Takahashi S, Lee M J
Anal Biochem. 1987 May 1;162(2):553-61. doi: 10.1016/0003-2697(87)90433-7.
A procedure for the quantification of various parameters of metabolism of collagen in fibrotic mouse liver has been developed. The method involves derivatization of hydroxyproline, a marker of collagen, with dansyl chloride, high-performance liquid chromatography of the derivative on an octadecyl C-18 column, and its detection by fluorescence. This assay improves upon existing procedures in several respects: It extends the analysis so that not only the collagen content of the tissue but also the metabolism of collagen is determined at levels found intracellularly. It is sensitive enough to quantify 0.1-10 nmol of hydroxyproline, and it includes three major amino acids (hydroxyproline, glycine, and proline) of collagen and two assay controls; it generates information on both the purity and quantity of collagen in each assay. The determination of specific activity of intracellular free [14C]proline, which is the precursor of protein-bound hydroxyproline, defines the specific activity of [14C]hydroxyproline of collagen converted from precursor residues of [14C]proline by the action of prolyl hydroxylase. The specific activity of [14C]hydroxyproline can be used for the evaluation of collagen synthesis and secretion and intracellular and extracellular degradation of the newly synthesized and secreted collagen in the tissue. The determination of specific activities of [14C]hydroxyproline and [14C]proline and of the ratio of [14C]hydroxyproline to [14C]proline of newly secreted collagen provides information concerning the extent of hydroxylation of [14C]proline residues of newly synthesized collagen.
已开发出一种用于定量纤维化小鼠肝脏中胶原蛋白各种代谢参数的方法。该方法包括用丹磺酰氯对胶原蛋白的标志物羟脯氨酸进行衍生化，在十八烷基C - 18柱上对衍生物进行高效液相色谱分析，并通过荧光检测。该测定法在几个方面改进了现有方法：它扩展了分析范围，不仅能测定组织中的胶原蛋白含量，还能在细胞内水平测定胶原蛋白的代谢。它足够灵敏，能够定量0.1 - 10 nmol的羟脯氨酸，并且包括胶原蛋白的三种主要氨基酸（羟脯氨酸、甘氨酸和脯氨酸）以及两个测定对照；它在每次测定中都能生成有关胶原蛋白纯度和数量的信息。测定细胞内游离[¹⁴C]脯氨酸的比活性，其是蛋白质结合羟脯氨酸的前体，可确定通过脯氨酰羟化酶的作用由[¹⁴C]脯氨酸的前体残基转化而来的胶原蛋白的[¹⁴C]羟脯氨酸的比活性。[¹⁴C]羟脯氨酸的比活性可用于评估组织中胶原蛋白的合成、分泌以及新合成和分泌的胶原蛋白在细胞内和细胞外的降解。测定新分泌胶原蛋白的[¹⁴C]羟脯氨酸和[¹⁴C]脯氨酸的比活性以及[¹⁴C]羟脯氨酸与[¹⁴C]脯氨酸的比率，可提供有关新合成胶原蛋白的[¹⁴C]脯氨酸残基羟化程度的信息。
