Chun L E, Koob T J, Eyre D R
Department of Orthopaedics, University of Washington, Seattle 98195.
Anal Biochem. 1988 May 15;171(1):197-206. doi: 10.1016/0003-2697(88)90142-x.
A method for quantifying hyaluronic acid in biological tissues and fluids is described. The assay uses ion-pair HPLC to resolve and quantify the oligosaccharide end products of Streptomyces hyaluronidase digestion. Tissue samples were solubilized by papain, and the nondiffusate after dialysis was exhaustively digested with Streptomyces hyaluronidase. The resulting tetrasaccharide and hexasaccharide cleavage products were resolved by reverse-phase high-performance liquid chromatography in the presence of the ion-pairing agent, tetrabutylammonium phosphate. The saccharides were detected and quantified by their absorbance at 232 nm due to the alpha, beta-unsaturated carboxyl group generated by the eliminase reaction. In control experiments 93 +/- 3% of a hyaluronic acid standard so treated was reproducibly recovered as its tetra- and hexasaccharide cleavage products. As little as 0.5 microgram of the oligosaccharides could be quantified with no interference from a vast excess of chondroitin sulfate or other tissue components. The assay was applied to various types of human, bovine, and rabbit cartilage and to samples of other tissues including nucleus pulposus, annulus fibrosus, skin, aorta, cervix, cockscomb, synovial fluid, and vitreous humor. Results on human articular cartilage showed a linear increase in the content of hyaluronate from 0.1 to 0.5% of tissue dry weight between birth and 80 years of age.
本文描述了一种定量生物组织和体液中透明质酸的方法。该测定法使用离子对高效液相色谱法来分离和定量透明质酸酶消化后的寡糖终产物。组织样本用木瓜蛋白酶溶解，透析后的非扩散物用透明质酸酶彻底消化。在离子对试剂磷酸四丁铵存在的情况下，通过反相高效液相色谱法分离得到的四糖和六糖裂解产物。由于消除酶反应产生的α,β-不饱和羧基，糖类在232nm处的吸光度被检测并定量。在对照实验中，如此处理的透明质酸标准品的93±3%可作为其四糖和六糖裂解产物被重复回收。低至0.5微克的寡糖可被定量，且不受大量硫酸软骨素或其他组织成分的干扰。该测定法应用于各种类型的人、牛和兔软骨以及其他组织样本，包括髓核、纤维环、皮肤、主动脉、子宫颈、鸡冠、滑液和玻璃体液。人关节软骨的结果显示，从出生到80岁，透明质酸盐含量从组织干重的0.1%线性增加到0.5%。
