Volume 41, Issue 1, Pages 59-63 (February 2007)

8 of 9

ABSTRACT

FULL TEXT

FULL-TEXT PDF (119 KB)

Diurnal and seasonal variation of cholecystokinin peptides in humans☆

Received 19 June 2006; accepted 15 September 2006. published online 09 January 2007.

Abstract

Cholecystokinin (CCK) was determined in plasma obtained from 10 female (aged 23.4±SD 2.3 years) and nine male (aged 22.0±SD 1.4 years) healthy volunteers. Blood samples were drawn three times (8.00a.m., 12 noon and 8.00a.m.) on each of two sessions, one in the winter (November–December) and one in the summer (April–July). The participants had fasted (and were nicotine-free) since midnight preceding the sampling. A standardized breakfast was served after the first sampling. CCK was determined by radioimmunoassay.

The area under the curve 0–24h (AUC)CCK Winter was lower than AUCCCK Summer (F1:17=4.73; P=0.0440) in the whole group of volunteers.

On comparing the CCK concentrations within each session, there was an overall difference in winter (F2:36=14.81; P<0.0001) as well in summer (F2:36=18.39; P<0.0001). Post hoc comparisons yielded a difference between the 8.00a.m. and 12 noon concentrations on the first day in winter (t=−3.96; P=0.0009) as well as in summer (t=−4.64; P=0.0002).

The difference between the summer and winter AUCsCCK correlated with the difference between AUCs for temperatures in summer and winter (r=0.58; P=0.0089). The correlation was accounted for by the females (r=0.73; P=0.0171).

The results are in accord with a diurnal and a seasonal variation of CCK in human plasma.

Keywords: Cholecystokinin, Diurnal variation, Seasonal variation

Article Outline

• Abstract

• 1. Introduction

• 2. Materials and methods

• 2.1. Subjects

• 2.2. Study design

• 2.3. Biochemical analyses

• 2.4. Statistical analyses

• 2.5. Ethics

• 3. Results

• 4. Discussion

• Acknowledgment

• References

• Copyright

1. Introduction

Cholecystokinin (CCK) is a gastrointestinal peptide that is released into the circulation from endocrine cells as well as neurons in the gastrointestinal tract. Together with gastrin and secretin, CCK constitutes a classical gut hormone triad. CCK regulates gallbladder contraction, pancreatic enzyme secretion, intestinal motility, satiety signalling and inhibition of gastric acid secretion (Rehfeld, 2004).

Like two other gut peptides, vasoactive intestinal polypeptide (VIP) and gastrin, CCK is also present in the central nervous system (Rehfeld and Kruse-Larsen, 1978), where it induces excitation of central neurons (Boden and Hill, 1988) but also has inhibitory postsynaptic effects (MacVicar et al., 1987). The CCK octapeptide (CCK-8) and tetrapeptide (CCK-4) are both implicated in behavioural and physiological functions such as satiety, anxiety and pain (Abelson, 1995) and might also play a role in the pathogenesis of panic disorder (Hösing et al., 2004), schizophrenia (Wang et al., 2002) and abuse (Crespi, 1998).

Whether or not CCK peptides are transferable between the central and peripheral compartments is a critical question. In animal experiments, CCK-8 injected in the lateral ventricle diffuses rapidly into the blood while intravenous administration does not affect the cerebrospinal fluid (Passaro et al., 1982). On the other hand, the fact that intravenous injection of CCK-4 induces panic-like attacks (Bradwejn, 1993) might be in accord with a transfer in the opposite direction.

Another field of interest is whether or not CCK peptides have a diurnal and/or seasonal variation. In the rat hypothalamus, CCK-8 has a diurnal rhythm with the lowest level at the onset of darkness (7.00p.m.) and the highest level at the time for lights on (7.00a.m.) (Nicholson et al., 1983). Interestingly, CCK-8 is present in the suprachiasmatic nucleus, which is known to be involved in body rhythm endogenous mechanisms (van den Pol and Tsujimoto, 1985).

The aim of the present study was to investigate whether CCK has a diurnal and/or seasonal variation in human plasma.

