Quantification of the Sulfated Cholecystokinin CCK-8 in Hamster Plasma Using Immunoprecipitation Liquid Chromatography-Mass Spectrometry/Mass Spectrometry

• Published in: Analytical chemistry (Washington) 2009-11, Vol.81 (21), p.9120-9128
• Main Authors: Young, Scott A, Julka, Samir, Bartley, Glenn, Gilbert, Jeffrey R, Wendelburg, Brian M, Hung, Shao-Ching, Anderson, W. H. Kerr, Yokoyama, Wallace H
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino acids; Analytical chemistry; Animals; Base Sequence; Biomarkers; Chemistry; Cholecystokinin - blood; Chromatographic methods and physical methods associated with chromatography; Chromatography; Chromatography, Liquid - methods; Cloning; Cricetinae; Dietary Fats; Exact sciences and technology; Immunoprecipitation - methods; Mass spectrometry; Mesocricetus; Molecular Sequence Data; Other chromatographic methods; Peptide Fragments - blood; Plasma; Rodents; Sequence Alignment; Spectrometric and optical methods; Tandem Mass Spectrometry - methods; Tyrosine - analogs & derivatives; Tyrosine - chemistry
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac9018318

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.