Neurotransmitter-mediated anxiogenic action of PACAP-38 in rats.

The action of PACAP-38 was studied by measuring the anxiogenic-anxiolytic behavior of rats in an elevated plus maze. PACAP-38 was administered into the lateral brain ventricle and the behavior of the animals was measured 3h later. The possible involvement of transmitters was measured by pretreating the animals with receptor blockers which alone did not influence the task, but in the doses used were effective with other neuropeptides. The receptor antagonist PACAP 6-38 (a PAC 1/VPAC2 receptor antagonist of PACAP-38 receptor), haloperidol (a non-selective dopamine receptor antagonist), phenoxybenzamine (an α1/α2ß-adrenergic receptor antagonist), propranolol(a ß-adrenergic receptor antagonist), bicuculline (a gamma-aminobutyric acid subunit A receptor antagonist), methysergide (a nonselective 5-HT2 serotonergic receptor antagonist), atropine (a nonselective muscarinic acetylcholine receptor antagonist), naloxone (a nonselective opioid receptor antagonist) and nitro-l-arginine which acts by blocking the enzyme nitric oxide synthase, thereby blocking the nitric oxide synthesis, were tested. The following parameters were measured: the time spent in open arms/the time spent in total entries. PACAP-38 decreased the ratio of time spent in open arms to the time spent in total entries, indicating anxiogenic action. The total number of entries was not altered significantly either by PACAP-38 or by the receptor blockers. The following receptor blockers diminished the action of PACAP-38: PACAP 6-38,haloperidol, methysergide, naloxone and nitro-l-arginine. Pretreatment with atropine, phenoxybenzamine, propranolol and bicuculline did not influence the action of PACAP-38 on the time spent in open arms. The results demonstrate that PACAP-38 administered into the lateral brain ventricle exerted anxiogenic action at 3 h following treatment. Pretreatment of the animals with various receptor blockers indicated that a nonselective dopaminergic receptor antagonist, 5HT2 serotonergic and opioid receptors, nitric oxide and PAC1 receptors are involved in the anxiogenic action induced by PACAP-38.

Mediators involved in the hyperthermic action of neuromedin U in rats.

Neuromedin U (NmU), first was isolated from the porcine spinal cord, has subsequently been demonstrated in a number of species, in which it is present in the periphery and also the brain. Two receptors have been identified: NmU1R is mainly present in peripheral tissues, and Nmu2R in the central nervous system. NmU, a potent endogenous anorectic, serves as a catabolic signaling molecule in the brain; it inhibits food uptake, increases locomotion, activates stress mechanism, having cardiovasscular effects and, causes hyperthermia. The mechanism of this hyperthermia is unknown. In the present experiments, the effects of NmU on the colon temperature following i.c.v administration were studied in rats. For an investigation of the possible role of receptors in mediating hyperthermia, the animals were treated simultaneously with CRF 9-41 and antalarmin, a CRH1 receptor inhibitors, astressin 2B, a CRH2 receptor antagonist, haloperidol a dopamine receptor antagonist, atropine a muscarinic cholinergic receptor antagonist, noraminophenazone a cyclooxygenase inhibitor or isatin, a prostaglandin receptor antagonist. NmU increased the colon temperature, maximal action being observed at 2-3h. CRF 9-41, antalarmin, astressin 2B haloperidol, atropine, noraminophenazone and isatin prevented the NmU-induced increase in colon temperature. The results demonstrated that, when injected into the lateral brain ventricle NmU increased the body temperature, mediated by CRHR1 and CRHR2, dopamine and muscarinic cholinergic receptors. The final pathway involves prostaglandin.

Anxiolytic action of neuromedin-U and neurotransmitters involved in mice.