2. Materials and methods

2.1. Subjects

Nineteen healthy subjects, nine male and 10 female students, were recruited through advertisements. Pregnancy, ongoing severe somatic/physical disease, a history of mental illness, ongoing medication or participation in contemporary experimental studies disqualified from participation. The volunteers were subjected to a medical check-up including physical examination and blood laboratory tests. The females had a regular normal menstrual cycle and were not using hormonal contraceptives.

2.2. Study design

The study comprised two sessions, one in the winter (November–December) and another in the summer (April–July). Each session comprised two days. Blood samples were drawn on the first day at 8.00a.m. and 12 noon and on the second day at 8.00a.m. The participants had fasted (and were nicotine-free) since 12 midnight prior to the sampling. After samples had been drawn at 8.00a.m. a standardized breakfast was served after which the subjects remained fasting until sampling at 12 noon. In female volunteers, blood samples were drawn in the follicular phase, within seven days from the onset of menstruation. Blood was collected in tubes containing ethylenediamine tetraacetic acid (EDTA), cooled immediately and centrifuged at 4°C and 2500g for 10min. Plasma was separated and frozen within one hour and stored at −70°C until analysed.

2.3. Biochemical analyses

CCK was analysed by radioimmunoassay. 0.5ml plasma was extracted with 1ml 96% ethanol, incubated, centrifuged, evaporated and resolved in 0.5ml assay buffer (0.11% bovine serum albumin in 0.05M barbiturate buffer, pH 8.6). Samples of 0.2ml were analysed in duplicates and calibrators of 0.2ml were analysed in triplicates. After adding 0.1ml CCK antiserum (92128) (Rehfeld, 1998a) with final dilution 1/10000 in assay buffer, samples were incubated at 6°C for 24h. A second incubation in the same manner was performed after adding 0.1ml 125I-CCK (diluted to 5000cpm ∗ 10% in assay buffer). Free and bound tracer was separated using 500μl sheep-anti-rabbit serum (Pharmacia Decanting Suspension III) with incubation at room temperature for 30min and centrifugation (1500g, 21°C, 10min). The supernatant was discarded and the precipitate was measured in a gamma counter. The limit of detection was 0.4pmol/l CCK using synthetically sulphated CCK-8 as a calibrator. For data on the specificity of high-affinity and high-titre CCK antisera, see Rehfeld (1998b). The inter-assay coefficient of variation (CV) was <13% for controls.

Blood samples for analyses of melatonin, cortisol and oestradiol were centrifuged at 3000rpm for 10min within 30min. Melatonin was analysed by a competitive immunoassay method (Bühlmann Laboratories AG, Switserland) with reagents provided by Skaffe Medlab (Onsala, Sweden). The interassay and intra-assay coefficients of variation were 6.6% and 7%, respectively. Cortisol was determined by a competitive fluoroimmunoassay method with reagents provided by Wallac Oy (Turku, Finland). Oestradiol was analysed by electrochemiluminescence immunoassay, ECLIA, on the Roche Elecsys 2010 immunoassay analyser, all supplied by Roche Diagnostics Scandinavia Inc.

2.4. Statistical analyses

The StatView 5 (SAS Institute Inc., Cary NC) program was used. All variables were considered to be normally distributed. Using the linear trapezoidal method, the area under the curve 0–24h (AUC) was calculated for CCK and for the temperature for each of the two sessions.

2.5. Ethics

Approval for the study was given by the Ethics Committee of the Linköping University Hospital. All subjects gave their informed consent after having been fully informed about the study. The study was conducted according to the principles embodied in the Declaration of Helsinki.

3. Results

Basal data are given in Table 1, Table 2, Table 3, Table 4. The concentrations of melatonin, cortisol and estradiol did not differ between sessions (Table 2).

Table 1.

Basal data on healthy volunteers (means±SD)


	Total (n=19)	Females (n=10)	Males (n=9)
Age (years)	22.7±2.0	23.4±2.3	22.0±1.4
Height (cm)	176.2±9.9	169.3±6.9	183.7±6.7
Body weight (kg)	69.9±11.1	64.0±8.1	76.6±10.5
Body mass index (BMI) (body weight (kg)/height (m2))	22.4±2.3	22.3±2.6	22.6±1.9

Table 2.

