Physiological effects of high-P50 erythrocyte transfusion on piglets

Rightward shifts of the O2 dissociation curve (ODC) were experimentally obtained in lysed and resealed erythrocytes following encapsulation of inositol hexaphosphate (IHP). This continuous lysing and resealing procedure led to in vitro P50 (Po2 at 50% hemoglobin saturation) increases up to 80 Torr (pH, 7.40; Pco2, 40 Torr; temp, 37 degrees C) for both human and pig erythrocytes. The Hill number of the transformed blood decreased when IHP was fixed on the hemoglobin, but the sigmoid shape of the ODC was maintained. The O2 hemoglobin binding capacity and the mean corpuscular hemoglobin content were found unchanged by the experimental procedure in human and pig erythrocytes. Isovolumic exchange transfusion of high-P50 erythrocytes in anesthetized and ambient air-ventilated piglets (n = 6) led to substantial in vivo P50 increases (range, 8-19 Torr). The rightward shift of the ODC was concomitant with an increase of the arterial Po2 and of the arteriovenous O2 content difference, 19 and 59% respectively above their control values. The mixed-venous Po2 (PVO2) remained unchanged. The cardiac output was shown to be inversely related to the P50 value. In spite of the O2-transport reduction (37%), O2 consumption was maintained due to enhanced O2 extraction.     

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.     

Chronic sodium cyanate treatment induces "hypoxia-like" effects in rats

Three weeks of sodium cyanate (NaCNO) intraperitoneal treatment in rats (n = 15) induced high hemoglobin O2 affinity, i.e., low PO2 at 50% hemoglobin saturation (P50), 20.5 +/- 1.4 Torr, in comparison with the mean control values, 34.5 +/- 1.6 Torr (n = 15). NaCNO rats showed a reduction in mean body weight, 376 +/- 27 g, in comparison with controls, 423 +/- 23 g (P less than 0.001). Despite arterial O2 partial pressure (PaO2) within normal limits NaCNO-treated rats had a higher systolic right ventricular pressure (SRVP), 33.7 +/- 3.1 Torr, in comparison with control value, 29.0 +/- 2.5 Torr (P less than 0.001). Right ventricle weights were significantly increased (P less than 0.001). After 60 min of an hypoxic challenge (fractional concentration of inspired O2 = 0.10) NaCNO-treated rats increased SRVP of only 7 +/- 4% compared with 46 +/- 9% in the control animals. Inducing high hemoglobin affinity in rats (n = 10; 6 wk NaCNO treatment) resulted in increases in hematocrit ratio and hemoglobin concentration (P less than 0.001). The characteristics of the red blood cell (RBC) itself changed; values of mean cell volume, mean cell hemoglobin, and mean cell hemoglobin concentration being significantly increased (P less than 0.001) when compared with mean control values. The count of nucleated RBC's appeared to be significantly higher from the 2nd wk of NaCNO treatment. Chronic NaCNO treatment was demonstrated to exert "hypoxia-like" effects since it induced prevention of normal growth, polycythemia, pulmonary hypertension, right ventricular hypertrophy, and blunted pulmonary pressor response to acute hypoxia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Red cell function at extreme altitude on Mount Everest

As part of the American Medical Research Expedition to Everest in 1981, we measured hemoglobin concentration, red cell 2,3-diphosphoglycerate (2,3-DPG), Po2 at which hemoglobin is 50% saturated (P50), and acid-base status in expedition members at various altitudes. All measurements were made in expedition laboratories and, with the exception of samples from the South Col of Mt. Everest (8,050 m), within 2 h of blood collection. In vivo conditions were estimated from direct measurements of arterial blood gases and pH or inferred from base excess and alveolar PCO2. As expected, increased 2,3-DPG was associated with slightly increased P50, when expressed at pH 7.4. Because of respiratory alkalosis, however, the subjects' in vivo P50 at 6,300 m (27.6 Torr) was slightly less than at sea level (28.1 Torr). The estimated in vivo P50 was progressively lower at 8,050 m (24.9 Torr) and on the summit at 8,848 m (19.4 Torr in one subject). Our data suggest that, at extreme altitude, the blood O2 equilibrium curve shifts progressively leftward because of respiratory alkalosis. This left shift protects arterial O2 saturation at extreme altitude.     