Peptide Neuromedin-U (NmU) is widely distributed in the central nervous system and the peripheral tissues. Its physiological effects include the regulation of blood pressure, heart rate, and body temperature, and the inhibition of gastric acid secretion. The action of NmU in rats is mediated by two G-protein-coupled receptors, NmU-1R and NmU-2R. NmU-2R is present mainly in the brain, and NmU-1R mainly in the periphery. Despite the great variety of the physiological action of NmU, little is known about its possible effects in different forms of behavior, such as anxiety. In the present work, NmU-23 (the rodent form of the peptide) was tested for its effect on anxiety in elevated plus maze test in mice. For detection of the possible involvement of neurotransmitters, the mice were pretreated with receptor blockers: haloperidol (a D2, dopamine receptor antagonist), propranolol (a ß-adrenergic receptor antagonist), atropine (a nonselective muscarinic acetylcholine receptor antagonist), phenoxybenzamine (a nonselective α-adrenergic receptor antagonist) or nitro-l-arginine (a nitric oxide synthase inhibitor). The peptide and nitro-l-arginine were administered into the lateral brain ventricle, while the receptor blockers were applied intraperitoneally. An NmU-23 dose 0.5µg elicited anxiolytic action, whereas this action is faded away when the dose was increased. For further testing therefore 0.5µg i.c.v. was used. Propranolol and atropine fully blocked the NmU-induced anxiolytic action, while haloperidol, phenoxybenzamine and nitro-l-arginine were ineffective. The results suggest that ß-adrenergic and cholinergic mechanisms are involved in the anxiolytic action of NmU.

Involvement of transmitters in the anxiolytic action of urocortin 3 in mice.

Urocortin 3 (Ucn 3) was tested for anxiolytic action in mice an elevated plus maze. For detection of the possible involvement of neurotransmitters, the mice were pretreated with receptor blockers: haloperidol, phenoxybenzamine, propranolol, atropine, methysergide, bicuculline or naloxone. The peptide was administered into the lateral brain ventricle; the receptor blockers were applied intra- peritoneally. Ucn 3 alone elicited dose-dependent bell-shaped anxiolytic action. The most effective dose was 0.5 µg. In the combined testing a 0.5 µg dose was used. Haloperidol, propranolol, atropine, methysergide, and naloxone, blocked the Ucn 3-induced anxiolytic action, while phenoxybenzamine and bicuculline were ineffective. The results suggest that dopaminergic, beta-adrenergic, cholinergic, serotonergic and opiate transmissions are involved in the anxiolytic action of Ucn 3.

Effects of the LHRH antagonist Cetrorelix on affective and cognitive functions in rats.

The decapeptide LHRH antagonist, Cetrorelix, inhibits gonadotropin and sex-steroid secretion. Cetrorelix is used for IVF-ET procedures and for the treatment of benign prostatic hyperplasia, endometriosis and leiomyomas. However little is known about the effects of Cetrorelix on brain functions. Previously we have tested Cetrorelix in mice on the impairment of the consolidation of a passive avoidance behavior caused by beta-amyloid 25-35, anxiolytic action in the plus-maze, antidepressive action in a forced swimming test, tail suspension and open-field behavior following its administration into the lateral brain ventricle. In the present study we repeated and extended the experiments in rats in order to determine whether there are species differences in the action of Cetrorelix between mice and rats. The effects of Cetrorelix evaluated included the methods used in mice without tail suspension test and extended by measuring core temperature. Cetrorelix fully blocked the impairment of the consolidation of passive avoidance learning when given icv 30 min following administration of beta-amyloid 25-35. If beta-amyloid 25-35 and Cetrorelix were given simultaneously, Cetrorelix was ineffective. Cetrorelix elicited slight anxiogenic and stronger anxiolytic action in the plus-maze, depending on the dose used. In the forced swimming tests, Cetrorelix showed antidepressive-like action. In open-field behavior tests Cetrorelix displayed a U-type action on locomotion with 0.5 and 2 microg increasing locomotion, and increase rearing but and had no effect on grooming at 0.5-2 microg. Cetrorelix had no action on core temperature. Our findings demonstrate that Cetrorelix is able to correct the impairment of the memory consolidation caused by beta-amyloid 25-35. Cetrorelix elicits anxiolytic and antidepressive action, slightly increases locomotion and rearing in open field, but it does not influence the core temperature. The results obtained in rats are similar to those reported previously by us in mice. Collectively our findings confirm the effects of Cetrorelix on brain function in two species and suggest the possible merit of a clinical trial with Cetrorelix in patients with anxiety, depression and Alzheimer's disease.

Involvement of CRH receptors in urocortin-induced hyperthermia.

