Experimental and theoretical investigation on the relative stability of the PdS2- and pyrite-type structures of PdSe2.

Under ambient condition PdSe2 has the PdS2-type structure. The crystal structure of PdSe2 under pressure (up to 30 GPa) was investigated at room temperature by X-ray diffraction in an energy-dispersive configuration using a diamond anvil cell with a mixture of water/ethanol/methanol as a pressure transmitting medium. A reversible structural transition from the PdS2-type to the pyrite-type structure occurs around 10 GPa, and the applied pressure reduces the spacing between adjacent 2/proportional to [PdSe2] layers of the PdS2-type structure to form the three-dimensional lattice of the pyrite-type structure. First principles and extended Hückel electronic band structure calculations were carried out to confirm the observed pressure-induced structural changes. We also examined why the isoelectronic analogues NiSe2 and PtSe2 adopt structures different from the PdS2-type structure on the basis of qualitative electronic structure considerations.

Chronopharmacologic aspects of continuous AcSDKP infusion in mice.

Inconsistent results characterized N-acetyl-Ser-Asp-Lys-Pro (AcSDKP or Goralatide) effects upon hematologic proliferation, possibly because its circadian organization had been overlooked. We investigated the circadian changes in AcSDKP disposition in plasma and in bone marrow during continuous infusion and AcSDKP effects upon the circadian rhythms in bone marrow granulomonocytic precursors (CFU-GM) and circulating blood cell counts. One hundred ninety-six male B6D2F1 mice received a constant infusion of AcSDKP (24 microg/ day) or 0.9% NaCl for 7 days, using an osmotic minipump. All mice were synchronized with an alternation of 12 hours of light and 12 hours of darkness for 3 weeks prior to study. Mice were sacrificed on the fifth or seventh infusional day at 3, 9, 15, or 21 hours after light onset (HALO) in order to assess plasma and bone marrow AcSDKP concentrations, CFU-GM, and/or circulating blood cell counts. In control mice, plasma and bone marrow AcSDKP concentrations displayed a circadian rhythm with a maximum level during the dark span, at 21 and 15 HALO respectively, while CFU-GM, leukocyte, lymphocyte, and monocyte counts peaked during early light. Continuous AcSDKP infusion increased fivefold mean plasma AcSDKP level at 3 or 9 HALO, thus inverted its physiologic rhythm and suppressed the CFU-GM peak that normally occurs at these times. This inhibition however, was indirect, because the rhythms in bone marrow AcSDKP concentration were similar with or without AcSDKP infusion. Conversely, mean leukocyte and lymphocyte counts were significantly reduced with AcSDKP infusion, while their circadian rhythms remained unaffected and were amplified. The results indicate that AcSDKP pharmacology displays circadian rhythmicity and warrant the exploration of chronopharmacologic schedules of AcSDKP delivery for further protecting bone marrow against chemotherapy insults.

Circadian-based effects of AcSDKP, with or without rhG-CSF on hematologic toxicity of chemotherapy in mice.

The hematologic toxicity of arabinosylcytosine (Ara-C) and carboplatin (CBDCA) as well as the stimulating effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on murine bone marrow vary according to their dosing time along the 24-h time scale. In the present study, we investigated whether the tolerability of Ara-C or CBDCA, given at their least toxic circadian time, could be improved further with AcSDKP, a negative regulator of hemopoiesis, rhG-CSF or both. A total of 228 B6D2F1 mice received once-daily injection of either Ara-C (42 mg/kg/d s.c.) for 7 d (d 0-6) at 8 hours after light onset - HALO) or CBDCA (40 mg/kg/d i.p.) for 5 d (d 2-6) at 16 HALO. AcSDKP (24 microg/d) was continuously infused for 7 d (d 0-6), using an osmotic minipump. rhG-CSF (400 microg/kg/d s.c.) was injected for 4 d (d 9-12) at 9 HALO. Subgroups of mice were sacrificed at 3 HALO on various days following treatment. AcSDKP significantly increased CFU-GM count on d 7 and leukocyte, neutrophil and monocyte counts on d 13 and d 16 compared to Ara-C alone. Also, rhG-CSF produced similar protective effects to those of AcSDKP with regard to leukocyte and CFU-GM counts. The combination of AcSDKP with rhG-CSF induced a further increase in total leukocytes and their subsets as compared to either agent alone, but did not alter the CFU-GM counts. Neither AcSDKP nor rhG-CSF nor their combination reduced CBD CA-induced hematological toxicity. In conclusion, AcSDKP or rhG-CSF administration further improved the tolerability of Ara-C beyond that already achieved with optimal circadian timing, while no such effect was observed in mice receiving CBDCA at the dose used. The results warrant further exploration of chronopharmacologic delivery schedules combining Ara-C with AcSDKP.

