Detection of cholecystokinin-58 in human blood by inhibition of degradation

Abstract

Although cholecystokinin-58 (CCK-58) is a major molecular form stored in the intestine, it has not yet been shown to be released into the circulation. This report describes in vitro degradation of CCK-58 in human blood and plasma and the molecular forms detected when this degradation is inhibited. After incubation of CCK-58 for 150 min between 20 and 24 degrees C, approximately 60% of immunoreactivity recovered was degraded to smaller immunoreactive forms. Storage of the 150-min incubate at -20 degrees C for 3 days greatly increased the observed degradation to 85%. When CCK-58 was added in vitro to blood, similar degradation occurred. Degradation of CCK-58 could be inhibited by addition of acid. Blood was obtained 1 h after a test meal designed to stimulate CCK release. The pH was lowered during collection and processing of blood and plasma to inhibit in vitro degradation of cholecystokinin. This method permitted the detection of significant amounts of CCK-58 in circulation.

CCK-58 is the only detectable endocrine form of cholecystokinin in rat

“…Our studies observe significant amounts of CCK-58 in the blood of dogs (5) and humans (2,4), but the proportions in humans are disputed (4,20). Previous reports on the endocrine forms of cholecystokinin in rats did not detect CCK-58.…”

“…CCK-58 added to human blood was degraded into smaller forms, and the degradation of CCK-58 could be inhibited by lowering the pH of blood and the plasma formed from the blood. These results led us to ask the question: is the predominance of these smaller molecular forms in rat truly a species difference in the endocrine forms of cholecystokinin compared with dogs (5) and humans (2,4,18,20), or does the detection of these smaller forms reflect degradation of endogenous forms of cholecystokinin in vitro during collection and processing of blood?…”

CCK-8 and CCK-58 differ in their effects on nocturnal solid meal pattern in undisturbed rats

“…At equimolar doses CCK-8 significantly reduces IMI while CCK-58 does not. This suggests that meal size and IMI are regulated at different sites or they have different actions at the same site, and the common idea that all molecular forms of CCK have the same physiological actions needs reconsideration, highlighting the importance of studying the actual endogenous molecular form of peptides, especially considering the finding that CCK-58 is the only circulating form of cholecystokinin (9,13,42).…”

“…Thus, endogenous endocrine CCK-8 detected by others (23,35) is most likely a product of peptide degradation during plasma formation. Importantly, CCK-58 is the major intestinal and endocrine form in dogs (8,13), rats (42), and humans (8,9,14), suggesting its biological action should be evaluated and the influence of CCK-58 on feeding patterns should be studied. The importance of this suggestion is indicated by the fact that CCK-8 and CCK-58 show different actions on various physiological processes.…”

Measurement of cholecystokinin

“…Another problem has been the appearance of various molecular forms of CCK in blood. Molecular forms ranging in size from CCK-8 to CCK-58 have been described in dogs, rats, and humans (3,4,16). To circumvent these problems reliable RIAs must be sensitive enough to detect blood CCK levels in the low picomolar range and not cross-react with gastrin with which CCK shares an identical carboxy-terminal pentapeptide.…”