Quantification of the Sulfated Cholecystokinin CCK-8 in Hamster Plasma Using Immunoprecipitation Liquid Chromatography-Mass Spectrometry/Mass Spectrometry
Quantification of the Sulfated Cholecystokinin CCK-8 in Hamster Plasma Using Immunoprecipitation Liquid Chromatography-Mass Spectrometry/Mass Spectrometry
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DOI：
10.1021/ac9018318
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发表时间：
2009-11-01
影响因子：
7.4
通讯作者：
Yokoyama, Wallace H.
中科院分区：
文献类型：
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作者：
Young, Scott A.;Julka, Samir;Yokoyama, Wallace H.
胆囊收缩素（CCK）和CCK的不同分子形式已被公认为饱腹感的生物标志物，但准确的分析受到多种天然存在形式和与胃泌素广泛相似性的限制。一种形式CCK-8（天然存在的八氨基酸CCK肽）水平的变化与饱腹感反应相关。内源性CCK-8在叙利亚金仓鼠中尚未得到很好的表征，叙利亚金仓鼠是研究脂肪摄取和消化的重要模型。我们已经克隆和测序仓鼠CCK和确定和特点内源性CCK-8从仓鼠血浆。CCK-8由8个氨基酸组成，在其他物种中高度保守。在准确鉴定和表征仓鼠CCK-8后，我们开发了一种高度特异性和灵敏度的基于免疫沉淀的LC-MS/MS测定法用于其定量。本试验能够测定仓鼠血浆样品中浓度范围为0.05 - 2.5 ng/mL的活性CCK-8。该范围涵盖基础和餐后CCK-8水平。在4天内以3个重复浓度检查方法性能验证样品。在这4天期间，所有样品的测定准确度（相对误差百分比，% RE）平均值为11.3%，精密度（变异系数百分比，% CV）为15.5%。此外，该方法用于测定高脂饮食激发的仓鼠内源性血浆CCK-8的增加。
Cholecystokinin (CCK) and the different molecular forms of CCK are well established as biomarkers for satiety but accurate analysis has been limited by the multiple naturally occurring forms and extensive similarities to gastrin. Changes in levels of one form, CCK-8, a naturally occurring eight amino acid peptide of CCK, have been correlated with satiety responses. Endogenous CCK-8 has not been well characterized in Syrian Golden hamsters, an important model in the study of fat uptake and digestion. We have cloned and sequenced hamster CCK and identified and characterized endogenous CCK-8 from hamster plasma. Hamster CCK-8 is composed of eight amino acid residues which are highly conserved among other species. Following accurate identification and characterization of hamster CCK-8, we have developed a highly specific and sensitive immunoprecipitation based LC-MS/MS assay for its quantification. The present assay enables determination of active CCK-8 over a concentration range from 0.05 to 2.5 ng/mL in hamster plasma samples. This range covers both the basal and postprandial levels of CCK-8. Method performance validation samples were examined at three concentrations replicated over the course of 4 days. The assay accuracy (percent relative error, % RE) average was 11.3% with a precision (percent coefficient of variation, % CV) of 15.5% over all samples in this 4 day period. Additionally, the method was used to determine increases of endogenous plasma CCK-8 in hamsters challenged with a high-fat meal.