The actions of individual corticotropin-releasing hormone (CRH) receptor (CRHR1 and CRHR2) were studied on the hyperthermia caused by urocortin 1, urocortin 2 and urocortin 3 in rats. Urocortin 1, urocortin 2 or urocortin 3 was injected into the lateral brain ventricle in conscious rats and the colon temperature was measured at different times following injection, up to 6h. In order to study the possible role of CRH receptors, the animals were treated with a urocortins together with the urocortin receptor inhibitors CRF 9-41, antalarmin and astressin 2B to influence the action of urocortins in initiating hyperthermia. Urocortin 1 at a dose of 2microg caused an increase in colon temperature, maximal action being observed in body temperature at 3h. CRH 9-41 and antalarmin, CRHR1 receptor antagonists, prevented the urocortin-induced increase in colon temperature while astressin 2B (CRHR2 receptor antagonist) was ineffective. Urocortin 2 at a dose of 2microg showed a byphasic action in increase in colon temperature having the first peak between 30 min and 1h and the second peak at 4h following treatment. CRF (9-41) and antalarmin was ineffective while astressin 2B fully blocked the action of urocortin 2. Urocortin 3 in a dose of lmicrog increased colon temperature; the maximal effect was observed at 2h. CRF (9-41) and antalarmin was ineffective while astressin 2B fully blocked the action of urocortin 3. The results demonstrated that urocortin 1, 2 or 3 when injected into the lateral brain ventricle caused increases in body temperature is mediated by urocortin receptors. The action of urocortin 1 is mediated by CRHR1 receptor, while in the action of urocortin 2 and urocortin 3 CRHR2 receptor is involved.

The action of urocortins on body temperature in rats.

The actions of individual urocortins on colon temperature were studied in rats. Urocortin 1, urocortin 2 or urocortin 3 was injected into the lateral brain ventricle in conscious rats and the colon temperature was measured at different times following injection, for up to 6 h. In order to study the possible role of prostaglandins, the animals were treated with either a urocortin together with the pyrazolone derivative noraminophenazone to inhibit the action of cyclooxygenase in initiating hyperthermia, or with noraminophenazone 30 min following urocortin administration to act on existing hyperthermia. Noraminophenazone was administered intramuscularly in a dose of 50 mg/kg. Urocortin 1 caused a dose-related increase in colon temperature, maximal action being observed at a dose of 2 microg with the maximal increase in body temperature at 4 h. Noraminophenazone prevented the urocortin-induced increase in colon temperature and attenuated the already existing elevated body temperature. Somewhat similar action was observed with urocortin 2. However, following treatment with 0.5 or 1.0 microg urocortin 2, the action was already over at 2 h, whereas 2 microg increased the colon temperature steadily, with a maximum at 4 h. Noraminophenazone blocked or diminished the action of urocortin 2. Urocortin 3 in a dose of 1 microg was the most effective in increasing the colon temperature; the maximal effect was observed at 2 h. Noraminophenazone blocked the development of urocortin 3-induced hyperthermia, or attenuated it when the hyperthermia was already present. The results demonstrated that urocortin 1, 2 or 3 caused increases in body temperature when injected into the lateral brain ventricle, though the optimal dose and the duration of hyperthermia differed for the individual urocortins. The cyclooxygenase inhibitor blocked or diminished the action of these urocortins, indicating the involvement of prostaglandins in urocortin-induced hyperthermia.

Involvement of neurotransmitters in urocortin-induced passive avoidance learning in mice.

The action of urocortin on one-way passive avoidance learning was tested in mice. Urocortin was administered into the lateral brain ventricle and the latency of the passive avoidance response was measured 24 h later. For study of the roles of various neurotransmitters in mediating the action of urocortin on the consolidation of memory, the animals were pretreated with different receptor antagonists. Urocortin facilitated the acquisition, consolidation and also retrieval of the passive avoidance response. The following receptor antagonists blocked the action of urocortin on consolidation: haloperidol, atropine, phenoxybenzamine, bicuculline, the CRF antagonist CRF9-41 and methysergide. Propranolol attenuated, but did not fully block the action of urocortin, while naloxone and nitro-L-arginine were ineffective. The results obtained demonstrate that urocortin is able to improve learning and memory and also retrieval processes in a passive avoidance learning in mice. D2, muscarinic cholinergic, alfa-adrenergic, CRF, serotonergic (5HT 1-2), GABA B receptors are involved in the consolidation of the passive avoidance response.

Involvement of different receptors in pituitary adenylate cyclase activating polypeptide induced open field activity in rats.

