Preparation and application of a novel molecularly imprinted solid-phase microextraction monolith for selective enrichment of cholecystokinin neuropeptides in human cerebrospinal fluid.

A novel molecularly imprinted polymer (MIP) monolith for highly selective extraction of cholecystokinin (CCK) neuropeptides was prepared in a micropipette tip. The MIPs were synthesized by epitope imprinting technique and the polymerization conditions were investigated and optimized. The synthesized MIPs were characterized by infrared spectroscopy, elemental analyzer and scanning electron microscope. A molecularly imprinted solid-phase microextraction (MI-µ-SPE) method was developed for the extraction of CCK neuropeptides in aqueous solutions. The parameters affecting MI-µ-SPE were optimized. The results indicated that this MIP monolith exhibited specific recognition capability and high enrichment efficiency for CCK neuropeptides. In addition, it showed excellent reusability. This MIP monolith was used for desalting and enrichment of CCK4, CCK5 and CCK8 from human cerebrospinal fluid prior to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis, and the results show that this MIP monolith can be a useful tool for effective purification and highly selective enrichment of multiple homologous CCK neuropeptides in cerebrospinal fluid simultaneously. By employing MI-µ-SPE combined with HPLC-ESI-MS/MS analysis, endogenous CCK4 in human cerebrospinal fluid was quantified.

Cholecystokinin antibody injected in cerebral ventricles stimulates feeding in sheep.

The role of brain cholecystokinin peptides in satiety was further assessed by using antibody to cholecystokinin to reduce cholecystokinin activity in the cerebrospinal fluid of sheep. Food intakes were increased approximately 100 percent during the 2-hour continuous injection of antibody into the cerebrospinal fluid. This supports the hypothesis that, during feeding, cholecystokinin is released into the cerebrospinal fluid, which transports it to the receptors that elicit satiety.

Cholecystokinin and Alzheimer's disease: a biomarker of metabolic function, neural integrity, and cognitive performance.

Cholecystokinin (CCK) is a satiety hormone that is highly expressed in brain regions like the hippocampus. CCK is integral for maintaining or enhancing memory and thus may be a useful marker of cognitive and neural integrity in participants with normal cognition, mild cognitive impairment, and Alzheimer's disease (AD). Cerebrospinal fluid (CSF) CCK levels were examined in 287 subjects from the Alzheimer's Disease Neuroimaging Initiative. Linear or voxelwise regression was used to examine associations between CCK, regional gray matter, CSF AD biomarkers, and cognitive outcomes. Briefly, higher CCK was related to a decreased likelihood of having mild cognitive impairment or AD, better global and memory scores, and more gray matter volume primarily spanning posterior cingulate cortex, parahippocampal gyrus, and medial prefrontal cortex. CSF CCK was also strongly related to higher CSF total tau (R2 = 0.342) and p-tau-181 (R2 = 0.256) but not Aß1-42. Tau levels partially mediated CCK and cognition associations. In conclusion, CCK levels may reflect compensatory protection as AD pathology progresses.

Decreased cholecystokinin levels in cerebrospinal fluid of patients with adult chronic hydrocephalus syndrome.

Cholecystokinin (CCK) levels were measured in cerebrospinal fluid (CSF) of patients with adult chronic hydrocephalus syndrome (ACHS) (n = 16) and compared with levels from a control group (n = 11). The CSF concentration of CCK in the ACHS group (0.79 +/- 0.53 fmol/mL) was significantly reduced (p = .002) with respect to the controls (1.55 +/- 0.54 fmol/mL). As CCK-8, the most prevalent from of CCK in the central nervous system, has been demonstrated to play a significant role in several physiological and behavioral actions, the reduced octapeptide values found in ACHS could be involved in the disturbances associated with this disorder. Continuous monitoring of intracranial pressure (ICP) demonstrated different ICP profiles in ACHS. We found that all patients with abnormal ICP records except one showed CCK values under the detection limit. Three of the 4 patients with normal ICP had CCK levels within the normal range. These preliminary studies could evidence that ICP alterations are responsible for part of the loss of brain neuropeptide levels in ACHS.

