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.     

Opposite control of O2 affinity for hemoglobin by donors and acceptors

The effects of electron donors and acceptors on O2 binding by hemoglobin were studied. 2,4-Dinitrophenol, levomycetin and pelargonidine-3,5-diglycoside which act as electron acceptors in free radical reactions, enhance this process. In contrast, N-propylajmalin which is known to be an electron donor in the above reactions, suppresses the O2 binding. Diphosphoglycerate and inositol hexaphosphate, the natural inhibitors of O2 binding, exhibit, similar to N-propylajamlin, the properties of electron donors, the latter being a more potent electron donor than the former.     

Muscle O2 deficit during hypoxia and two levels of O2 demand

We have examined the relative deficits in tension development and O2 uptake in contracting skeletal muscle during severe hypoxic hypoxia. Anesthetized mongrel dogs were ventilated to maintain an end-tidal PCO2 between 35 and 40 Torr. Venous outflow from the gastrocnemius muscle was measured using an electromagnetic flow probe. The tendon was cut and attached to a strain gauge. The muscle was stimulated to contract isometrically at 2 or 4 Hz for 20 min. Hypoxia (9% O2 in N2) was then imposed for 30 min, followed by 30 min of normoxia. Blood flow first increased in proportion to the contraction frequency and then increased further a similar amount in both groups during hypoxia. O2 extraction and blood flow reached maximal levels during hypoxia in the 2-Hz group. The further O2 deficit that was accumulated during 4 Hz and hypoxia was, therefore, a result of the greater discrepancy between O2 supply and demand. O2 uptake decreased more in hypoxia than did developed tension. These results are best explained by ATP supplementation from nonaerobic energy sources that was promoted by the free-flow condition of hypoxic hypoxia.     

Polymerized bovine hemoglobin decreases oxygen delivery during normoxia and acute hypoxia in the rat

Hemoglobin-based oxygen carriers (HBOC) have been primarily studied for blood loss treatment. More recently infusions of HBOC in euvolemic subjects have been proposed for a wide variety of potential therapies in which increased tissue oxygenation would be beneficial. However, compared with the exchange transfusion models to study blood loss, less is known about HBOC oxygen delivery and vasoacitvity when it is infused in euvolemic subjects. We hypothesized that HBOC [polymerized bovine hemoglobin (PBvHb)] infusion creating hypervolemia would increase oxygen delivery to tissues during acute global hypoxia. Vascular oxygen content and hemodynamics were determined after euvolemic rats were infused with 3 ml of either lactated Ringer or PBvHb solution (13 g/dl, 1.3 g/kg) during acute hypoxia (FIO2 = 10%, 4 h) or normoxia (FIO2 = 21%) exposure. Our data demonstrated that compared with Ringer-infused animals, in hypoxia and normoxia, PBvHb treatment improved oxygen content but raised mean arterial pressure, lowered stroke volume, heart rate, and cardiac index, which resulted in a net reduction in blood flow and oxygen delivery to the tissues. The PBvHb vasoactive effect was similar in magnitude and direction as to the Ringer-infused animals treated with a nitric oxide synthase inhibitor nitro-l-arginine, suggesting the PBvHb effect is mediated via nitric oxide scavenging. We conclude that infusion of PBvHb is not likely to be useful in treating global hypoxia under these conditions.     

Oxygen affinity of hemoglobin regulates O2 consumption,metabolism, and physical activity

The oxygen affinity of hemoglobin is critical for gas exchange in the lung and O(2) delivery in peripheral tissues. In the present study, we generated model mice that carry low affinity hemoglobin with the Titusville mutation in the alpha-globin gene or Presbyterian mutation in the beta-globin gene. The mutant mice showed increased O(2) consumption and CO(2) production in tissue metabolism, suggesting enhanced O(2) delivery by mutant Hbs. The histology of muscle showed a phenotypical conversion from a fast glycolytic to fast oxidative type. Surprisingly, mutant mice spontaneously ran twice as far as controls despite mild anemia. The oxygen affinity of hemoglobin may control the basal level of erythropoiesis, tissue O(2) consumption, physical activity, and behavior in mice.     