The action of pituitary adenylate cyclase activating polypeptide (PACAP) 38 was tested in an open field 30 min, 3 h, 6 h and 24 h after icv PACAP 38 administration in rats. The effects on locomotion, rearing and grooming were measured. The possible roles of different receptors were tested in animals that had been pretreated with different receptor blockers followed by PACAP 38 administration. The locomotion, rearing and grooming activities were increased at 30 min, after PACAP 38 administration, whereas at 3 and 6 h there was no change in grooming, while the locomotion and rearing activities were sharply decreased. At 24 h after PACAP administration, there was no change in any of the parameters studied. PACAP antiserum, a PACAP antagonist (PACAP 6-38), haloperidol, phenoxybenzamine, propranolol and naloxone each prevented the changes observed at 30 min and 3 h. Atropine, nitro-l-arginine, bicuculline and methysergide were ineffective. The data demonstrate that the action of PACAP 38 on the open-field activity is regulated by D2, alpha- and beta-adrenergic and opiate receptors.

The action of orexin A on passive avoidance learning. Involvement of transmitters.

The action of orexin A on one-way passive avoidance learning was studied in rats. Orexin A administered into the lateral brain ventricle in conscious rats facilitated learning, the consolidation of learning and also retrieval processes in a dose-dependent manner in a passive avoidance paradigm. The involvement of transmitters was studied by pretreating the animals with receptor antagonists, which had proved to be effective with other neuropeptides in attenuating or blocking the action of orexin A. The following receptor blockers were used: haloperidol, phenoxybenzamine, propranolol, atropine, bicuculline, naloxone and nitro-L-arginine, which can block nitric oxide synthase. In the doses used all of the receptor blockers attenuated, but none of them fully blocked the action of orexin A on the consolidation of passive avoidance learning. The results demonstrate that orexin A is able to facilitate learning, consolidation of learning and also retrieval processes in a passive avoidance paradigm. A number of transmitters could be involved in the action of consolidation, but none of them is absolutely essential.

Cyclic GMP excretion blocked by isatin administration under conditions of fluid overload.

Isatin is a potent inhibitor of atrial natriuretic peptide (ANP) receptors and ANP-induced generation of cGMP in vitro. This study was designed to determine whether it had a similar effect in vivo, using a model of fluid overload known to induce ANP. We confirmed that this model increased urinary output of cGMP 3 hr after volume loading, and showed that this effect was blocked by i.p. injection of isatin (50 mg/kg). Isatin had no effect on urine volume or sodium output. However, isatin did have an effect on plasma protein concentration, both compared with control values, compatible with shifting fluid to the vascular compartment, and after volume overload, in which it normalised such a shift. Isatin thus affected both the generation of cGMP and fluid balance in vivo.

The action of isatin (2,3-dioxoindole) an endogenous indole on brain natriuretic and C-type natriuretic peptide-induced facilitation of memory consolidation in passive-avoidance learning in rats.

Isatin is an endogenous indole which has been shown to counteract some of the effects of atrial natriuretic peptide (ANP) both in vitro and in vivo. The present study was designed to determine whether it could antagonise in vivo effects of the related peptides brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP). The model used was consolidation of memory in a one-trial step-through passive-avoidance paradigm in the rat. Previous studies have shown that all three peptides (1 microg intracerebroventricular) can consolidate such learning and increase latency to entry a dark box. Isatin was given intraperitoneally at doses of 5, 10 and 50 mg/kg before the peptide, or a saline control. Both BNP and CNP significantly increased the latency of entry. Isatin alone had no effect. Isatin reduced the effect of both BNP and CNP; this was significant for its effect on BNP at 50 mg/kg and on CNP at both 10 and 50 mg/kg. These results show that isatin can inhibit behavioural effects of BNP and CNP as well as ANP.

Isatin (Indole-2, 3-dione) inhibits natriuretic peptide-induced hyperthermia in rats.

The effects of an endogenous indole, isatin (indole-2, 3-dione), on the hyperthermia induced by atrial natriuretic peptide (ANP-28), brain natriuretic peptide (BNP-32), and C-type natriuretic peptide (CNP-22) were investigated in rats. Intracerebroventricular administration of each peptide in a dose of 1 microg caused elevations in colon temperature 30 and 60 min after injection. An intraperitoneal (i.p.) injection of isatin (50 mg/kg) abolished the natriuretic peptide-induced hyperthermia. These data reinforce the possible involvement of natriuretic peptides in thermoregulatory processes in the central nervous system, and suggest that isatin might counteract their hyperthermic effect in vivo.