Effect of increased Hb-O2 affinity on VO2max at constant O2 delivery in dog muscle in situ

We investigated the effect of increasing hemoglobin- (Hb) O2 affinity on muscle maximal O2 uptake (VO2max) while muscle blood flow, [Hb], HbO2 saturation, and thus O2 delivery (muscle blood flow X arterial O2 content) to the working muscle were kept unchanged from control. VO2max was measured in isolated in situ canine gastrocnemius working maximally (isometric tetanic contractions). The muscles were pump perfused, in alternating order, with either normal blood [O2 half-saturation pressure of hemoglobin (P50) = 32.1 +/- 0.5 (SE) Torr] or blood from dogs that had been fed sodium cyanate (150 mg.kg-1.day-1) for 3-4 wk (P50 = 23.2 +/- 0.9). In both conditions (n = 8) arterial PO2 was set at approximately 200 Torr to fully saturate arterial blood, which thereby produced the same arterial O2 contents, and muscle blood flow was set at 106 ml.100 g-1.min-1, so that O2 delivery in both conditions was the same. VO2max was 11.8 +/- 1.0 ml.min-1.100 g-1 when perfused with the normal blood (control) and was reduced by 17% to 9.8 +/- 0.7 ml.min-1.100 g-1 when perfused with the low-P50 blood (P less than 0.01). Mean muscle effluent venous PO2 was also significantly less (26 +/- 3 vs. 30 +/- 2 Torr; P less than 0.01) in the low-P50 condition, as was an estimate of the capillary driving pressure for O2 diffusion, the mean capillary PO2 (45 +/- 3 vs. 51 +/- 2 Torr). However, the estimated muscle O2 diffusing capacity was not different between conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Low-O(2) affinity erythrocytes improve performance of ischemic myocardium.

O(2) transport and O(2) diffusion interact in providing O(2) to tissue, but the extent to which diffusion may be critical in the heart is unclear. If O(2) diffusion limits mitochondrial oxygenation, a change in blood O(2) affinity at constant total O(2) transport should alter cardiac O(2) consumption (VO(2)) and function. To test this hypothesis, we perfused isolated isovolumically working rabbit hearts with erythrocytes at physiological blood-gas values and P(50) (PO(2) required to half-saturate hemoglobin) values at pH of 7.4 of 17 +/- 1 Torr (2,3-bisphosphoglycerate depletion) and 33 +/- 5 Torr (inositol hexaphosphate incorporation). When perfused at 40 and 20% of normal coronary flow, mean VO(2) decreased from the control value by 37 and 46% (P < 0.001), and function, expressed as cardiac work, decreased by 38 and 52%, respectively (P < 0.001). Perfusion at higher P(50) during low-flow ischemia improved VO(2) by 20% (P < 0.001) and function by 36% (P < 0.02). There was also modest improvement at basal flow (P < 0.02 and P < 0.002, respectively). The improvement in VO(2) and function due to the P(50) increase demonstrates the importance of O(2) diffusion in this cardiac ischemia model.     

A technique to continuously measure arteriovenous oxygen content difference and P50 in vivo

In hypoxemic high-altitude polycythemic natives whose arterial O2 saturation (SaO2) normally ranges between 70 and 80%, three polyurethane catheters with both optical and polarographic sensors were inserted into the radial artery to measure SaO2 and O2 tension (PaO2), and three thermodilution fiber-optic balloon-tipped catheters were floated into the pulmonary artery to measure mixed venous O2 saturation (SvO2). Correlation of the in vivo SaO2, PaO2, and SvO2 values with the in vitro measurements was high (r = 0.97, 0.99, and 0.98, respectively). Both catheters were inserted in one polycythemic subject before and 4 days after isovolemic hemodilution. Data from the sensors were used to calculate arteriovenous O2 content difference (CaO2 - CvO2) and the O2 half-saturation pressure of hemoglobin (P50). The mean +/- 1 SD of the in vivo and in vitro P50 calculated with the Hill equation was 27.61 +/- 2.15 Torr and 27.35 +/- 1.60 Torr, respectively. The mean +/- 1 SD of the absolute difference between the in vivo and in vitro measurements was 1.16 +/- 1.21 Torr. The in vivo CaO2 - CvO2 correlated well with the in vitro measurements (r = 0.93), and the mean +/- 1 SD of the error in the catheter CaO2 - CvO2 measurements was 0.47 +/- 0.50 ml/dl. This technique appears to provide a useful measurement of blood gas exchange parameters and should be applicable to the study of exercise physiology and clinical regulation of O2 transport.     