Increasing plasmalogen levels protects human endothelial cells during hypoxia

Supplementation of cultured human pulmonary arterial endothelial cells (PAEC) with sn-1-O-hexadecylglycerol (HG) resulted in an approximately twofold increase in cellular levels of plasmalogens, a subclass of phospholipids known to have antioxidant properties; this was due, primarily, to a fourfold increase in the choline plasmalogens. Exposure of unsupplemented human PAEC to hypoxia (PO(2) = 20-25 mmHg) caused an increase in cellular reactive oxygen species (ROS) over a period of 5 days with a coincident decrease in viability. In contrast, HG-supplemented cells survived for at least 2 wk under these conditions with no evidence of increased ROS. Hypoxia resulted in a selective increase in the turnover of the plasmalogen plasmenylethanolamine. Human PAEC with elevated plasmalogen levels were also more resistant to H(2)O(2), hyperoxia, and the superoxide generator plumbagin. This protection was seemingly specific to cellular stresses in which significant ROS were generated because the sensitivity to lethal heat shock or glucose deprivation was not altered in HG-treated human PAEC. HG, by itself, was not sufficient for protection; HG supplementation of bovine PAEC had no effect upon plasmalogen levels and did not rescue these cells from the cytotoxic effects of hypoxia. This is the initial demonstration that plasmalogen content can be substantially enhanced in a normal cell. These data also demonstrate that HG can protect cells during hypoxia and other ROS-mediated stress, likely due to the resulting increase in these antioxidant phospholipids.     

O2 delivery to contracting muscle during hypoxic or CO hypoxia

The consequences of a decreased O2 supply to a contracting canine gastrocnemius muscle preparation were investigated during two forms of hypoxia: hypoxic hypoxia (HH) (n = 6) and CO hypoxia (COH) (n = 6). Muscle O2 uptake, blood flow, O2 extraction, and developed tension were measured at rest and at 1 twitch/s isometric contractions in normoxia and in hypoxia. No differences were observed between the two groups at rest. During contractions and hypoxia, however, O2 uptake decreased from the normoxic level in the COH group but not in the HH group. Blood flow increased in both groups during hypoxia, but more so in the COH group. O2 extraction increased further with hypoxia (P less than 0.05) during concentrations in the HH group but actually fell (P less than 0.05) in the COH group. The O2 uptake limitation during COH and contractions was associated with a lesser O2 extraction. The leftward shift in the oxyhemoglobin dissociation curve during COH may have impeded tissue O2 extraction. Other factors, however, such as decreased myoglobin function or perfusion heterogeneity must have contributed to the inability to utilize the O2 reserve more fully.     

Regional O2 uptake during hypoxia and recovery in hypermetabolic dogs

The regional distribution of O2 deficit in muscle and nonmuscle tissues was measured in hypermetabolic dogs ventilated with a low inspired O2 fraction and was compared with excess O2 used in these regions during normoxic recovery. O2 uptake was stimulated by 2,4-dinitrophenol (DNP). Arterial, mixed venous, and muscle venous blood samples were drawn before, during, and after severe hypoxia (9% O2-91% N2) for the calculation of hindlimb O2 uptake and cardiac output. The O2 deficit and excess O2 uptake in recovery were calculated as the cumulative differences between normoxic control and respective hypoxic and recovery O2 uptake values. The DNP data were compared with data previously obtained in our laboratory. A greater whole-body O2 deficit was incurred in the DNP group during hypoxia and was associated with a larger O2 use in recovery. The total O2 deficit was equally distributed between muscle and nonmuscle tissues, but more excess O2 use occurred in nonmuscle tissues. The greater excess O2 used by nonmuscle tissues may have been associated with the restoration of intracellular ion concentrations brought about by the increased activity of energy-using membrane pumps.     

Acute vs. chronic effects of elevated hemoglobin O(2) affinity on O(2) transport in maximal exercise.

These studies were conducted to compare the effects on systemic O(2) transport of chronically vs. acutely increased Hb O(2) affinity. O(2) transport during maximal normoxic and hypoxic [inspired PO(2) (PI(O(2))) = 70 and 55 Torr, respectively] exercise was studied in rats with Hb O(2) affinity that was increased chronically by sodium cyanate (group 1) or acutely by transfusion with blood obtained from cyanate-treated rats (group 2). Group 3 consisted of normal rats. Hb O(2) half-saturation pressure (P(50); Torr) during maximal exercise was approximately 26 in groups 1 and 2 and approximately 46 in group 3. In normoxia, maximal blood O(2) convection (TO(2 max) = cardiac output x arterial blood O(2) content) was similar in all groups, whereas in hypoxia TO(2 max) was significantly higher in groups 1 and 2 than in group 3. Tissue O(2) extraction (arteriovenous O(2) content/arterial O(2) content) was lowest in group 1, intermediate in group 2, and highest in group 3 (P < 0.05) at all exercise PI(O(2)) values. In normoxia, maximal O(2) utilization (VO(2 max)) paralleled O(2) extraction ratio and was lowest in group 1, intermediate in group 2, and highest in group 3 (P < 0.05). In hypoxia, the lower O(2) extraction ratio values of groups 1 and 2 were offset by their higher TO(2 max); accordingly, their differences in VO(2 max) from group 3 were attenuated or reversed. Tissue O(2) transfer capacity (VO(2 max)/mixed venous PO(2)) was lowest in group 1 and comparable in groups 2 and 3. We conclude that lowering Hb P(50) has opposing effects on TO(2 max) and O(2) extraction ratio, with the relative magnitude of these changes, which varies with PI(O(2)), determining VO(2 max). Although the lower O(2) extraction ratio of groups 2 vs. 3 suggests a decrease in tissue PO(2) diffusion gradient secondary to the low P(50), the lower O(2) extraction ratio of groups 1 vs. 2 suggests additional negative effects of sodium cyanate and/or chronically low Hb P(50) on tissue O(2) transfer.     