Pituitary adenylate cyclase-activating polypeptide induces hyperthermia in the rat.

The effects of centrally administered pituitary adenylate cyclase-activating polypeptide (PACAP-38) on body temperature were investigated in rats. Intracerebroventricular (i.c.v.) administration of PACAP-38 in doses of 500 and 1000 ng induced a dose-related elevation in colon temperature 2, 3, 4, 5 and 6 h after injection. The i.c.v. pretreatment of the animals with different dilutions of PACAP-38 antiserum prevented the development of hyperthermia in PACAP-38-treated animals, whereas PACAP-38 antiserum alone did not modify the colon temperature. An intramuscular injection of noraminophenazone (a cyclooxygenase inhibitor) abolished the PACAP-38-induced hyperthermia. Our data indicate that PACAP may induce hyperthermia via the central nervous system, and this hyperthermic effect may be mediated via a cyclooxygenase-involved pathway.

[Changes in platelet concentrates from dogs due to storage. I. Platelet count in in vitro function]. / Lagerungsbedingte Veränderungen im Thrombozytenkonzentrat des Hundes. Teil I: Zahl und In-vitro-Funktionsfähigkeit der Thrombozyten.

The possibility of storage of canine platelet concentrates (PC) was investigated using PC from dogs which were obtained with an automatic cell separator in C4-cell separation sets with low gasdiffusionable Polyvinylchlorid (PVC) storage containers or in C4L-sets developed for storage with high gasdiffusionable Polyolefin (PO) containers, respectively. The storage was carried out for a period of 10 days under permanent agitation at 22 degrees C (C4/22 degrees C, n = 10; C4L/22 degrees C, n = 11) or at 4 degrees C (C4L/4 degrees C, n = 6), respectively. Measurements were done directly after production of the PC, after 6 hours and then daily during the 10-day storage period. In the first part of this paper the results of platelet count (determined automatically with a blood cell differentiation automat and visually), the number of platelet aggregates, the mean platelet volume (MPV) as well as the platelet function with regard to the platelet aggregation induced by collagen or ADP and the resonance-thrombogram (RTG) are presented. The platelet count, measured automatically as well as visually, remained preponderantly constant over the complete storage time in all storage conditions. Dependent on the storage conditions--especially under storage at 22 degrees C--an increase of the number of platelet aggregates and a decrease of MPV was determined. In addition, the loss of platelet function measured by aggregation induced by collagen as well as by ADP showed a significant dependency of storage conditions. The stored platelets lost their ability to aggregate under C4/22 degrees C-conditions after a storage period of 2 days, under C4L/22 degrees C-conditions after 4 days and under C4L/4 degrees C-conditions not before 8 days of storage. Previous resuspending of platelets in fresh plasma delayed the loss of platelet function. Because the loss of platelet function described in the RTG became significant at nearly the same point in time, a storage of canine PC under corresponding conditions can be recommended for upto 2 days (C4/22 degrees C), for 4 days (C4L/22 degrees C) or 8-10 days (C4L/4 degrees C), respectively.

[Changes in platelet concentrates from dogs due to storage. II. Biochemical changes in concentrate plasma]. / Lagerungsbedingte Veränderungen im Thrombozytenkonzentrat des Hundes. Teil II: Biochemische Veränderungen im Konzentratplasma.