Unchanged in vivo P50 at high altitude despite decreased erythrocyte age and elevated 2,3-diphosphoglycerate

We measured hematological and erythrocyte O2 transport parameters in whole blood and density-separated erythrocytes in 11 mountaineers before and during 5 days of exposure to high altitude (4,559 m). We determined the in vivo (arterial pHblood and PCO2) and standard (pHblood = 7.4, PCO2 = 40 Torr) O2 tension at 50% O2 saturation of hemoglobin and (P50,vv and P50,st) and Bohr coefficients (BC) for fixed acid (H+) and CO2 and examined the contribution of the altered average age of circulating erythrocytes due to the stimulation of erythropoiesis on whole blood 2,3-diphosphoglycerate (2,3-DPG) and P50,st. At altitude, whole blood P50,vv remained almost unchanged, whereas P50,st and 2,3-DPG increased significantly (+4 Torr; 3.5 mumol/g hemoglobin). BCCO2 was elevated significantly at altitude. Serum erythropoietin increased transiently fourfold, iron utilization increased, and serum iron decreased by 66%. Reticulocyte counts increased, but other hematological parameters were unchanged. In density-separated erythrocytes, P50,st and 2,3-DPG increased with decreasing cell density but were higher in fractions with comparable reticulocyte counts in cells prepared at altitude than in those from control studies. Our data show that, despite the increase in 2,3-DPG and the decrease in average erythrocyte age, the in vivo hemoglobin-O2 affinity remains unchanged. P50,st values reflect the elevation of 2,3-DPG, and approximately 50% of the increase in both parameters can be ascribed to the increase in the number of reticulocytes and young erythrocytes.     

The multigenic family of the extracellular hemoglobin from the annelid polychaete Arenicola marina

The extracellular hemoglobin of the lugworm Arenicola marina which inhabits on the intertidal area, a sulfide-rich environment, comprises eight globin chains previously determined by mass spectrometry. We have cloned and sequenced five of the globin components. The deduced amino-acid sequences exhibit an extracellular signal peptide and two cysteine residues involved in an internal disulfide bond. The molecular weights calculated from the globin primary structures obtained from complete cDNA sequences are in good agreement with the mass spectrometry values obtained with the native hemoglobin. Phylogenetic analysis has allowed assigning the five A. marina sequences to the different globin sub-families. Two of the globins were found to be A2 globin chains lacking the cysteine residues proposed to be involved in the binding of hydrogen sulfide by such hemoglobin. We discuss the unusual absence of these cysteines in the light of their invariant occurrence in the A2 subfamily of hemoglobins from annelids inhabiting sulfide-rich environments.     

Chronic administration of sodium cyanate decreases O2 extraction ratio in dogs

It has been proposed that an increase in the affinity of hemoglobin for O2 may be beneficial in severe hypoxemia. To test this hypothesis, we compared the response to progressive hypoxemia in dogs with normal hemoglobin affinity (P50 = 32.4 +/- 0.7 Torr) to dogs with a left shift of the oxyhemoglobin dissociation curve (P50 = 21.9 +/- 0.5 Torr) induced by chronic oral administration of sodium cyanate. Animals were anesthetized, paralyzed, and mechanically ventilated. The inspired O2 fraction was progressively lowered by increasing the inspired fraction of N2. The lowest level of O2 transport required to maintain base-line O2 consumption (VO2) was 9.3 +/- 0.8 ml.min-1.kg-1 for control and 16.5 +/- 1.1 ml.min-1.kg-1 for the sodium cyanate-treated dogs (P less than 0.01). Other measured parameters at this level of O2 transport were, for experimental vs. control: arterial PO2 19.3 +/- 2.4 (SE) Torr vs. 21.8 +/- 1.6 Torr (NS); arterial O2 content 10.0 +/- 1.2 ml/dl vs. 4.9 +/- 0.4 ml/dl (P less than 0.01); mixed venous PO2 14.0 +/- 1.5 Torr vs. 13.8 +/- 1.0 Torr (NS); mixed venous O2 content 6.8 +/- 1.0 ml/dl vs. 2.3 +/- 0.2 ml/dl (P less than 0.01); and O2 extraction ratio 32.7 +/- 2.8% vs. 51.2 +/- 3.8% (P less than 0.01). We conclude that chronic administration of sodium cyanate appears to be detrimental to O2 transport, since the experimental dogs were unable to increase their O2 extraction ratios to the same level as control, thus requiring a higher level of O2 transport to maintain their base-line VO2 values.     

