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Gastroentérologie Clinique et Biologique
Vol 22, N° 1  - février 1998
p. 25
Doi : GCB-02-1998-22-1-0399-8320-101019-ART76
Plasma cholecystokinin and neurotensin after an ordinary meal in humans
 

Lucio GULLO, Raffaele PEZZILLI, Paola TOMASSETTI, Roberto DE GIORGIO
Plasma cholecystokinin and neurotensin after an ordinary meal in humans
A prolonged time study

Lucio GULLO, Raffaele PEZZILLI, Paola TOMASSETTI, Roberto DE GIORGIO

Unit for the Study of Pancreatic Disease, Department of Internal Medicineand Gastroenterology, St. Orsola Hospital, University of Bologna, Bologna,Italy.

S

UMMARY

Background/Aim. -- Ingestion of a meal causes the release of cholecystokininand neurotensin into the circulation, but little is known about the durationof this release.

Methods. -- Six healthy volunteers were studied. Blood samples forcholecystokinin, neurotensin and gastrin assessment were drawn before andafter consumption of a typical Italian lunch. Postprandial samples wereobtained every hour for a total of 10 hours. All peptides were measuredusing previously validated radioimmunoassays.

Results. -- Ingestion of the meal caused a prompt and significantincrease in plasma levels of all three peptides. Cholecystokinin remainedelevated for about 7 hours and then tended to return towards basalvalues, whereas the increase of neurotensin persisted for the entire periodof the study (10 hours). Gastrin remained elevated for about 5 hoursand then declined. The integrated CCK and gastrin responses during the initialpostprandial hours were greater than those in the late hours, whereas theintegrated neurotensin response during the initial hours was lower thanthat in the late hours.

Conclusions. -- The results indicate that, after an ordinary meal,cholecystokinin is released into the circulation for about 7 hours,much longer than previously reported (3-4 hours). The release of neurotensinbegins soon after the meal and persists even longer, for at least 10 hours.Possible physiological implications of these findings are discussed.

Key words:

Gut peptides -- Cholecystokinin-- Neurotensin -- Gastrin -- Postprandial release.

R

ÉSUMÉ

Evolution des concentrations plasmatiques de cholécystokinineet de neurotensine chez l'homme à la suite d'un repas standard.

But de l'étude. -- L'ingestion d'un repas entraîne lalibération dans le sang de cholécystokinine et de neurotensine.L'étude a pour but d'établir la cinétique de cettelibération.

Méthodes. -- L'étude a porté sur 6 volontairessains. Des échantillons de sang ont été prélevésà jeun, puis à la suite d'un repas italien standard, toutesles heures pendant 10 h. Les concentrations de cholécystokinine,de neurotensine et de gastrine ont été estimées dansles prélèvements à l'aide de dosages radio-immunologiquesvalidés précédemment.

Résultats -- Le repas a provoqué une élévationrapide et significative des taux plasmatiques des trois peptides. Les tauxde cholécystokinine et de gastrine sont restés élevéspendant 7 h et 5 h, respectivement, avant de revenir progressivementà leur valeur basale. En revanche, le taux de neurotensine est restéélevé pendant les 10 h de l'étude. La réponseintégrée de gastrine et de cholécystokinine a étéplus élevée au début de la période postprandiale,alors que celle de la neurotensine a été plus élevéedans les 5 dernières heures de l'étude.

Conclusions. -- Après un repas standard, la cholécystokinineest libérée dans le sang pendant environ 7 h, alors quedes résultats antérieurs suggéraient que la libérations'interrompait après 3-4 heures. La libération de neurotensineest plus longue et dépasse 10 heures. Ces données pourraientaider à mieux comprendre le rôle physiologique de la cholécystokinineet de la neurotensine.

Mots-clés :

Peptides intestinaux-- Cholécystokinine -- Neurotensine -- Gastrine -- Libérationpostprandiale.