CSF cholecystokinin concentrations in patients with panic disorder and in normal comparison subjects.

Cholecystokinin concentrations in the CSF of 25 patients with panic disorder and 16 normal comparison subjects were ascertained by radioimmunoassay. The patients with panic disorder had significantly lower CSF concentrations of cholecystokinin, which may reflect increased CNS cholecystokinin receptor sensitivity, reduced numbers of receptors, or a compensatory reduction in cholecystokinin octapeptide secondary to theoretically increased central cholecystokinin tetrapeptide activity.

Cholecystokinin and neurotensin gradients in human CSF.

In successive samples of human lumbar CSF, concentrations of two neurally active peptides, cholecystokinin (CCK) and neurotensin (NT), were compared with levels of homovanillic acid (HVA), the major metabolite of dopamine. Although HVA values progressively increased between the first and 20th milliliter samples, no significant change occurred in the concentration of either peptide. Thus, lumbar CSF levels of CCK and NT, unlike levels of HVA, may not closely reflect amounts of these peptides in supraspinal CSF or brain.

Neuronal cholecystokinin and schizophrenia: pathogenic and therapeutic studies.

Neuroleptic-free schizophrenic patients received caerulein, a potent analogue of cholecystokinin octapeptide, in a fixed- and rising-dose schedule. In addition, neuroleptic-treated patients received a single dose of the peptide with a 4-week follow-up. No significant change in mental status was observed after any of these administration schedules. Peak plasma levels of caerulein were noted at 20-30 min after IM administration; at this time no changes in cortical evoked potential were demonstrated. Furthermore, levels of cholecystokinin were not found to be reduced, but were in fact elevated in lumbar cerebrospinal fluid of schizophrenic patients. These data argue against the antipsychotic efficacy of systemic caerulein administration and, because evidence of CNS response to CCK is lacking, suggest that other pharmacologic strategies may be necessary to effectively modify central peptide systems with systemically administered drugs.

Increased level of cholecystokinin in cerebrospinal fluid is associated with chronic pain-like behavior in spinally injured rats.

Cholecystokinin (CCK) is a physiological antagonist of opioid-mediated antinociception and may be involved in some chronic pain states where opioids have reduced effect. We have previously shown in a rat model of central neuropathic pain after spinal cord injury that blockade of CCK-B receptors lead to marked pain relief. In the present study, we showed that spinally injured rats exhibiting chronic pain-like behaviors (aversive reaction to innocuous mechanical and cold stimulation) had significantly elevated level of CCK-like immunoreactivity in cerebrospinal fluid compared to normal rats or spinally injured rats which did not exhibit pain-like behaviors. The increased level of circulating CCK in the cerebrospinal fluid may thus contribute to the maintenance of chronic pain in these rats by reducing the endogenous inhibitory tone provided by opioid peptides and may be involved in the phenomenon of opioid insensitivity.

CCK-octapeptide injected in CSF decreases meal size and daily food intake in sheep.

Cholecystokinin octapeptide (CCK-OP) is a potent and specific suppressor of feeding when administered as a continuous lateral cerebral ventricular injection in fasted sheep, and we have proposed that endogenous CCK-OP in the brain is released during meals and acts to terminate feeding. In previous studies, however, only relatively short-term effects of CCK-OP on feeding were examined. In the first experiment of the present series sheep were adapted to a 6-hr feeding period per day. CCK-OP injected continuously for 6 hr into the lateral ventricles reduced feeding during the entire feeding period (809 +/- 72 g, sham; 695 +/- 71 g, carrier; 505 +/- 69 g, CCK-OP; p less than 0.05). In addition mean feed intake for the two days (injection + first post injection day) was significantly reduced by CCK-OP; thus with CCK-OP, sheep did not compensate by the day after injection for the decreased feed intake on injection day. In a second experiment CCK-OP was injected into the lateral ventricles only during four consecutive 15 min meals 2 hours apart. With a dose of 0.159 pmoles/min CCK-OP, size of the second meal was reduced, but with 0.638 pmoles/min CCK-OP feeding during each of the first two meals was reduced and cumulative intake for the four meals was decreased. These results indicate that CCK-OP administered centrally can have long-term effects on feeding, and under appropriate conditions, could result in negative energy balance.