Differential effects of fedotozine compared to other kappa agonists on diuresis in rats.

The aim of our study was to test the effect of fedotozine (0.1-30 mg/kg, s.c.), a novel kappa opioid agonist, on water diuresis in the conscious hydrated rat. Its effect was compared to that of morphine (2.5-10 mg/kg), a mu opioid agonist and to some of the recognized kappa agonists described in the literature: bremazocine (0.3-30 micrograms/kg), tifluadom (0.1-3 mg/kg), Cl 977 1-1000 micrograms/kg), (-)-cyclazocine (0.01-1 mg/kg), PD 117,302 (0.03-3 mg/kg), U-50,488h (0.25-10 mg/kg) and U-69,593 (0.3-3 mg/kg). The effect of fedotozine was also tested after intracerebroventricular administration (100 micrograms/kg) and compared to that of U-50,488h (10-30 micrograms) and dynorphins A(1-17), A(1-13) and B(1-13) (2.5-10 micrograms). All the reference kappa agonists administered by the s.c. route induced water diuresis, whereas morphine inhibited diuresis and electrolyte excretion. However, fedotozine (0.1-30 mg/kg s.c.) had no effect on diuresis, even after low doses of naloxone (0.1 mg/kg s.c.) or nor-BNI (10 mg/kg s.c.), and at 1 mg/kg had inconsistent effects on electrolyte elimination. When administered in the lateral ventricle of the brain, U-50,488h and dynorphin A(1-17) induced water diuresis, unlike fedotozine (100 micrograms), DYN A(1-13) and DYN B(1-13) that had no effect on urine output. Furthermore, fedotozine did not alter the diuretic effects of U-50,488h. These results suggest that fedotozine is an atypical kappa agonist, lacking activity on the kappa receptor subtypes regulating diuresis.

Pharmacological evaluation of JO 1870: relation to the potential treatment of urinary bladder incontinence.

The effect of p.o. administered JO 1870 [(-)-1-(p-chlorophenyl)-N,N- dimethyl-1-ethyl(4-phenyl)-but-3-en-1-ylamine, hydrochloride] on the urinary bladder cystometrogram has been investigated in the unanesthetized rat. The effects of JO 1870 have been compared with those of morphine and reference drugs currently used for the treatment of urinary incontinence in humans. JO 1870 (2.5-25 mg/kg) dose-dependently increased the bladder capacity and the threshold pressure responsible for urination. These effects were antagonized by the i.v. administration of naloxone (0.2 mg/kg). In comparison, morphine (2.5-25 mg/kg) moderately increased urinary volume and threshold pressure. Different anticholinergic drugs (propantheline bromide, 10 mg/kg; terodiline, 25 mg/kg; dicyclomine, 25 mg/kg) had no significant effects on either parameter and the antidepressant clomipramine (25 mg/kg) decreased both. In vitro, JO 1870 specifically displaced [3H]-[D-Ala2,MePhe4,Gly5-(ol)]enkephalin from binding sites in guinea pig whole-brain membranes and rat thalamus; the sodium shift ratio obtained from [3H]naloxone binding was 16. These results suggest that JO 1870 has some opioid agonist activity. JO 1870 (2.5-300 mg/kg) had few effects on cardiorespiratory, gastrointestinal or nociceptive systems in rats. Together, these results indicate that JO 1870 is a nonanticholinergic agent that potently increases bladder capacity, likely through an opioid mechanism, and may have potential use in the treatment of urinary incontinence.

Effects of endothelin on the lethality induced by platelet-activating factor or endotoxin in the mouse.