Salvage of focal cerebral ischemic damage by transfusion of high O2-affinity recombinant hemoglobin polymers in mouse.

Cell-free hemoglobin solutions with high oxygen affinity might be beneficial for selectively delivering oxygen to ischemic tissue. A recombinant hybrid hemoglobin molecule was designed using the human alpha-subunit and the bovine beta-subunit, with placement of surface cysteines to permit disulfide bond polymerization of the tetramers. The resulting protein generated from an Escherichia coli expression system had a molecular mass >1 MDa, a P50 of approximately 3 Torr, and a cooperativity of n = 1.0. Anesthetized mice were transfused during 2-h occlusion of the middle cerebral artery. Compared with transfusion with 5% albumin, cerebral infarct volume was reduced by 41% with transfusion of a 3% solution of the high oxygen-affinity hemoglobin polymer and by 50% with transfusion of a 6% solution of the polymer. Transfusion of a 6% solution of a 500-kDa polymer possessing a P50 of 17 Torr and a cooperativity of n = 2.0 resulted in a 66% reduction of infarct volume. These results indicate that cell-free Hb polymers with P50 values much lower than that of red blood cell hemoglobin are highly capable of salvaging ischemic brain. The assumption that the P50 of blood substitutes should be similar to that of blood might not be warranted when used during ischemic conditions.     

Severe hemodilutional anemia increases cerebral tissue injury following acute neurotrauma.

Anemia may worsen neurological outcomes following traumatic brain injury (TBI) by undefined mechanisms. We hypothesized that hemodilutional anemia accentuates hypoxic cerebral injury following TBI. Anesthetized rats underwent unilateral TBI or sham injury (n > or = 7). Target hemoglobin concentrations between 50 and 70 g/l were achieved by exchanging 40-50% of the blood volume (1:1) with pentastarch. The effect of TBI, anemia, and TBI-anemia was assessed by measuring brain tissue oxygen tension (Pbr(O(2))), regional cerebral blood flow (rCBF), jugular venous oxygen saturation (Sjv(O(2))), cerebral contusion area, and nuclear staining for programmed cell death. Baseline postinjury Pbr(O(2)) values in the TBI and TBI-anemia groups (9.3 +/- 1.3 and 11.3 +/- 4.1 Torr, respectively) were lower than the uninjured controls (18.2 +/- 5.2 Torr, P < 0.05 for both). Hemodilution caused a further reduction in Pbr(O(2)) in the TBI-anemia group relative to the TBI group without anemia (7.8 +/- 2.7 vs. 14.8 +/- 3.9 Torr, P < 0.05). The rCBF remained stable after TBI and increased comparably after hemodilution in both anemia and TBI-anemia groups. The Sjv(O(2)) was elevated after TBI (87.4 +/- 8.9%, P < 0.05) and increased further following hemodilution (95.0 +/- 1.6%, P < 0.05). Cerebral contusion area and nuclear counts for programmed cell death were increased following TBI-anemia (4.1 +/- 3.0 mm(2) and 686 +/- 192, respectively) relative to TBI alone (1.3 +/- 0.3 mm(2) and 404 +/- 133, respectively, P < 0.05 for both). Hemodilutional anemia reduced cerebral Pbr(O(2)) and oxygen extraction and increased cell death following TBI. These results support our hypothesis that acute anemia accentuated hypoxic cerebral injury after neurotrauma.     