Cholecystokinin and panic disorder--three unsettled questions.

The serendipitously discovered panicogenic effect of the cholecystokinin fragment, the C-terminal tetrapeptide amide (CCK-4), has suggested that the widespread network of CCK neurons and corresponding CCK-B receptors in the brain are in some way involved in pathogenesis panic disorders in man. Two decades of research have now established that exogenous CCK-4 in a reproducible, dose-dependent and sensitive manner indeed evokes panic attacks in both healthy subjects and at even lower doses in anxiety patients. But several questions about the molecular mechanisms by which endogenous CCK peptides may precipitate panic attacks remain to be answered. This review focuses on three immediate questions. (1) Does endogenous CCK-4 exist? (2) Is the panicogenic effect mediated only through CCK-B receptors? (3) Are measurements of CCK peptides in cerebrospinal fluid of use in elucidating the pathogenesis and/or diagnosis? This review concludes that the answers to these questions may further the understanding of panic disorder substantially, and hence contribute to improved diagnosis and therapy of the disease.

How to measure cholecystokinin in tissue, plasma and cerebrospinal fluid.

This review examines a major problem for an old hormone. Hormones are defined by the ability to reach their targets via blood. Consequently, knowledge about a hormone requires measurement of its behaviour in blood. So far, however, it has proven exceptionally difficult to measure the classical gut hormone, cholecystokinin (CCK), in circulation. The review therefore describes the premises for reliable plasma CCK measurements as compared to the premises for measurement in tissue extracts and cerebrospinal fluid. The critical plasma premises comprise equimolar quantitation of the bioactive CCK peptides in circulation (CCK-83, -58, -33, -22 and -8) without interference from homologous gastrin peptides. The latter may appear nearly impossible, because the bioactive epitopes of CCK and gastrin are almost identical, and because the plasma concentrations of gastrin are more than tenfold above those of CCK. In comparison, measurement of CCK in tissue is considerably simpler, especially in extracts of the two main production sites, the brain and jejunoileal mucosa. For cerebrospinal fluid, degradation, low levels and shortage of material constitute major problems so that the molecular nature and biological/clinical relevance of CCK measurements in CSF still remain to be settled. The review finally enlists the reports on plasma CCK measurements published so far. A multitude of different immuno- and bioassays have been used with corresponding variation in the results. The theory for different types of assays in combination with general assay experience suggest that accurate CCK measurements require radioimmunoassay technology based on high-affinity antibodies. These antibodies have to be exquisitely specific for the 0-sulfated C-terminal heptapeptide amide of CCK without binding the similar gastrin epitope. Only few of such antibodies have been raised.

Effect of sulpiride or paroxetine on cerebrospinal fluid neuropeptide concentrations in patients with chronic tension-type headache.

In lumbar cerebrospinal fluid (CSF) obtained from patients with chronic tension-type headache (CTH), the concentrations of beta-endorphin, met-enkephalin, dynorphin, cholecystokinin (CCK), calcitonin gene-related peptide (CGRP), and somatostatin were measured before and after 8 weeks of treatment with sulpiride or paroxetine. We previously reported higher than normal met-enkephalin concentrations in CTH. The present study reveals normal basal concentrations of CCK, CGRP and somatostatin and slightly decreased dynorphin in the same patients. Treatment with sulpiride or paroxetine did not change the concentration of any of the neuropeptides measured. These data suggest central changes in opioid systems but not in other peptide systems (CCK, CGRP, somatostatin) involved in nociceptive processing at the level of the spinal cord dorsal horn/nucleus caudalis of the trigeminal nerve in CTH. Such central changes might be pathophysiologically important or merely secondary to other more important occurrences. The lack of changes in neuropeptide concentrations during drug treatment makes planning of studies involving CSF analysis easier, but also limits the probability of obtaining information on specific neuropeptide systems through CSF analysis.