Platelet-activating factor (PAF) is a mediator that decreases cardiac output and total peripheral resistances leading to profound hypotension. It seems to be involved in shock states and in the deleterious effects of endotoxin. As the natural peptide endothelin (ET) shows potent vasoconstrictor properties, we evaluate its activity towards PAF and endotoxin-induced lethality in mice. ET, at doses which per se did not exert any effects on mice vitality, produced a dose-dependent decrease in the PAF-induced lethality with a total protection at 5 micrograms/kg. But conversely to these results, ET potentiated the mortality induced by endotoxin. These results suggest that ET or endothelin analogs could be worthy for therapeutic use in some types of shock, at least when endotoxin is not involved. Complementary studies are necessary to strengthen these preliminary results.

Biochemical and pharmacological evaluation of the novel antidepressant and serotonin uptake inhibitor 2-(3,4-Dichlorobenzyl)-2-dimethylamino-1-propanol hydrochloride.

2-(3,4-Dichlorobenzyl)-2-dimethylamino-1-propanol, hydrochloride (JO 1017) is a novel antidepressant drug. Its biochemical and pharmacological properties were investigated in mice, rats, dogs, rabbits and guinea pigs. In vitro, it selectively inhibited serotonin uptake and had a high affinity for the 3H-paroxetine and 3H-imipramine binding sites. Biochemical studies demonstrated the lack of MAO-A and MAO-B inhibition and the absence of marked affinity for muscarinic, histaminic or other conventional brain receptors. Chronic treatment with JO 1017 induced a decrease in the Bmax values for imipramine sites but did not modify the Bmax for beta-adrenergic and 5-HT2 receptors. The neuropsychopharmacological profile of JO 1017 is characterized by a decrease of the immobility times in behavioural despair tests with mice, a decrease of the escape failures in the rat learned helplessness test, a strong potentiation of L-5-HT P-induced head-twitches in mice and an antagonism of reserpine-induced ptosis in rabbits. It weakly antagonized oxotremorine-induced hypothermia and did not influence the hypothermia induced by apomorphine. In contrast to most other antidepressants, a high dose of JO 1017 induced hypermotility in mice placed in an activity meter without producing stereotyped behaviour and group toxicity. Unlike tricyclic antidepressants, JO 1017 was devoid of severe cardiotoxicity in guinea pigs and had no central anticholinergic nor antihistaminic properties. These results suggest that JO 1017 is a selective serotonin uptake inhibitor with a high safety margin. JO 1017 may have a potential clinical utility both in the treatment of depression and for indications where serotonin transmission is involved, e.g., anxiety, panic attack, obsessive compulsive disorder, obesity and alcohol consumption.

Modulation of eosinophil recruitment in the rat by the platelet-activating factor (PAF) antagonist, BN 52021, the somatostatin analog, BIM 23014, and by cyclosporin A.

The factors responsible for in vivo eosinophil recruitment are poorly defined, although T-lymphocytes appear to be involved in the etiology of eosinophilia. In order to clarify this relationship, we studied the modulation of eosinophil mobilization in the rat after immune challenge, by chronic treatment with the PAF-antagonist, BN 52021, the somatostatin analog, BIM 23014 and with Cyclosporin A (CsA). In rats made hypereosinophilic by pretreatment with cyclophosphamide or sephadex, a significant increase of the eosinophil count in blood and peritoneal fluid was induced by anaphylactic reaction. CsA totally abolished both hypereosinophilia and peritoneal eosinophil infiltration. BIM 23014 also, significantly reduced the circulating eosinophils (-68%, p less than 0.001) and cell infiltration (-86%, p less than 0.05). In contrast, BN 52021 decreased peritoneal eosinophil recruitment, while having relatively little effect on circulating cells. CsA and somatostatin are known to affect T-cell proliferation, and as T-cells are involved in the differentiation of hematopoietic cells into eosinophils, these drugs could decrease eosinophil availability for recruitment. In contrast, the PAF antagonist may act by inhibiting PAF-induced eosinophil chemotaxis, providing a more specific inhibition of this process than that exerted by CsA, BIM 23014 and other immunosuppressive agents.

Modulation by drugs of eosinophil recruitment induced by immune challenge in the rat. Possible roles of interleukin 5 and platelet-activating factor.

