Department of Endocrinology, Metabolism and Internal Diseases, Poznan University of Medical Sciences, Poznan, Poland.
Endokrynol Pol. 2011;62(4):336-9.
In recent years we have performed more than 1,000 radioimmunoassays of ghrelin and obestatin. In these assays, we have encountered several technological obstacles. Another difficulty was the enormous discrepancy of plasma ghrelin results published by different authors. The aim of this article is to comment on these problems. Not all peptides of the hypothalamus and intestines are present in blood circulation. Several neuropeptides do not cross the blood-brain barrier, and several gastrointestinal peptides are present in extremely low concentrations in the blood. That requires time-consuming and laborious extraction. In these procedures, considerable amounts of peptides may be lost. In addition, these peptides are very unstable and prone to enzymatic degradation. This makes it mandatory to add enzymatic inhibitors to plasma samples. The peptides are also unstable in elevated temperatures, hence the assays should be performed in air-conditioned laboratories and the kits should be transported in proper low temperature conditions. Peptides may appear in several isoforms of different biological activity, but antibodies routinely used in these assays are polyclonal and do not differentiate between these forms. This complicates clinical evaluation of the results. To date, there are no international standards of ghrelin, obestatin or other active peptides, probably because of their extreme instability. Because of technological difficulties, the results of peptide assays performed in different scientific research institutions vary greatly and cannot be compared to each other. This disadvantage may be partially diminished by including samples of healthy subjects in each assay run to check whether the peptide concentrations of the patients differ significantly from that of control subjects.
近年来，我们已经进行了超过 1000 次的生长激素释放肽和肥胖抑制素的放射免疫分析。在这些分析中，我们遇到了一些技术障碍。另一个困难是不同作者发表的血浆生长激素释放肽结果存在巨大差异。本文的目的是对这些问题进行评论。并非所有下丘脑和肠道的肽类都存在于血液循环中。一些神经肽不能穿过血脑屏障，而一些胃肠道肽在血液中的浓度极低。这需要耗时且费力的提取。在这些过程中，可能会丢失相当数量的肽类。此外，这些肽类非常不稳定，容易被酶降解。这就要求向血浆样本中添加酶抑制剂。这些肽类在高温下也不稳定，因此，检测应在空调实验室中进行，试剂盒应在适当的低温条件下运输。肽类可能以不同生物活性的几种同工型出现，但这些检测中常规使用的抗体是多克隆的，不能区分这些形式。这使得对结果进行临床评估变得复杂。迄今为止，还没有生长激素释放肽、肥胖抑制素或其他活性肽的国际标准，可能是因为它们的极端不稳定性。由于技术困难，不同科研机构进行的肽类检测结果差异很大，无法相互比较。通过在每次检测中包含健康受试者的样本，可以部分减少这种不利情况，以检查患者的肽浓度是否与对照受试者的肽浓度有显著差异。