Platelet concentrate (PC) obtained from dogs with an automatic cell separator was stored in C4-cell separation sets with low gasdiffusionable Polyvinylchlorid (PVC) storage containers or in C4L-sets developed for storage with high gasdiffusionable Polyolefin(PO) containers, respectively. PC were stored for 10 days under permanent agitation at 22 degrees C (C4/22 degrees C, n = 10; C4L/22 degrees C, n = 11) or at 4 degrees C (C4L/4 degrees C, n = 6), respectively. Measurements were carried out directly after production of the PC, after 6 hours and then daily during the 10-day storage period. In the second part of this paper the results of pH, the concentration of bicarbonate, glucose, lactate and potassium ions as well as the activity of lactate dehydrogenase (LDH) are presented. The varying duration and intensity of the energy metabolism of the platelets and different part of glycolysis became obvious by the consumption of glucose and production of lactate, which differed significantly between the different storage conditions. Resulting from this, the mean pH decreased under the limit prescribed for human PC (pH = 6.3) already after a storage period of 3 days due to the slight capacity of gas diffusion in PVC-containers (C4/22 degrees C). In the PO-containers the pH fell below this limit at 22 degrees C (C4L/22 degrees C) after a storage period of 5 days and at 4 degrees C (C4L/4 degrees C) after 10 days. The latter reflects the high gas diffusion capacity of the PO-containers and the decreased metabolism activity at 4 degrees C. The increase of activity of LDH and of the concentration of potassium ions, which are localized in the cytosol of platelets, depended also on the different storage conditions and, thereby, reflected the different rapidity of increasing membrane permeability or the destruction of the cell membrane, respectively. The results of this study nearly are in agreement with the changes of platelet function shown in part I. Biochemical changes occur in canine platelet concentrates similar to those in human platelet concentrates during storage in dependency of the storage conditions, in part even with a higher rate or in a higher extent.

[Effect of the evaluation parameter on sensitivity of resonance thrombography of thrombocytopenia in dogs]. / Einfluss des Auswertungsverfahrens auf die Sensitivität der Resonanzthrombographie bei der Thrombozytopenie des Hundes.

Based on 109 blood samples taken from 36 dogs suffering from thrombocytopenia resonance thrombography with the resonance thrombograph RTG 801 (von Hoerner und Sulger Electronic GmbH, Schwetzingen; manufacturer: Fresenius AG, Bad Homburg) was distinctly more sensitive and more closely correlated to the platelet count using an optimized parameter of the resonance thrombogramm (RTG) in comparison to usual parameters. Nevertheless, clinical requirements regarding samples with platelet counts > 25,000/microliter were not fulfilled. Out of 13 samples with reduced platelet count and simultanous extended capillary bleeding time, depending on the used parameter a maximum of 9 samples could be detected as pathological by the RTG. The normal RTG in part of the cases with clearly altered primary haemostasis contrasts to the exclusive use of RTG in the screening of thrombocytopenia in dogs.

[Sensitivity of resonance thrombograms in thrombocytopenia of dogs]. / Sensitivität des Resonanzthrombogramms bei der Thrombozytopenie des Hundes.

Based on results of 84 blood samples, taken from 28 dogs suffering from thrombocytopenia, the resonance thrombography turned out as a method with low sensitivity (S) to detect thrombocytopenia in dogs. It was insignificant which one of the two tested resonance thrombographs was taken for measurement and which parameter of the resonance thrombogram (RTG) was used for the thrombocytic potential of haemostasis, the amplitude (RTG-P) or descending time of the platelet side. Only samples containing < or = 25,000 platelets/microliter were reliably measured (S > or = 0.90), whereas thrombocytopenias with > 50,000 platelets/microliter usually resulted in false negative results. The correlation between the platelet count and RTG-P could be almost expressed by a geometrical regression (rs = -0.709). The low sensitivity of RTG mirrors the multifactorial influences and contrasts to the exclusive use of RTG in the screening of thrombocytopenia.

[Technical aspects of extraction of platelet concentrates from dogs with an automated cell separator]. / Technische Aspekte bei der Gewinnung von Thrombozytenkonzentraten vom Hund mit einem automatischen Zellseparator.

The results of this work point out that the cell separator AS 104 (Fresenius AG, Bad Homburg), which is developed to obtain platelet concentrates from human beings, can be used for dogs. The base adjustment of the cell separator for human beings (adjustment I) is useful, because of the high yield [platelet count (median): 1.75 x 10(11)] and quality [low leucocyte- (40/microliter) and erythro count (10 x 10(3)/microliter)]. In addition, there was no clear difference between the base adjustment (adjustment I) and an empirical modification (adjustment II). The platelet count of the donor dog decreased during the separation by approximately 140,000/microliter and reached the initial value after four days. Supplementary, the time course of the haematocrit and the concentrations of albumin, total protein and total calcium of the donor dog were measured. It should be mentioned that the concentrations of ionized calcium distinctly decreased during the separation. Synchronously, the concentrations of ionized calcium and of citrat reached the starting-counts 3-4 hours after the ending of the separation.