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.

The time-dependent release of molecular variants of cholecystokinin (CCK) into the circulation was studied before and 1, 2, and 4 h after a test meal in six healthy volunteers. At each time period, 100 ml of blood were drawn in a manner to inhibit CCK degradation. Plasma was formed and CCK concentrated by Sep-Pak C18 cartridge chromatography. Molecular variants of CCK and gastrin were well separated from each other by high-performance liquid chromatography (HPLC). Molecular forms of CCK and gastrin were measured by radioimmunoassay using an antibody that requires the presence of the carboxyl-terminal phenylalanine amide for full recognition, implying that biologically active forms were detected. HPLC elution positions of gastrin forms were determined using a gastrin-specific antibody. Chromatographic separation of CCK from gastrin forms was complete, allowing separate integration of gastrin and CCK forms. Therefore no subtraction of gastrin-like immunoreactivity from CCK-like immunoreactivity (CCK-LI) was necessary and CCK-LI could be directly determined. Peaks of CCK-LI were integrated in the column eluates and the plasma concentrations were calculated. Total plasma CCK-LI rose from a value of 2.4 +/- 0.6 pM before the test meal to 6.4 +/- 0.8, 6.6 +/- 0.9, and 5.8 +/- 1.2 pM 1, 2, and 4 h postprandially. The major molecular forms released into the circulation eluted on HPLC in the position of CCK-58 and CCK-39 (which coelutes with CCK-33). Minor amounts were detected in the position of CCK-8. There was no significant difference in the relative proportions of the molecular forms released at the different time periods.(ABSTRACT TRUNCATED AT 250 WORDS)

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