Hosoda Hiroshi, Kangawa Kenji
Department of Regenerative Medicine and Tissue Engineering, National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka, Japan.
Methods Enzymol. 2012;514:113-26. doi: 10.1016/B978-0-12-381272-8.00008-8.
Octanoyl modification of ghrelin is rapidly hydrolyzed to des-acyl ghrelin in blood samples. Owing to the increased interest in ghrelin measurement, a standardized method of sample collection is required.
This chapter investigates the effect of a variety of anticoagulants and storage conditions on ghrelin stability. Experiment 1 evaluates the effects of anticoagulants on ghrelin measurements. Experiment 2 evaluates the effect of plasma pH on ghrelin stability. Experiment 3 evaluates the mechanisms of degradation of the active form of ghrelin in plasma. Experiment 4 investigates the kinetics of ghrelin following intravenous injection of rat ghrelin.
In whole blood and plasma, octanoylated ghrelin is highly unstable. The collection of blood samples with EDTA-aprotinin under cooled conditions was appropriate to maintain ghrelin stability. Acidification of plasma to pH 3-4 and storage at 4°C maintained ghrelin stability. The degradation of ghrelin was shown to be due to the hydrolysis to des-acyl ghrelin. After intravenous administration to rats, plasma ghrelin levels rapidly decreased with a half-life of 8 min.
The results showed that the ghrelin values measured in human blood samples were markedly affected by the conditions of collection and storage, the pH, and the RIA method in measurement. Measuring the ghrelin values of the active form is useful for studying plasma ghrelin changes over short time periods. As ghrelin is highly unstable, it is necessary to standardize the preparation of samples to ensure reliable ghrelin measurements.
血液样本中胃饥饿素的辛酰化修饰会迅速水解为去酰基胃饥饿素。由于对胃饥饿素测量的兴趣增加，需要一种标准化的样本采集方法。
本章研究了多种抗凝剂和储存条件对胃饥饿素稳定性的影响。实验1评估抗凝剂对胃饥饿素测量的影响。实验2评估血浆pH对胃饥饿素稳定性的影响。实验3评估血浆中胃饥饿素活性形式的降解机制。实验4研究静脉注射大鼠胃饥饿素后胃饥饿素的动力学。
在全血和血浆中，辛酰化胃饥饿素非常不稳定。在冷却条件下用乙二胺四乙酸 - 抑肽酶采集血样适合维持胃饥饿素的稳定性。将血浆酸化至pH 3 - 4并在4°C储存可维持胃饥饿素的稳定性。胃饥饿素的降解显示是由于水解为去酰基胃饥饿素。给大鼠静脉注射后，血浆胃饥饿素水平迅速下降，半衰期为8分钟。
结果表明，在人体血样中测得的胃饥饿素值受采集和储存条件、pH以及测量中的放射免疫分析方法的显著影响。测量活性形式的胃饥饿素值对于研究短时间内血浆胃饥饿素的变化很有用。由于胃饥饿素高度不稳定，有必要规范样本制备以确保可靠的胃饥饿素测量。
