（HHL）是皮肤中的一种三价交联，它为胶原蛋白提供稳定性和其他生理特性。用9-氟甲基氯甲酸甲酯标记皮肤水解物，建立了一种高灵敏度的反相高效液相色谱法测定HHL的方法。我们用这种方法测量了系统性硬化症患者皮肤标本中HHL的含量，并与年龄和部位匹配的对照皮肤标本中的HHL进行了比较。一半的皮肤样本进行常规组织学检查，并选择具有SSc特征性组织学变化，整个真皮层胶原增厚透明的皮肤样本。以HHL与胶原的分子比表达时，SSc中HHL的含量较对照显著增加。胶原分子中HHL量的增加可能与疾病的病理性纤维化有关。值得进一步研究的是，这种增加是否是SSc所特有的，或者是哪种因素调节了体内分子间交联的形成。我们研究了缺氧和低血清浓度对真皮成纤维细胞细胞外基质相关基因表达和I型胶原三功能分子间交联形成的影响。在逆转录酶聚合酶链反应（RT-PCR）研究中，缺氧和低血药浓度均显示正常和系统性硬化症患者真皮成纤维细胞单层培养中转化生长因子- β mRNA表达升高，金属蛋白酶-1 mRNA表达降低。在补充抗坏血酸2-磷酸的三维培养中，缺氧显著抑制了I型胶原三功能分子间交联的成熟型组织二羟基赖氨酸的形成。转化生长因子- β和金属蛋白酶- 1 mRNA表达的改变可能是系统性硬化症炎症后阶段的病理机制。在低氧或低血药浓度的培养条件下，组氨酸羟基赖氨酸氨基亮氨酸的形成受到抑制，这与之前在体内的发现不一致。需要进一步的研究来阐明调节I型胶原三功能分子间交联形成的因子。少

Histidinohydroxylysinonorleucine (HHL) is a trivalent type of cross-link in skin, which provides collagen for stability and other physiological properties. We established a novel highly sensitive method to measure HHL by using reversed phase HPLC after labeling skin hydrolysates with 9-fluorenylmethyl chrolformate. We measured the content of HHL in the skin specimens of patients with systemic sclerosis and compared it with the HHL of age- and site-matched control skin specimens using this method. Half of the skin sample was processed for a conventional histological examination and chose skin samples that showed the characteristic histological change of SSc, thickened and hyalinized collagens in the entire dermis. The content of HHL in the SSc was significantly increased compared with that of controls when expressed as the molecular ratio of HHL to collagen. The increased amount of HHL in collagen molecules may be associated with the pathological fibrosis of the disease. It is worth inv … More estigating if this increase is specific for SSc or not, or which facto (s) regulates the formation of intermolecular cross-links in vivo.We investigated the effects of hypoxia and low serum concentartion on the expression of extracellular matrix-related genes and the formation of trifunctional intermolecular cross-link of type I collagen by dermal fibroblasts. In the studies of reverse transcriptase polymerase chain reaction (RT-PCR), both hypoxia and low serum concentration revealed the increased mRNA expression of transforming growth factor-beta and the decreased mRNA expression of metalloproteinase-1 in the monolayr culture of dermal fibroblasts from normal individuals and patients with systemic sclerosis. In the three-dimensional culture supplemented with ascorbic acid 2-phosphate, hypoxia remarkably suppressed the formation of histidinohydroxylysinonorleucine, a mature type of trifunctional intermolecular cross-link of type I collagen.The altered mRNA expression of transforming growth factor-beta and metalloproteinase- 1 may well account for the pathoetiology of the post inflammatory stage of systemic sclerosis. The suppressed formation of histidinohydroxylysinonorleucine under the culture condition of hypoxia or low serum concentartion disagreed with the previous in vivo finding. Further studies are required to elucidate factor (s) to regulate the formation of trifunctional intermolecular cross-link of type I collagen. Less