Oestradial (pmol/l), melatonin (nmol/l) and cortisol (nmol/l) in plasma (mean ±SD)


	Total (n=19)	Females (n=10)	Males (n=9)
Oestradiol in plasma
Winter
Day 1 8.00a.m.	106.0±49.4	113.4±66.6	97.6±18.9
Summer
Day 1 8.00a.m..	117.3±59.0	128.2±79.5	105.1±19.7

Melatonin in plasma
Winter
Day 1 8.00a.m.	0.40±0.23 (n=17)a	0.44±0.27 (n=9)a	0.37±0.20 (n=8)a
Day 2 8.00a.m	0.41±0.28	0.41±0.38	0.42±0.18
Summer
Day 1 8.00a.m.	0.32±0.18 (n=18)a	0.29±0.17 (n=9)a	0.35±0.29 (n=9)
Day 2 8.00a.m.	0.31±0.20	0.33±0.25	0.29±0.16

Cortisol in plasma
Winter
Day 1 8.00a.m.	440.8±94.1	487.5±82.4	389.0±80.9
Summer
Day 1 8.00a.m.	472.8±85.6	492.2±109.4	451.2±45.1

Some values missing owing to technical problems.

Table 3.

Data on CCK concentrations (mean±SD in plasma; pmol/l) and on AUCCCK (pmol×l−1×h)


	Total (n=19)	Females (n=10)	Males (n=9)
CCK in plasma
Winter
Day 1
8.00a.m.	0.65±0.20	0.66 ±0.18	0.64±0.24
12 noon	1.10±0.44	0.94±0.28	1.28±0.53
Day 2
8.00a.m.	0.71±0.20	0.71±0.18	0.70±0.24
AUCCCK	20.8±5.8	19.2±2.1	22.5±8.0

Summer
Day 1
8.00a.m.	0.72±0.20	0.70±0.12	0.74±0.28
12 noon	1.23±0.40	1.19±0.42	1.28±0.40
Day 2
8.00a.m.	0.75±0.17	0.78±0.18	0.72±0.16
AUCCCK	22.8±4.0	22.7±3.8	22.9±4.3

Table 4.

Data on atmospheric pressure (hPa) and ambient temperature (°C) (mean±SD)


	Total (n=19)	Females (n=10)	Males (n=9)
AP12.00(hPa)
Winter	1007.1±5.4	1004.3±6.0	1010.3±2.0
Summer	1004.6±5.4	1002.4 ±5.9	1007.0±5.4

Ambient temperature (°C)
Winter
8.00a.m.	0.9±4.0	0.3±3.7	1.6±4.4
12 noon	3.3±3.5	2.1±3.5	4.7±3.2
8.00a.m.	2.4±2.5	2.1±2.0	2.7±3.1
AUCtemp winter	65.6 ±56.6	46.8±55.5	86.6±53.0
Summer
8.00a.m.	4.7±4.9	5.5±4.7	3.9±5.2
12 noon	5.3±4.9	6.3±5.5	4.2±4.2
8.00a.m.	9.1±6.8	12.0±4.4	5.8±7.8
AUCtemp summer	119.4±111.4	140.5±110.8	96.0±113.7

Using a repeated measures ANOVA, with gender as the between-group factor, AUC CCK Winter was lower than AUCCCK Summer (F1:17=4.73; P=0.0440; Table 3) in the whole group of volunteers. There was no effect of gender (F1:17=0.071; NS).

On comparing the CCK concentrations within each session (Table 3), there was an overall difference in winter (F2:36=14.81; P<0.0001) as well in summer (F2:36=18.39; P<0.0001). Post hoc comparisons using the paired t-test yielded a difference between 8.00a.m. and 12 noon concentrations on the first day in winter (t=−3.96; P=0.0009) as well as in summer (t=−4.64; P=0.0002). The four 8.00a.m. levels did not differ (F3:54=1.58; NS).

The difference between the summer and winter AUCsCCK correlated with the difference between AUCs for temperature in summer and winter. (r=0.58; P=0.0089; Fig. 1). The correlation was accounted for by the females (r=0.73; P=0.0171), while there was no correlation in males (r=0.45; NS). No influence of air pressure (Table 4) was found.