Gastrin and cholecystokinin in human cerebrospinal fluid. Immunochemical determination of concentrations and molecular heterogeneity.

To determine whether gastrin and cholecystokinin (CCK), recently found in the central nervous sytem, were present in cerebrospinal fluid (CSF), we studied human specimens by sensitive and specific radioimmunoassays for the two related polypeptide hormones. The concentration of gastrin in cerebrospinal fluid from 10 neurologically normal persons ranged from 1.5 to 8.0 pM (mean 3.4 pM), whereas the concentration of CCK ranged from 4 to 55 pM (mean 14 pM). The molecular heterogeneity of gastrin and CCK in CSF was determined by gel chromatography of concentrated fluid monitored by 3 gastrin radioimmunoassays specific for different sequences of gastrin17 and 3 CCK radioimmunoassays specific for different sequences of CCK33. Chromatography revealed that gastrin was present in molecular forms corresponding to gastrin34 ('big gastrin') and gastrin17. CCK was present in molecular forms corresponding to the COOH-terminal octapeptide amide of CCK33 and a fragment corresponding to sequence 25-29 of CCK33. Also, a peptide corresponding to COOH-terminal tetrapeptide amide common to both gastrin and CCK was found. The results indicate that true gastrin as well as CCK are present in CSF, and that both hormones display a molecular heterogeneity similar to that found in extracts of brain tissue.

Rapid appearance of intraventricularly administered neuropeptides in the peripheral circulation.

125I-Labeled cholecystokinin octapeptide ([125I]CCK-OP) diffuses rapidly by a non-carrier-mediated mechanism into the peripheral blood following injection into the lateral ventricle of a rabbit. In contrast, no [125I]CCK-OP was observed in the CSF following intravenous injection. This unidirectional free transport mechanism from CSF to blood may apply to other neuropeptides as well.

Altered neuropeptide concentrations in cerebrospinal fluid of psychiatric patients.

Immunoreactive somatostatin, bombesin, and cholecystokinin were measured in cerebrospinal fluid of normal subjects and patients with anorexia nervosa, depression, mania, and schizophrenia. Somatostatin-like immunoreactivity was decreased in anorexic and depressed patients. Bombesin-like immunoreactivity tended to be decreased in schizophrenics. Cholecystokinin-like immunoreactivity did not differ between groups. These data suggest a possible function for neuropeptides in regulation of human behavior.

Cholecystokinin in human cerebrospinal fluid: concentrations, dynamics, molecular forms and relationship to fasting and feeding in health, depression and alcoholism.