The factors responsible for eosinophil recruitment are poorly defined, although both platelet-activating factor (PAF) and cytokines appear to be involved in regulating this process. We compared eosinophil mobilization induced by PAF or antigen injection in the peritoneal cavity of hypereosinophilic rats and the effects of the PAF antagonist BN 52021, the somatostatin analog BIM 23014, and cyclosporin A on this process. PAF induced a significant increase of both peritoneal and circulating eosinophil count. Cyclosporin A almost totally abrogated these variations, whereas BN 52021 reduced the peritoneal increase. Similarly to PAF, peritoneal antigen challenge in actively sensitized animals increased peritoneal and circulating eosinophil counts. Cyclosporin A abolished both hypereosinophilia and peritoneal eosinophil infiltration. BIM 23014 reduced the circulating eosinophils and cell infiltration. In contrast, BN 52021 primarily decreased peritoneal eosinophil recruitment, while having little effect on circulating cells. The different mechanisms of action of these drugs and the involvement of interleukin 5 in eosinophil recruitment are discussed.

Chemotactic effect of PAF-acether on peritoneal eosinophils from normal rats.

The chemotactic activity of PAF-acether was compared with that of tetrapeptide eosinophil chemotactic factors of anaphylaxis (ECF-A, Ala-Gly-Ser-Glu and Val-Gly-Ser-Glu) using eosinophils obtained from the peritoneal cavity of normal rats. Cells were isolated by separation over discontinuous metrizamide gradients which resulted in eosinophil suspensions of 80 to 90% purity. PAF-acether produced a dose-dependent effect which, at its maximum, was more than 30-fold greater than control and about 5- to 7-fold greater than the maximal activity obtained with the ECF-A-tetrapeptides. BN 52021 and WEB 2086 inhibited PAF-acether-induced eosinophil chemotaxis in a dose-dependent manner, suggesting that this phenomenon is mediated by specific PAF-acether receptors.

Chemotactic effect of PAF-acether on peritoneal eosinophils from normal rats

The chemotactic activity of PAF-acether was compared with that of tetrapeptide eosinophil chemotactic factors of anaphylaxis (ECF-A, Ala-Gly-Ser Glu and Val-Gly-Ser-Glu) using eosinophils obtained from the peritoneal cavity of normal rats. Cells were isolated by separation over discontinuous metrizamide gradients which resulted in eosinophil suspensions of 80 to 90% purity. PAF-acether produced 7-fold greater than the maximal activity obtained with the ECF-A-tetrapeptides. BN 52021 and WEB 2086 inhibited PAF-acether-induced eosinophil chemotaxis in a dose-dependent manner, suggesting that this phenomenon is mediated by specific PAF-acether receptors

Inhibition of rat endotoxin-induced lethality by BN 52021 and BN 52063, compounds with PAF-acether antagonistic effect and protease-inhibitory activity.

Since proteolytic enzymes play a key role in endotoxaemia and anaphylaxis, diseases in which PAF-acether may be involved, we have investigated the activity of BN 52021 and BN 52063, two potent and specific PAF-acether antagonists, on plasma protease activity in the rat. After oral administration, both drugs dose-dependently decreased plasma trypsin-like activity (PTLA) although they did not show any inhibitory effect in vitro. PAF-acether and endotoxin injected into rats increased PTLA, the increase with endotoxin appearing more slowly but to a higher extent than with PAF-acether. Inhibition of the endotoxin-induced increase in PTLA was obtained with BN 52021, BN 52063 and dexamethasone, whereas aprotinin was ineffective. Since BN 52021 and BN 52063 reduced also the endotoxin-induced lethality in the rat, their antiprotease activity may be a consequence of their PAF-antagonistic effect. Further studies will be required with other PAF antagonists to assess fully this assumption.

The relative role of PAF-acether and icosanoids in septic shock.

Endotoxemia and gram negative sepsis remain a clinically important problem since mortality rate is still high in these diseases. Recently, the participation of some new potential mediators in this pathology is beginning to be demonstrated but the results obtained on animal models with specific inhibitors are contradictory. In order to clarify the pathological importance of icosanoids and PAF-acether in the septicemic process, we investigated the effects of indomethacin (IND) a cyclooxygenase inhibitor, NDGA and EP 10045 two lipoxygenase inhibitors, dexamethasone (DXM) a phospholipase A2 inhibitor and BN 52021 a PAF-acether receptor antagonist, on the Salmonella enteritidis-induced endotoxic shock (E.S.) in the rat. Injected subcutaneously 15 min before the test, NDGA, EP 10045 and IND were moderately effective when DXM completely prevented the endotoxin lethality. BN 52021 decreased the death rate in a dose-related manner and exerted at a non-active dose a synergistic effect on IND treatment. Furthermore, given orally 1 hour before endotoxin, it provided a potent protective effect. Our results seem to confirm that PAF-acether exerted alone, or in conjunction with products of the cyclooxygenase pathway, a key role in E.S. when LTs seem to play a role of minor importance.