Characterization and functional properties of the extracellular coelomic hemoglobins from the deep-sea, hydrothermal vent scaleworm Branchipolynoe symmytilida

Polychaete species belonging to the genus Branchipolynoe are commensal with mussels from deep-sea hydrothermal vents and cold-seeps. Possessing hemoglobins (Hbs), the species B. symmytilida, which is found in the mussel Bathymodiolus thermophilus on the East Pacific Rise, is exceptional in a family normally devoid of respiratory pigments. In a previous paper we described two major coelomic extracellular hemoglobins with unique quaternary structures. Aiming to discern respiratory adaptations to the highly variable hydrothermal environment, this paper characterizes the functional properties of these Hbs and the coelomic fluid. The two major hemoglobins (C1 and C2) exhibit spectrophotometric characteristics of both intra- and extracellular hemoglobins. However, their amino acid content is very different from other known hemoglobins and is characterized by a high proportion of alanine and glycine (up to 40% cumulated in C1). C1 and C2 differ markedly by their cysteine content (0.8% and 13% respectively). The coelomic fluid exhibits a strong buffer capacity due to the high hemoglobin content (3 mM heme). In vitro, CO2 accumulation (up to 10-12 mM CO2 for PCO2 = 7.5 Torr) occurs with limited pH changes and is only partly accounted for by carbamino-Hb formation. The two hemoglobins exhibit high oxygen-affinities (P50 0.4 Torr for C1 and 0.9 Torr for C2, at 10 degrees C, pH 8) and a normal Bohr effect (phi values ranging from -0.54 and -0.37 at 10 degrees C, to -0.24 and -0.28 at 30 degrees C, for C1 and C2, respectively). Cooperativity values range from 0.8 to 1.9 for C1 and from 0.8 to 1.7 for C2. The temperature sensitivity of O2 affinity reflect deltaH values that decrease from -30 to -60 kJ x mol(-1) with increasing pH. C2 exhibits a slight specific effect of CO2 on oxygenation properties.     

Increased hemoglobin O2 affinity does not improve O2 consumption in hypoxemia

We perfused an isolated rabbit hindlimb preparation with suspensions of human erythrocytes (RBC) having different O2 affinities. Our objective was to compare the effect of changes in P50, the PO2 at which hemoglobin is 50% saturated, on tissue O2 consumption during severe hypoxemia. A high-affinity (HA) group (n = 9) was perfused with RBC incubated in NaCNO (P50 = 21.4 +/- 1.9 Torr). This was compared with a low-affinity (LA) group (n = 9) perfused with rejuvenated RBC (P50 = 31.1 +/- 1.8 Torr). The arterial PO2 of the perfusate was decreased to approximately 24 Torr in both preparations. Perfusion flow and hemoglobin concentration were maintained constant. During hypoxemia arterial O2 saturation and total O2 transport (TO2) were greater in the HA than the LA group (P less than 0.05). O2 consumption and effluent venous PO2 decreased with hypoxemia in both groups to similar levels. Consequently, the LA group showed a greater O2 extraction ratio than the HA group (P less than 0.05). The ratio of phosphocreatine to inorganic phosphate, measured with 31P magnetic resonance spectroscopy, decreased at a comparable rate in both groups. As shown by a mathematical model of peripheral O2 transport, these experimental results can be explained on the basis of peripheral limitation to O2 diffusion. We conclude that increased hemoglobin affinity does not appreciably improve tissue oxygenation in hypoxemia, since the increase in TO2 is offset by diffusion limitation at the tissues.     

Oxygen delivery and utilization in hypothermic dogs

Hypothermia produces a decrease in metabolic rate that may be beneficial under conditions of reduced O2 delivery (Do2). Another effect of hypothermia is to increase the affinity of hemoglobin for O2, which can adversely affect the release of O2 to the tissues. To determine the overall effect of hypothermia on the ability of the peripheral tissues to extract O2 from blood, we compared the response to hypoxemia of hypothermic dogs (n = 8) and of normothermic controls (n = 8). The animals were anesthetized, mechanically ventilated, and paralyzed to prevent shivering. The inspired concentration of O2 was progressively reduced until the dogs died. The core temperatures of the control and hypothermic dogs were 37.7 +/- 0.3 and 30.5 +/- 0.1 degree C, respectively (P less than 0.01). The O2 consumption (VO2) of the control dogs was significantly greater than that of the hypothermic dogs (P less than 0.05), being 4.7 +/- 0.4 and 3.2 +/- 0.3 ml X min-1 X kg-1, respectively. Hypothermia produced a left shift of the oxyhemoglobin dissociation curve (ODC) to a PO2 at which hemoglobin is half-saturated with O2 of 19.8 +/- 0.7 Torr (control = 32.4 +/- 0.7 Torr, P less than 0.01). The O2 delivery at which the VO2 becomes supply dependent (DO2crit) was 8.5 ml X min-1 X kg-1 for control and 6.2 ml X min-1 X kg-1 for hypothermia. The hypothermic dogs maintained their base-line VO2's at lower arterial PO2's than control.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationship of extracellular dopamine in striatum of newborn piglets to cortical oxygen pressure