View Large Image
Download to PowerPoint

Fig. 1. Correlation between ΔAUCCCK and ΔAUCtemp (summer–winter) in healthy volunteers (df=18; r=0.58; P=0.0089; Y=1.1+0.2X).

4. Discussion

The difference between AUCCCK in winter and summer (Table 3) is indicative of a seasonal variation. An elevated density of CCK (and 5-HT2) receptors in the frontal cortex of rats has been found in summer (Kõks et al., 2000). Furthermore, the thermoregulatory responses to changes in the ambient temperature in rats and mice lacking the CCK A-receptor are disturbed (Nomoto et al., 2004). Whether or not these findings are relevant to our results is obscure. At any rate, the correlation between ΔAUCCCK and ΔAUCtemperature might indicate a sensitivity to ambient temperature variation in females (but not in males).

The increased CCK concentrations at 12 noon compared with 8.00a.m. are in accord with a presumed diurnal rhythm. The fact that food intake increases the plasma CCK levels within 80min with a subsequent decline to the basal level (Rehfeld, 2004) might have influenced the results. A similar influence of food intake on other gut hormones has been reported previously (Hornnes et al., 1980). As our volunteers were given a standardized breakfast, Rehfeld’s observation can hardly explain the seasonal difference observed in our volunteers. We could not find any influence of atmospheric pressure on the plasma CCK levels. Such an influence has previously been reported for CCK in cerebrospinal fluid (Gunnarsson et al., 1999). A previous study (Eklundh et al., 2000) provided no indication of a correlation between CSF and serum levels as far as the sulphated CCK-8S is concerned. This might at least partly explain why atmospheric pressure did not exert an effect in the present study.

In conclusion, there seems to be a seasonal variation in plasma CCK with the highest levels in the summer. Evidence for an influence of the ambient temperature has been found but the mechanism behind it remains unexplained so far. Previous findings of a diurnal rhythm have been confirmed. Further research is warranted in this field.

Acknowledgements

We thank Gösta Karlsson, Department of Neurochemistry, Sahlgrenska University Hospital/Mölndal, Sweden, Capio Inc. at St Göran Hospital, Stockholm, Sweden and the Department of Clinical Chemistry at the Linköping University Hospital, Sweden for biochemical analyses. We also thank our research nurses, Mrs Gunilla Johansson, Mrs Hazel Holmberg-Forsyth and Mrs Maud Mansfield, for their most excellent assistance.

References

Abelson, 1995. 1.Abelson JL. Cholecystokinin in psychiatric research: a time for cautious excitement. J. Psychiatr. Res. 1995;29:389–396. MEDLINE | CrossRef

Boden and Hill, 1988. 2.Boden P, Hill RG. Effects of cholecystokinin and related peptides on neuronal activity in the ventromedial nucleus of the rat hypothalamus. Br. J. Pharmacol. 1988;94:246–252. MEDLINE

Bradwejn, 1993. 3.Bradwejn J. Neurobiological investigations into the role of cholecystokinin in panic disorder. J. Psychiatry Neurosci. 1993;18:178–188. MEDLINE

Crespi, 1998. 4.Crespi F. The role of cholecystokinin (CCK), CCK-A or CCK-B receptor antagonists in the spontaneous preference for drugs of abuse (alcohol or cocaine) in naïve rats, Methods Find. Exp. Clin. Pharmacol. 1998;20:679–697.

Eklundh et al., 2000. 5.Eklundh T, Gunnarsson T, Örnhagen H, Nordin C. Cerebrospinal fluid levels of monoamine compounds and cholecystokinin peptides after exposure to standardized barometric pressure. Aviat. Space Environ. Med. 2000;71:1131–1136. MEDLINE

Gunnarsson et al., 1999. 6.Gunnarsson T, Eklundh T, Eriksson M, Sjöberg S, Nordin C. Cholecystokinin peptides in cerebrospinal fluid: a study in healthy male subjects lumbar-punctured without preceding strict bed-rest. J. Neural Transm. 1999;106:275–282. MEDLINE | CrossRef

Hornnes et al., 1980. 7.Hornnes PJ, Kühl C, Holst JJ, Lauritsen KB, Rehfeld JF, Schwartz TW. Simultaneous recording of the gastro-entero-pancreatic hormonal peptide response to food in man. Metabolism. 1980;29:777–779. MEDLINE | CrossRef

Hösing et al., 2004. 8.Hösing VG, Schirmacher A, Kuhlenbaumer G, Freitag C, Sand P, Schlesiger C, et al. Cholecystokinin- and cholecystokinin-B-reeptor gene polymorphisms in panic disorder. J. Neural Transm. 2004;68:147–156.