It is well established that ingestion of a meal causes the releaseof cholecystokinin (CCK) and neurotensin, among other gut hormones, intothe circulation (1-13) ; however, very limited information is availableabout the duration of the release of these gastrointestinal peptides. Moreover,there is also some discordance in the literature regarding CCK : whilesome investigators (4) have found that the postprandial plasma elevationof this peptide peaks at about 60 min and then declines rapidly tonear basal values after 3 to 4 hours, others (1-3, 5) have reporteda persistence of the elevation during the same 3 to 4 hour period.Since none of the authors have studied the postprandial pattern of plasmaCCK beyond 4 hours (1-5), plasma levels of this hormone during thelate postprandial phase have not been delineated. Similarly, most studiesof the postprandial plasma levels of neurotensin have not exceeded 4-5 hoursin duration (6-13) ; thus, it is not well defined whether this peptideis released into the circulation by the arrival of chyme in the distal ileum,where neurotensin cells are maximally concentrated (14) and, if so, to whatextent.

The present study was undertaken to more preciselydetermine the duration of CCK and neurotensin release into plasma afteran ordinary meal in healthy volunteers. In addition, plasma levels of gastrinwere also measured.

MATERIALS AND METHODS

S

TUDY SUBJECTS

Six healthy volunteers, 4 males and 2 females(aged : 24-28 years, mean : 25 years) were studied.They were university students, free of any digestive disorders, who werefully informed about the nature and scope of the study and who gave writtenconsent. None of them was either smoker or drinker.

E

XPERIMENTAL DESIGN

On the study day, subjects had their usual breakfast(cappuccino and croissant) at 7 :30 a.m. ; subsequently,they fasted until lunch, which was eaten at 12 :30 pm. A typicalItalian meal, consisting of spaghetti with tomato sauce, beef steak, salad,cheese, bread, fruit and water (protein : 54 g, fat : 50 g,carbohydrates : 111 g, total calories : 1 090), wasserved to them in a good quality restaurant located near the hospital wherethe studies were performed. This type of meal was decided by volunteersthemselves according to their usual dietary habits. Consumption of the mealtook about 40 minutes. After lunch, subjects were taken to a quietand comfortable room at our Institute, where they spent the remainder ofthe study period in reading and conversation. During the postprandial period,none of them complained of epigastric fullness or other digestive symptoms.Studies were performed on two subjects per day on three separate days.

Blood samples for hormone assessment were drawnat - 30 and 0 min before lunch, and every hour aftercompletion of the meal, for a total of 10 hours. In the postprandialperiod, an indwelling catheter was inserted into an antecubital vein tofacilitate blood drawing. All blood samples were collected into ice-coldheparinized tubes containing 4 000 kallikrein inhibitory unitsof aprotinin (Trasylol, Bayer, Italy). The samples were immediately centrifugedat 4°C and the separated plasma was frozen and stored at - 20°Cwithin 5 minutes.

R

ADIOIMMUNOASSAY

CCK and neurotensin were measured using previouslydescribed radioimmunoassays which have been widely validated (12,15). Briefly,CCK was assessed using the rabbit polyclonal T204 antibody which bindsthe sulphated tyrosine region of all CCK peptides, including CCK-8 andother larger biologically active molecular forms (CCK-33, CCK-39 andCCK-58). T204 antibody does not bind to unsulphated gastrins, showsless than 2 % cross-reactivity with sulphated gastrins whereas it doesnot cross-react with structurally unrelated peptides (e. g. insulin, glucagon,pancreatic polypeptide, peptide YY, neuropeptide Y and neurotensin) (15).The intraassay and interassay variations were 8 % and 11 %, respectively.The detection limit was 0.5 pmol/L.

