Quantitation of procholecystokinin and its products in plasma by processing-independent analysis.
A procedure for processing-independent quantitation of procholecystokinin (proCCK) and its products has been applied to plasma. The procedure is based on tryptic cleavage after Lys61 and Arg71 with subsequent monospecific radioimmuno-analysis of fragment 62-71 of human proCCK, which again corresponds to fragment 1-10 of CCK-22. The detection limit of the analysis was 0.2 pmol/l. Plasma was extracted with ethanol. In plasma from 13 healthy volunteers the basal concentration with the above-mentioned radioimmunoassay was 1.1 +/- 0.1 pmol/l (mean +/- S.E.M.) before, and 13.7 +/- 0.6 pmol/l after, incubation with trypsin. Two hours after ingestion of a mixed meal, the plasma concentration was 2.0 +/- 0.1 pmol/l before, and 21.7 +/- 1.2 pmol/l after tryptic cleavage. With a conventional CCK radioimmunoassay specific for the C-terminally amidated and O-sulfated bioactive epitope, the concentration was 1.0 +/- 0.1 pmol/l in the basal state and 4.2 +/- 0.4 pmol/l 2 h after a meal. Tryptic cleavage did not increase the concentrations of amidated, bioactive CCK peptides. In plasma from 37 patients with the carcinoid syndrome, the basal concentration of proCCK and its products was 14.1 (2.8-150.4) pmol/l (median (range)), compared with 0.3 (0-18.8) pmol/l for carboxyamidated CCK. Only two patients had significantly elevated CCK concentrations. We conclude that processing-independent analysis is useful for quantitation of proCCK and its products in plasma, since it quantitates CCK cell secretion more accurately than conventional CCK assays.
Paloheimo
L I
LI
Department of Clinical Biochemistry, Rigshospitalet, University of Copenhagen, Denmark.
Rehfeld
J F
JF
D000328:Adult; D002766:Cholecystokinin / Q000032:analysis / Q000097:blood*; D002850:Chromatography, Gel; D004386:Duodenum / Q000737:chemistry; D005260:Female; D006801:Humans; D006868:Hydrolysis; D007413:Intestinal Mucosa / Q000737:chemistry; D007414:Intestinal Neoplasms / Q000378:metabolism; D008297:Male; D008303:Malignant Carcinoid Syndrome / Q000378:metabolism; D008875:Middle Aged; D010455:Peptides / Q000097:blood; D010949:Plasma / Q000737:chemistry*; D011498:Protein Precursors / Q000032:analysis / Q000097:blood*; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D011863:Radioimmunoassay; D014357:Trypsin / Q000737:chemistry
D010455:Peptides; D011498:Protein Precursors; C060249:procholecystokinin; D002766:Cholecystokinin; D014357:Trypsin