The present studies describes the relationship between extracellular dopamine in striatum of newborn piglets and cortical oxygen pressure. The extracellular level of dopamine was measured by in vivo microdialysis and the oxygen pressure in the cortex was measured by phosphorescence lifetime of oxygen probe in the blood. Controlled, graded levels of hypoxic insult to the brain of animals were generated by decreasing of the oxygen fraction in the inspired gas (FiO₂) from 21% to 14%, 11%, and 9%. This resulted in decrease in the cortical oxygen pressure from 31–35 Torr to about 24 Torr, 15 Torr and 4 Torr, respectively. The changes in extracellular level of dopamine, DOPAC and HVA were dependent on changes in cortical oxygen pressure. Stepwise decrease in the cortical oxygen pressure (see above) caused increases in extracellular dopamine of about 80%, 200% and 550%, respectively. The levels of DOPAC and HVA progressively decreased and when cortical oxygen decreased to 4–6 Torr were about 50% and 70% of control. respectively. After return of FiO₂ to control (21%), the cortical oxygen pressure rapidly increased to above normal, then returned to control values. The extracellular levels of dopamine, DOPAC, and HVA recovered more slowly, attaining control values in about 30 minutes. The data show that extracellular levels of dopamine increase with even very small decreases in oxygen pressure. Thus, there is no oxygen reserve which protects dopamine release and metabolism from decrease in oxygen pressure.     

A nematode hemoglobin gene contains an intron previously thought to be unique to plants

Summary Hemoglobin genes from plants and animals both have a characteristic chromosomal organization. Plant hemoglobin genes contain a unique intron inserted into the heme-binding domain of exon 2. This intron has not been previously reported in animal globin genes, and its loss was hypothesized to have occurred early in the evolution of hemoglobins. We report here a unique six-intron, seven-exon internally duplicated nematode hemoglobin gene that contains an intron equivalent to the plant central intron in its first repeat. This nematode hemoglobin gene has lost both the central and the normal third intron in its second repeat. The nematode globin also contains a unique intron between its secretory peptide leader sequence and its coding sequence, which is absent in other extracellular invertebrate globin genes. Possible models to explain the head-to-tail duplication of this gene are discussed. Offprint requests to: B. Pohajdak     

Tissue oxygen extraction during hypovolemia: role of hemoglobin P50

When the delivery of O2 to tissues (QO2 = blood flow X O2 content) falls below a critical threshold, tissue O2 uptake (VO2) becomes limited by QO2. The mechanism responsible for this extraction limitation is not understood but may involve molecular diffusion limitation as mean capillary PO2 drops below a critical minimum level in some capillaries. We tested this hypothesis by measuring the critical QO2 necessary to maintain VO2 independent of QO2 in anesthetized, paralyzed normal dogs (n = 7) and in a second group in which PO2 at 50% saturation of hemoglobin (P50) was reduced by exchange transfusion with low-P50 erythrocytes (n = 7). QO2 was reduced in stages by removing blood volume to reduce blood flow while VO2 was measured by spirometry at each step. To the extent that O2 extraction was limited by a critical capillary PO2, we reasoned that the onset of diffusion limitation should occur at a higher QO2 with low P50, since a lower end-capillary PO2 is required to achieve the same O2 extraction. The critical QO2 (7.8 +/- 1.2 ml X min-1 X kg-1) and extraction ratio (0.63 +/- 0.06) in dogs with reduced P50 were not different from controls. At the critical delivery, mixed venous PO2 was lower in low P50 (16.1 +/- 2.9 Torr) than controls (29.9 +/- 2.3 Torr). We concluded that diffusion limitation does not initiate the early fall in VO2 below the critical QO2 and offer an alternative model to explain the onset of supply dependency.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of hemoglobin P50 in O2 transport during normoxic and hypoxic exercise in the dog