Neurotensin was measured by using the rabbitC-terminally directed antibody (NT58) (12). The cross-reactivity of NT58 withneurotensin fragments was as follows : 66 % with neurotensin 6-13,83 % with neurotensin 7-13, 10 % with neurotensin 8-13, and lessthan 0.2 % with neurotensin 1-8 and 1-9. The antibody showed nocross-reactivity with other regulatory peptides, including peptide YY andneuropeptide Y. The intraassay and interassay variations were 4.3 %and 9 %, respectively. The detection limit was 1.8 pmol/L.

Plasma gastrin was measured using a commerciallyavailable radioimmunoassay (Becton Dickinson kit, USA). The assay fullydetected gastrin-34, but showed < 1 % cross-reactivitywith CCK. The intraassay and interassay variations were 4 % and 9.6 %,respectively. The detection limit was 2.0 pmol/L.

The radioimmunoassays were performed on non-extractedplasma. Plasma hormone concentrations were expressed in pmol/L.

S

TATISTICAL ANALYSIS

For each subject the mean of the two hormoneconcentrations at - 30 and 0 min before lunch was comparedwith the concentrations after lunch by Student's t-test for paired data.The areas under the curve for each hormone were evaluated for initial (0-5 hours)and final (5-10 hours) postprandial periods by means of the trapezoidalrule after subtraction of the basal value. Results are presented as means ±SEM. P values < 0.05 were considered statistically significant.

RESULTS

Figure 1 shows the mean values ± SEMof plasma CCK, gastrin and neurotensin concentrations before and after theingestion of the meal in the 6 healthy volunteers. The hormone concentrationsat - 30 and 0 min before the meal were not significantlydifferent. The meal induced a prompt and statistically significant increasein all three hormones studied. CCK remained significantly elevated for about7 hours and then showed an evident tendency to decrease. Gastrin persistedelevated for about 5 hours, and then declined towards the basal value.By contrast, the increase in plasma neurotensin persisted for the entireperiod of the study (10 hours). (Voir Figure)

The areas under the curve of CCK and gastrinfor the initial 5 postprandial hours (23.7 ± 7.6 pmol/Land 40.9 ± 7.1 pmol/L, respectively) were significantlygreater (P < 0.05) than those of the final 5 postprandialhours (15.5 ± 4.8 pmol/L and 6.2 ± 3.7 pmol/L,respectively). For neurotensin, the area under the curve during the final5 postprandial hours (188.4 ± 24.0 pmol/L)was greater than that in the initial 5 hours (138.4 ± 16.3 pmol/L),although the difference was not statistically significant.

DISCUSSION

The specific purpose of this work was to studythe time course of plasma CCK and neurotensin for a lengthy postprandialperiod under strict physiological conditions. The results have shown thatthe rise in plasma concentrations of these gut peptides produced by an ordinarymeal is much more prolonged than previously reported (1-13).

With regard to CCK, this peptide increased rapidlyand significantly after the meal and the increase persisted about 7 hours ;moreover, the integrated plasma secretion during the first 5 hourswas greater than that of the subsequent 5 hours, which is in keepingwith the distribution of CCK cells mainly in the upper portion of the smallintestine (16).

Our findings do not agree with those of Friedet al. (4) who emphasized that plasma levels of CCK after a mixed solidmeal peak at 30-60 min and then decline rapidly, reaching almost basalvalues 3-4 hours postprandially, despite continued entry of chyme intothe duodenum. These investigators speculated that this early postprandialdecrease in CCK was due to the release of factors that inhibit CCK release,in particular peptide YY. The reason for the discrepancy is not clear, thoughmarked differences in experimental conditions between the two studies areprobably partly responsible ; indeed, while we reproduced normal eatingconditions as closely as possible, Fried's volunteers were subjected tointubation with multilumen intestinal tubes and duodenal aspiration, whichare far from physiological conditions. Moreover, differences in the mealsused and also in the time of day that the meal was taken could have playeda role. However, the early postprandial decrease in plasma CCK describedby Fried et al. (4) was not seen by any of the other investigators, allof whom found a persistent elevation for the 2 to 4 hour durationof their studies, whether the meal was solid (5) or liquid (1-3). In a studyin the dog (17), plasma CCK was determined for 5 hours postprandially,and the rise persisted for the whole 5 hours.

