Yon Lisa, Faulkner Brian, Kanchanapangka Sumolya, Chaiyabutr Narongsak, Meepan Sompast, Lasley Bill
University of Nottingham, School of Veterinary Medicine and Science, Sutton Bonington, Loughborough, United Kingdom.
Zoo Biol. 2010 Nov-Dec;29(6):760-6. doi: 10.1002/zoo.20309.
Noninvasive hormone assays provide a way to determine an animal's health or reproductive status without the need for physical or chemical restraint, both of which create unnecessary stress for the animal, and can potentially alter the hormones being measured. Because hormone metabolism is highly species-specific, each assay must be validated for use in the species of interest. Validation of noninvasive steroid hormone assays has traditionally required the administration of relatively high doses of radiolabelled compounds (100 µCi or more of (14)C labeled hormone) to permit subsequent detection of the excreted metabolites in the urine and feces. Accelerator mass spectrometry (AMS) is sensitive to extremely low levels of rare isotopes such as (14)C, and provides a way to validate hormone assays using much lower levels of radioactivity than those traditionally employed. A captive Asian bull elephant was given 1 µCi of (14)C-testosterone intravenously, and an opportunistic urine sample was collected 2 hr after the injection. The sample was separated by HPLC and the (14)C in the fractions was detected by AMS to characterize the metabolites present in the urine. A previously established HPLC protocol was used, which permitted the identification of fractions into which testosterone sulfate, testosterone glucuronide, and the parent compound testosterone elute. Results from this study indicate that the majority of testosterone excreted in the urine of the Asian bull elephant is in the form of testosterone sulfate. A small amount of testosterone glucuronide is also excreted, but there is no parent compound present in the urine at all. These results underscore the need for enzymatic hydrolysis to prepare urine samples for hormone assay measurement. Furthermore, they highlight the importance of proper hormone assay validation in order to ensure accurate measurement of the desired hormone. Although this study demonstrated the utility of AMS for safer validation of noninvasive hormone assays in nondomestic species, this methodology could also be applied to studies of nutrient metabolism and drug pharmakokinetics, both areas in great need of further study in wildlife species.
非侵入性激素检测提供了一种在无需物理或化学约束的情况下确定动物健康或生殖状态的方法，这两者都会给动物造成不必要的压力，并可能改变所测激素的水平。由于激素代谢具有高度的物种特异性，每种检测方法都必须针对所关注的物种进行验证。传统上，非侵入性类固醇激素检测的验证需要给予相对高剂量的放射性标记化合物（100 μCi或更多的¹⁴C标记激素），以便随后检测尿液和粪便中排泄的代谢物。加速器质谱（AMS）对极低水平的稀有同位素如¹⁴C敏感，并提供了一种使用比传统方法低得多的放射性水平来验证激素检测的方法。给一头圈养的亚洲雄象静脉注射1 μCi的¹⁴C-睾酮，并在注射后2小时采集一份随机尿液样本。通过高效液相色谱（HPLC）分离样本，并通过AMS检测各馏分中的¹⁴C，以表征尿液中存在的代谢物。使用了先前建立的HPLC方案，该方案允许鉴定硫酸睾酮、睾酮葡萄糖醛酸苷和母体化合物睾酮洗脱的馏分。这项研究的结果表明，亚洲雄象尿液中排泄的大部分睾酮是以硫酸睾酮的形式存在。也排泄少量的睾酮葡萄糖醛酸苷，但尿液中完全没有母体化合物。这些结果强调了在进行激素检测测量时需要进行酶水解来制备尿液样本。此外，它们突出了正确的激素检测验证的重要性，以确保准确测量所需的激素。尽管这项研究证明了AMS在更安全地验证非家养物种的非侵入性激素检测方面的实用性，但这种方法也可应用于营养代谢和药物药代动力学研究，这两个领域在野生动物物种中都非常需要进一步研究。