In vivo inhibition of plasma protein leakage and Salmonella enteritidis-induced mortality in the rat by a specific paf-acether antagonist: BN 52021.

The effects of BN 52021, a new specific paf-acether receptor antagonist and the total Ginkgo Biloba extract (GBE 761) from which this product was isolated, were studied in the rat on paf-acether-induced permeability and cell number changes and on endotoxin-induced lethality. Their activities were compared to those of cyclooxygenase, 5-lipoxygenase and phospholipase A2 inhibitors. BN 52021 given s.c. or orally exerted a dose-related inhibition of paf-acether deleterious effects as well as of endotoxin lethality whereas the other drugs tested were poorly effective. These results strongly suggest paf-acether involvement in endotoxic and septic shock.

In vivo anti-allergic and anti-inflammatory effects of drugs that inhibit lipoxygenase activity in vitro.

Studies in vitro have already shown that the chemotactic effect of leukotriene LTB4, as well as its pro-aggregatory effect on a suspension of leukocytes, could be inhibited by the lipoxygenase (LO) inhibitor ETYA; moreover a beneficial effect of another LO-inhibitor, diethylcarbamazine, has been shown on experimental asthma. To explore a possible therapeutic application of substances that inhibit 15-LO of soybean in vitro, we have tested several active compounds, namely nordihydroguaiaretic acid (NDGA), phenidone, diethylcarbamazine and mefenamic acid, on the following in vivo inflammation models in the rat: plantar oedema induced by carrageenin; cutaneous vascular hyperpermeability induced by pafacether; pleurisy induced by carrageenin. The correlation between the LO-inhibitor effect of these compounds in vitro and their beneficial effects in the different tests that were performed, as well as the mechanisms of action induced in the different experimental models, are discussed.

Induced low P50 in anesthetized rats: blood gas, circulatory and metabolic adjustments.

In anesthetized, normoxic or hypoxic rats the hemodynamic, metabolic and O2 transport characteristics following exchange transfusion with human erythrocytes containing a high O2 affinity hemoglobin (Hemoglobin Creteil, beta 89 Ser----Asp) have been studied. The in vivo oxygen partial pressure at 50% oxygen hemoglobin saturation (P50) decreased from 37.4 +/- 2.1 to 12.7 +/- 0.7 mm Hg; the arterial oxygen tension was reduced significantly from 109.9 +/- 7.7 to 87.3 +/- 12.0 mm Hg. There was a decrease in right ventricular partial pressure of oxygen (PvO2), (P less than 0.001), oxygen consumption (VO2), (P less than 0.001), arterio-venous difference, (P less than 0.001), and peripheral vascular resistance index, (P less than 0.01). Exchange transfusion with normal rat blood (P50 = 37.2 +/- 2.4 mm Hg) or with 2,3-diphosphoglycerate-enriched human red blood cells (P50 = 34.7 +/- 2.2 mm Hg), did not modify these variables in normoxic rats. In hypoxia, the reduction in P50 was associated with a further decrease in PvO2 an increase in serum lactate concentration and a VO2 decrease.

Pulmonary vasoconstrictor response to acute decrease in blood P50.

The O2 sensor that triggers hypoxic pulmonary vasoconstriction may be sensitive not only to alveolar hypoxia but also to hypoxia in mixed venous blood. A specific test of the blood contribution would be to lower mixed venous PO2 (PvO2), which can be accomplished by increasing hemoglobin-O2 affinity. When we exchanged transfused rats with cyanate-treated erythrocytes [PO2 at 50% hemoglobin saturation (P50) = 21 Torr] or with Créteil erythrocytes (P50 = 13.1 Torr), we lowered PvO2 from 39 +/- 5 to 25 +/- 4 and to 14 +/- 4 Torr, respectively, without altering arterial blood gases or hemoglobin concentration. Right ventricular systolic pressure increased from 32 +/- 2 to 36 +/- 3 Torr with cyanate erythrocytes and to 44 +/- 5 Torr with Créteil erythrocytes. Cardiac output was unchanged. Control exchange transfusions with normal rat or 2,3-diphosphoglycerate-enriched human erythrocytes had no effect on PvO2 or right ventricular pressure. Alveolar hypoxia plus high O2 affinity blood caused a greater increase in right ventricular systolic pressure than either stimulus alone. We concluded that PvO2 is an important determinant of pulmonary vascular tone in the rat.