The prolonged postprandial rise of plasma CCKseen in this study is consistent with a continued release of the peptideduring jejunal transit of nutrients, even after gastric emptying of chymeinto the duodenum has ceased, which usually takes about 4-5 hours (18, 19).

With regard to neurotensin, the results of thisstudy clearly showed that ingestion of an ordinary meal causes a promptand very prolonged rise in plasma levels of this peptide, lasting at least10 hours ; moreover, the integrated plasma neurotensin secretionduring the late postprandial hours was greater than that in the initialhours. These data would be consistent with the greater concentration ofneurotensin cells in the distal ileum (14) and with a marked release ofthis peptide by the arrival of nutrients in this intestinal segment. Thereason for the rather rapid postprandial increase in plasma neurotensin,which has also been reported by others (6-13), is unclear. One possibilityis that some neurotensin can be released by the action of other releasingpeptides and/or by nervous reflexes originating in the upper gastrointestinaltract.

As for gastrin, plasma levels of this hormoneincreased rapidly and significantly after the meal in agreement with previousstudies (20, 21). The rise in gastrin levels remained fairly stablefor about 5 hours and then declined, providing indirect evidence thatgastric emptying of chyme in our subjects was completed within this period,as would be expected (18, 19).

We believe that our results may have some implicationsfor the understanding of the regulation of digestive functions. For example,we (22) and others (23, 24) have shown, in humans or in dogs, thatpancreatic stimulation by a meal lasts much longer than the 3-4 hoursthat had generally been reported in the past (20, 25) ; whileit is known that the stimulation during the first 3 to 4 hoursis mediated by neurohormonal mechanisms, including CCK, originating in thegastroduodenal tract (26), the mechanisms involved from the fourth houron are unknown. In light of the present results, it is reasonable to supposethat CCK and especially neurotensin could have a role in the control ofthe late phase of pancreatic stimulation by a meal. Clearly, this remainsto be verified because we did not measure pancreatic secretion. This measurement,as well as evaluation of gastrointestinal transit of food, was not donesince our study was intentionally designed to reproduce physiological conditionsas closely as possible, thus precluding the use of tubes in the intestinallumen.

Another implication of this study regards appetiteregulation : elevated plasma CCK has been reported to produce feelingof satiety (27, 28) ; thus, it is possible that the 6 to7 hours of satiety typically produced by an abundant meal could havesome relationship with the persistence of plasma CCK elevation for thisprolonged period.

In conclusion, our study provides new informationabout the time course of plasma CCK and neurotensin under physiologicalconditions after an ordinary meal. The significance of the prolonged persistenceof elevated postprandial plasma levels of CCK and neurotensin remains tobe clarified.

A

CKNOWLEDGEMENTS

The present work was partially supported by researchfunds from University of Bologna (Fondi 60 %). We wish to thank ProfessorJ.B.M.J. J

ANSEN

for radioimmunoassays. Dr. R. D

E

G

IORGIO

is the recipient of a « Dottoratodi Ricerca » in Pancreatic Pathophysiology from the Italian Ministryof University Research, Science and Technology (MURST).

Fig. 1. -- Plasma CCK, gastrin and neurotensin concentrations beforeand after an ordinary meal in 6 healthy volunteers. Means ± SEM.B1 and B2 indicate - 30 and 0 min before themeal. * P < 0.05 ; ** P < 0.005,compared with the basal value (mean of B1 and B2).

Concentrations plasmatiques de CKK, gastrine et neurotensine, observéesavant et après un repas ordinaire chez 6 sujets volontairessains (moyenne ± ESM). B1 et B2 correspondent aux temps - 30min et 0 min avant le repas.

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