Very little is known about the physiologic significance of the gut-brain hormone cholecystokinin (CCK) in the human central nervous system, although the hormone has been hypothesized to be involved in the regulation of both appetite and anxiety. We continuously collected lumbar cerebrospinal fluid (CSF) via indwelling subarachnoid catheters in ten normal volunteers, ten patients with major depression and five abstinent alcoholic humans, while fasting and after eating. Five other healthy subjects were fasted throughout the experiment. We quantified CSF immunoreactive cholecystokinin (IR-CCK) and glucose concentrations at 10-min intervals from 11.00 to 17.00 h. No difference in CSF IR-CCK concentration, half-life or rhythm was observed between normal volunteers and either depressed or alcoholic patients. Fasting CSF IR-CCK concentrations were 1.3 +/- 0.18, 1.3 +/- 0.21 and 1.2 +/- 0.21 fmol/ml (mean +/- S.E.M.) in normal volunteers, depressed patients and alcoholic patients, respectively. After eating, CSF IR-CCK concentrations rose to 1.5 +/- 0.21, 1.5 +/- 0.24 and 1.4 +/- 0.26 fmol/ml, respectively. Normal volunteers who did not eat had similar basal CSF IR-CCK concentrations (1.1 +/- 0.1 fmol/ml) which similarly rose to 1.4 +/- 0.13 fmol/ml during the sampling interval. In contrast, CSF glucose concentrations rose only in the subjects who ate, beginning to rise after about 1 h and remaining elevated for at least 3 h after eating. These data suggest the existence of a diurnal rhythm of IR-CCK release into CSF, as opposed to a response to feeding. The disappearance half-time of CCK in human CSF is less than 13 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Concentration of cholecystokinin in cerebrospinal fluid is decreased in psychosis: relationship to symptoms and drug response.

1. Cholecystokinin (CCK) is a neuropeptide which is co-localized within some mesolimbic and mesocortical dopamine neurons. 2. CCK resembles an antipsychotic drug in some pharmacological and behavioral tests. 3. Levels of CCK in the cerebrospinal fluid (CSF) are reduced in eleven drug-free schizophrenics in comparison with six controls. 4. Schizophrenic males have lower CSF CCK levels than females. 5. Rapidity of antipsychotic response to haloperidol appeared to be inversely related to drug-free baseline CSF CCK levels.

Neuropeptides in the cerebrospinal fluid (CSF) in psychiatric disorders.

Concentrations of vasoactive intestinal polypeptide (VIP), cholecystokinin (CCK) and gastrin in the cerebrospinal fluid (CSF) was studied in patients with endogenous depression, non-endogenous depression, mania, schizophrenia and a control group. All patients were classified according to various diagnostic systems. In the group of non-endogenously depressed patients CSF-VIP levels (median 16 pmol/l) were found significantly lowered compared to controls (median = 32 pmol/l) and endogenous depression (26 pmol/l). Going through the non-endogenous group it appeared that the low CSF-VIP was due to a group of patients with a former diagnosis of endogenous depression or a present diagnosis of possible endogenous depression. Moreover, this group was clinically characterized by 'dysphoric/hysterical features', 'reversed diurnal variation' (i.e. worst in the evening), and 'lack of clearly circumscribed episode'. In many aspects this group seems similar to the atypical depressions described as monoamineoxidase responders. Concerning CSF-CCK and CSF-gastrin no significant differences between the examined groups were demonstrated.

Cerebrospinal fluid cholecystokinin, bombesin and somatostatin in schizophrenia and normals.

Cerebrospinal fluid from 31 normals and two groups of phenomenologically similar schizophrenics (n = 72) were collected by identical methods. Radioimmunoassay of CSF was carried out for somatostatin, bombesin, and cholecystokinin. One group of schizophrenics had increased baseline somatostatin and cholecystokinin, and decreased bombesin. No CSF gradient effect was found for the peptides nor were their levels affected by probenecid or pimozide treatment. An inverse correlation was found between bombesin and psychosis rating. Intercorrelation between the peptides and HVA, 5-HIAA, and MHPG were not significant.

Cholecystokinin in CSF from depressed patients: possible relations to severity of depression and suicidal behaviour.

Levels of cholecystokinin (CCK) peptides were measured in the CSF from 105 patients suffering from major depressive disorders admitted to a research psychiatric ward for diagnostic evaluation, by a radioimmunoassay method using two different antibodies. Relations between CCK levels and parameters of depression, anxiety, and suicidal behaviour were investigated. Significant inverse correlations were found between CCK levels and certain depression and anxiety parameters. Patients who had made one or more suicide attempts tended to have higher CSF CCK levels than those who had not. No correlations were found between CSF CCK and 5-HIAA or HVA, or with plasma cortisol.