Determination of the PO2 temperature blood factor from oxygen dissociation curves.

The variation with saturation of the temperature coefficient of PO2 in human blood (delta log PO2/delta T) was determined by continuous recording of the oxygen dissociation curve (ODC), at 37 degrees C and 25 degrees C, on the same blood samples. PCO2 and pH were held constant through an ODC run, and PCO2 was reduced at 25 degrees C to the value measured by anaerobic cooling of the same sample. delta log PO2/delta T was calculated from isosaturation points on the 37 and 25 degrees C curves. The temperature coefficient was also computed as an independent check on this method by determination of the effects of temperature (25, 30, 37 and 40 degrees C) on hemoglobin ligand interaction: fixed acid Bohr effect (delta log PO2/delta pH), carbamino-formation (delta log PO2/delta log PCO2) and hemoglobin oxygen affinity. The values of delta log PO2/delta T ratio obtained from the two different approaches were found to be in good agreement. The coefficient decreased when [H+] concentration was increased. A linear relationship between the Bohr factor and the temperature was found: delta log PO2/delta pH = 0.00267 T-0.520 (r = 0.85; n = 40) At 25 degrees C, the carbamino-formation was one order of magnitude lower than at 37 degrees C. Acid-base state and saturation value appeared to be major determinant factors for the temperature correction coefficient to be applied to blood PO2 values measured at standard (37 degrees C) temperature.

Acute circulatory collapse caused by platelet-activating factor (PAF-acether) in dogs.

Synthetic platelet-activating factor (PAF-acether) was shown to act as a shock inducer when a dose of 9 or 36 nmol . kg-1 was injected i.v. into dogs anesthetized with 30 mg . kg-1 i.v. sodium pentobarbitone. Nine nmol . kg-1 PAF-acether caused portal vein and pulmonary artery hypertension but only transiently; blood TXB2 and 6 keto PGF1 alpha levels rose; there was a lasting fall of 77% in systemic blood pressure, cardiac output fell by 86%, heart rate by 17%, plasma volume by 43%, and femoral artery blood flow by 66%, whereas the hematocrit rose by 33%. A dose of 36 nmol . kg-1 PAF-acether reduced coronary artery blood flow by 56%, diminished myocardial O2 consumption, raised O2 extraction and caused metabolic acidosis leading to death in 2 out of 7 animals. All these changes displayed the typical features of common acute circulatory collapse with distributive and hypovolemic etiologies, suggesting that PAF-acether might be an endogenous mediator in the early and late stages of shock.

Consequences of an acute increase in P50 in anaesthetized guinea pigs.

In anaesthetized guinea pigs, ventilated with ambient air, the peripheral haemodynamics and oxygen transport characteristics have been studied following a blood exchange transfusion with rat erythrocytes suspended in guinea pig plasma. Since the rat haemoglobin exhibited a lower oxygen affinity than guinea pig haemoglobin, the oxygen partial pressure at 50% of oxygen haemoglobin saturation (P50) increased from 25.2 +/- 1.1 to 37.2 +/- 0.9 mm Hg (n = 10). This increase in P50 was accompanied by a significant increase in arterial oxygen partial pressure (PaO2) and in arterio-venous difference (AVDO2). Cardiac output (Q) was decreased significantly, but oxygen consumption (VO2) remained within control values. The increase in P50 was associated with a venous oxygen partial pressure (P-VO2) which remained constant but an increase in blood lactate concentration was observed. Control exchange transfusion with fresh guinea pig blood had no effect on acid-base status, on oxygen transport, or on peripheral resistance. The sudden reduction in haemoglobin oxygen affinity induced an increase in peripheral resistance with a decrease in cardiac output, the arterial systemic pressure being maintained. These results suggested that an acute decrease in haemoglobin oxygen affinity was compensated for by a simultaneous diminution of overall tissue blood flow and reduction of capillary recruitment.