HPLC测定驼鹿和狍雪尿肌酐及嘌呤衍生物

Determination of creatinine and purine derivatives from snow-urine of moose and roe deer by HPLC

反刍动物尿液中嘌呤衍生物的含量可以间接反映其营养状态。高效液相色谱技术（HPLC）是测定反刍动物尿液中嘌呤衍生物含量的重要技术手段之一。本文通过优化的液相色谱条件（依利特-C18（200mm×4.6mm,5μm）色谱柱;流动相：0.01mol/LKH2PO4、20%乙腈＋80%0.01mol/LKH2PO4;流速1.0ml/min;柱温20℃;检测波长220nm,254nm）,测定出野生驼鹿和狍雪尿中肌酐及嘌呤衍生物的含量。测定结果显示,尿囊素、肌酐、尿酸、次黄嘌呤、黄嘌呤在0.0010-0.2000mg/ml范围内线性关系良好（R^2=0.9999）,标准品回收率均在96%-105%范围内;2006和2007年冬季在黑龙江省胜山和沾河两个林场所采集的所有驼鹿以及近三分之一的狍雪尿样本中,未检测到尿囊素的存在,所有未检测到尿囊素的驼鹿和狍雪尿样本中,黄嘌呤和次黄嘌呤之和占嘌呤衍生物总量的20.7%-55.2%,而所有可检测到尿囊素的尿液样品中,黄嘌呤和次黄嘌呤之和占嘌呤衍生物总量的15.2%-36.1%,尿囊素占嘌呤衍生物总量的40.6%-85.6%,表明所有驼鹿和部分狍个体越冬时可能存在一定程度的营养限制,且驼鹿和狍的营养代谢存在着种间差异。因此,这种非损伤性的测定方法可以快速、有效地评估冬季鹿类动物营养状况,对其营养状况进行动态监测,揭示出不同物种之间营养动态的差异性,并为野生有蹄类种群和生境的有效保护管理提供科学依据。

Currently the content detection of purine derivatives in urine of ruminant was used broadly to reflect their nutritional conditions indirectly world wide. In this paper a optimized High Performance Liquid Chromatography was performed to determine the content of creatinine and purine derivatives in snow-urine of moose（Alces alces） and roe deer（Capreolus capreolus）. The results showed that there were good linearities from 0.0010mg/ml to 0.2000mg/ml （R^2=0.9999） for allantoin, uric acid, hypoxanthine, xanthine and creatinine; the recovery ratio of the standards were 96%-105%.Allantoin could not be detected in all the samples of moose and almost 1/3 roe deer, which were collected from Shengshan and Zhanhe forest farms of Heilongjiang Province in the winter of 2006 and 2007. Moreover, in these samples, hypoxanthine and xanthine took up 20.7%-55.2%of the total purine derivatives. In all the samples in which the allantoin could be detected, hypoxanthine and xanthine took up 15.2%-36.1% of the total purine derivatives and the allantoin took up 40.6%-85.6%. These results suggested that all the moose and some roe deer individuals were in the situation of nutritional restriction and there were differences of metabolism between moose and roe deer. The noninvasive method could be used to evaluate the nutritional conditions of deer in winter in a fast and effective way and to take dynamic inspect for the nutritional conditions of deer; And it also could be used to show the differences of nutrition to provide scientific evidences for population protection and effective management of habitat of ungulates.