An Improved Method for Estimating Cholecystokinin in Human Serum

Abstract

Because of difficulties encountered in setting up radioimmunoassays for cholecystokinin (CCK) a sensitive and reliable biological method for estimating this hormone is still needed. The principles of such a biological technique and an improvement to it have already been described, but the serum levels of CCK reported were high and the technique required further refinement and validation. The strips of rabbit gall-bladder used to estimate the concentration of CCK increased in sensitivity to standard solutions of CCK over a 6--8 period before stabilizing, but a single sample of serum increased the sensitivity of the strips of gall-bladder to their maximum immediately. These two problems were eliminated by 'priming' the strips of gall-bladder by exposure to two serum samples before exposure to the standard solutions used for production of a dose--response curve. Thirdly, it was discovered that some non-peptide substances in serum possessed CCK-like activity; by extracting all the small peptides from serum with dextran-coated charcoal the residual activity could be measured and subtracted from the total CCK activity. Finally, the activity of CCK in the serum increased during processing before freezing. This increase was eliminated by taking the blood samples into aprotinin which has been shown to cause dramatic reduction in CCK activity in some experiments. When all these factors were taken into account and the technique suitably modified, the mean level of CCK in the serum of ten normal fasting subjects was found to be 28 milli Ivy Dog units/ml (2.4 pmol/ml), which is only one third of that reported previously.

Feedback regulation of pancreatic enzyme secretion. Suppression of cholecystokinin release by trypsin.

“…Evaluation of the role ofCCK in the feedback regulation ofpancreatic enzyme secretion has been impeded by nonspecific and insensitive assays for CCK. Bioassays for CCK, based on gallbladder contractions (26), are too insensitive to detect plasma CCK levels, and radioimmunoassays for CCK lack specificity due to their cross-reactivity with gastrin. Our measurements of CCK are based on release of amylase from isolated rat pancreatic acini.…”

Cholecystokinin bioactivity in human plasma. Molecular forms, responses to feeding, and relationship to gallbladder contraction.

“…In general, prior bioassays for CCK have not been sensitive enough to measure circulating levels of the hormone (12,13), and radioimmunoassays have been hampered by crossreactivity with gastrinlike substances. Estimations of circulating CCK levels by radioimmunoassay have been useful, but to distinguish CCK from gastrin, either two antibodies that differ in their ability to recognize CCK and gastrin must be used, or the plasma must be chromatographed to separate CCK from gastrin (14)(15)(16).…”

Effects of some drugs and peptide hormones on the responsiveness of the rabbit isolated gall‐bladder to cholecystokinin.

“…Each experiment therefore yielded duplicate results, and was performed at least twice. All studies were performed using the automated superfusing apparatus already described (Marshall et al 1978).…”

“…The effect of neurotransmitter blocking drugs on CCK-induced contractions For reasons already described (Marshall et al 1978), despite its impurity (0-1 % CCK) Boots Pancreozymin was used as the standard preparation of cholecystokinin throughout these experiments. The effects of the anticholinergic drug atropine sulphate, the fl-adrenergic antagonist 416 PEPTIDE HORMONES AND CCK BIOACTIVITY propranolol hydrochloride, the a-adrenoceptor antagonist phentolamine mesylate and the local anaesthetic lignocaine, on the response of the rabbit isolated gall-bladder to CCK, were studied using the automatic superfusing apparatus previously described (Marshall et al 1978).…”

“…The effects of the anticholinergic drug atropine sulphate, the fl-adrenergic antagonist 416 PEPTIDE HORMONES AND CCK BIOACTIVITY propranolol hydrochloride, the a-adrenoceptor antagonist phentolamine mesylate and the local anaesthetic lignocaine, on the response of the rabbit isolated gall-bladder to CCK, were studied using the automatic superfusing apparatus previously described (Marshall et al 1978).…”