High hemoglobin affinity for O2 [low PO2 at 50% saturation of hemoglobin (P50)] could degrade exercise performance in normoxia by lowering mean tissue PO2 but could enhance O2 transport in hypoxic exercise by increasing arterial O2 saturation. We measured O2 transport at rest and at graded levels of steady-state exercise in tracheostomized dogs with normal P50 (28.8 +/- 1.8 Torr) and again after P50 was lowered (19.5 +/- 0.7 Torr) by sodium cyanate infusions. Measurements were made during ventilation with room air (RA), 12% O2 in N2, or 10% O2 in N2. Cardiac output (QT) as a function of O2 consumption (VO2) was not altered by low P50 at any inspired O2 fraction (P greater than 0.05). With RA exercise, arterial content (CaO2) and O2 delivery (QT X CaO2) were unchanged at low P50, whereas mixed venous PO2 was reduced at each level of VO2. With exercise in hypoxia, CaO2 and O2 delivery were significantly improved at low P50 (P less than 0.05). Mixed venous PO2 was lower than control during 12% O2 (P less than 0.05) but not different from control during 10% O2 exercise at low P50. Despite a presumed decrease in tissue PO2 during RA and 12% O2 exercise, exercise performance and base excess decline were not significantly worse than control levels. We conclude that, in canine steady-state exercise, hemoglobin P50 is not an important determinant of tissue O2-extraction capacity during normoxia or moderate hypoxia. In extreme hypoxia, low P50 may help to maintain tissue PO2 by enhancing systemic O2 delivery at each level of QT.     

Synthesis and physicochemical characterization of a series of hemoglobin-based oxygen carriers: objective comparison between cellular and acellular types

A series of hemoglobin (Hb)-based O(2) carriers, acellular and cellular types, were synthesized and their physicochemical characteristics were compared. The acellular type includes intramolecularly cross-linked Hb (XLHb), polyoxyethylene (POE)-conjugated pyridoxalated Hb (POE-PLP-Hb), hydroxyethylstarch-conjugated Hb (HES-XLHb), and glutaraldehyde-polymerized XLHb (Poly-XLHb). The cellular type is Hb-vesicles (HbV) of which the surface is modified with POE (POE-HbV). Their particle diameters are 7 +/- 2, 22 +/- 2, 47 +/- 17, 68 +/- 24, and 224 +/- 76 nm, respectively, thus all the materials penetrate across membrane filters with 0.4 microm pore size, though only the POE-HbV cannot penetrate across the filter with 0.2 microm pore size. These characteristics of permeability are important to consider an optimal particle size in microcirculation in vivo. POE-PLP-Hb ([Hb] = 5 g/dL) showed viscosity of 6.1 cP at 332 s(-1) and colloid osmotic pressure (COP) of 70.2 Torr, which are beyond the physiological conditions (human blood, viscosity = 3-4 cP, COP = ca. 25 Torr). XLHb and Poly-XLHb showed viscosities of 1.0 and 1.5 cp, respectively, which are significantly lower than that of blood. COP of POE-HbV is regulated to 20 Torr in 5% human serum albumin (HSA). HES-XLHb and POE-HbV/HSA showed comparable viscosity with human blood. Microscopic observation of human red blood cells (RBC) after mixing blood with POE-PLP-Hb or HES-XLHb disclosed aggregates of RBC, a kind of sludge, indicating a strong interaction with RBC, which is anticipated to modify peripheral blood flow in vivo. On the other hand, XLHb and POE-HbV showed no rouleaux or aggregates of RBC. The acellular Hbs (P(50) = 14-32 Torr) have their specific O(2) affinities determined by their structures, while that of the cellular POE-HbV is regulated by coencapsulating an appropriate amount of an allosteric effector (e.g., P(50) = 18, 32 Torr). These differences in physicochemical characteristics between the acellular and cellular types indicate the advantages of the cellular type from the physiological points of view.