Kõks et al., 2000. 9.Kõks S, Männistö PT, Bourin M, Shlik J, Vasar V, Vasar E. Cholecystokinin-induced anxiety in rats: relevance of pre-experimental stress and seasonal variations. J. Psychiatry Neurosci. 2000;25:33–42. MEDLINE

MacVicar et al., 1987. 10.MacVicar BA, Kerrin JP, Davison JS. Inhibition of synaptic transmission in the hippocampus by cholecystokinin (CCK) and its antagonism by a CCK-analog (CCK27-33). Brain Res. 1987;406:130–135. MEDLINE | CrossRef

Nicholson et al., 1983. 11.Nicholson SA, Adrian TE, Bacarese-Hamilton AJ, Gillham B, Jones MT, Bloom SR. 24-hour variation in content and release of hypothalamic neuropeptides in the rat. Regul. Pept. 1983;7:385–397. MEDLINE | CrossRef

Nomoto et al., 2004. 12.Nomoto S, Ohta M, Kanai S, Yoshida Y, Takiguchi S, Funakoshi A, et al. Absence of the cholecystokinin-A receptor deteriorates homeostasis of body temperature in response to changes in ambient temperature. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2004;287:R556–R561. MEDLINE | CrossRef

Passaro et al., 1982. 13.Passaro E, Debas H, Oldendorf W, Yamada T. Rapid appearance of intraventricularly neuropeptides in the peripheral circulation. Brain Res. 1982;241:335–340. MEDLINE

Rehfeld and Kruse-Larsen, 1978. 14.Rehfeld JF, Kruse-Larsen C. Gastrin and cholecystokinin in human cerebrospinal fluid. Immunochemical determination of concentrations and molecular heterogeneity. Brain Res. 1978;155:19–26. MEDLINE | CrossRef

Rehfeld, 1998a. 15.Rehfeld JF. Accurate measurement of cholecystokinin in plasma. Clin. Chem. 1998;44:991–1001. MEDLINE

Rehfeld, 1998b. 16.Rehfeld JF. How to measure cholecystokinin in tissue, plasma and cerebrospinal fluid. Regul. Pept. 1998;78:31–39. MEDLINE | CrossRef

Rehfeld, 2004. 17.Rehfeld JF. Cholecystokinin. Best. Pract. Res. Clin. Endocrinol. Metab. 2004;18:569–586. | CrossRef

van den Pol and Tsujimoto, 1985. 18.van den Pol AN, Tsujimoto KL. Neurotransmitters of the hypothalamic suprachiasmatic nucleus: immunocytochemical analysis of 25 neuronal antigens. Neuroscience. 1985;15:1049–1086. MEDLINE | CrossRef

Wang et al., 2002. 19.Wang Z, Wasslink T, Andreasen N, Crowe RR. Possible association of a cholecystokinin promoter variant in schizophrenia. Am. J. Med. Genet. 2002;114:479–482. MEDLINE | CrossRef

a Department of Neuroscience and Locomotion, Psychiatry Section, Faculty of Health Sciences, Linköping University, SE-581 85 Linköping, Sweden

b Department of Molecular and Clinical Medicine, Division of Obstetrics and Gynaecology, Faculty of Health Sciences, Linköping University, SE-581 85 Linköping, Sweden

Corresponding author. Tel.: +46 13 22 38 64; fax: + 46 13 22 33 92.

☆ This study was supported by Grants from the County Council of Östergötland, Sweden.

PII: S0143-4179(06)00114-4

doi:10.1016/j.npep.2006.09.049

8 of 9