High O2 affinity hemoglobin-based oxygen carriers synthesized via polymerization of hemoglobin with ring-opened 2-chloroethyl-beta-D-fructopyranoside and 1-o-octyl-beta-D-glucopyranoside

Second generation hemoglobin-based O(2) carriers (HBOCs) are being developed with high O(2) affinity (low P(50)) in order to suppress vasoconstriction elicited by over-oxygenating tissues, a problem associated with low O(2) affinity first generation HBOCs. Our group has previously investigated the polymerization of hemoglobin (Hb) with dialdehydes as a strategy for engineering high O(2) affinity HBOCs. In this study, two novel reactive dialdehydes were synthesized by ring-opening 2-chloroethyl-beta-D-fructopyranoside (2-CEFP) and 1-o-octyl-beta-D-glucopyranoside (1-OGP) at the 1,2-diol position, respectively, to yield novel Hb polymerizing reagents. High-affinity polymerized HBOCs were synthesized by reacting R-state bovine hemoglobin (bHb) with ring-opened 2-CEFP and 1-OGP at cross-linker to bHb molar ratios ranging from 10:1 to 30:1. The resulting polymerized bovine HBOCs (bHBOCs) displayed P(50)s ranging from 7 to 18 mmHg, cooperativities ranging from 0.8 to 1.4, and methemoglobin (metHb) levels ranging from 3% to 10%. The cross-linking reaction also stabilized the third stepwise Adair coefficient for bHbs reacted with ring-opened 1-OGP at cross-linker to bHb molar ratios of 20:1 and 30:1 and for bHbs reacted with ring-opened 2-CEFP at molar ratios of 30:1. Additionally, the number-averaged molecular weight, M(n), of each polymerized bHBOC was larger compared to bHb. Molecular weight distributions leaning towards larger molecular weight bHBOCs were obtained by increasing the cross-linker to bHb molar ratio. Taken together, the results of this study have identified novel Hb polymerization reagents that are easy to synthesize, and that are capable of yielding bHBOCs with higher O(2) affinities and weight-averaged molecular weights compared to bHb.     

Increased blood ammonia in hypoxia during exercise in humans

The effect of acute hypoxia on blood concentration of ammonia ([NH3]b) and lactate (la-]b) was studied during incremental exercise(IE), and two-step constant workload exercises (CE). Fourteen endurance-trained subjects performed incremental exercise on a cycle ergometer under normoxic (21% O2) and hypoxic (10.4% O2) conditions. Eight endurance-trained subjects performed two-step constant workload exercise at sea level and at a simulated altitude of 5000 m (hypobaric chamber, P(B)=405 Torr; P(O2)=85 Torr) in random order. In normoxia, the first step lasted 25 minutes at an intensity of 85 % of the individual ventilatory anaerobic threshold (AT(vent), ind) at sea level. This reduced workload was followed by a second step of 5 minutes at 115% of their AT(vent), ind. This test was repeated into a hypobaric chamber, at a simulated altitude of 5,000 m. The first step in hypoxia was at an intensity of 65 % of AT(vent), ind., whereas workload for the second step at simulated altitude was the same as that of the first workload in normoxia (85 % of AT(vent), ind). During IE, [NH3]b and [la-]b were significantly higher in hypoxia than in normoxia. Increases in these metabolites were highly correlated in each condition. The onset of [NH3]b and [la-]b accumulation occurred at different exercise intensity in normoxia (181W for lactate and 222W for ammonia) and hypoxia (100W for lactate and 140W for ammonia). In both conditions, during CE, [NH3]b showed a significant increase during each of the two steps, whereas [la-]b increased to a steady-state in the initial step, followed by a sharp increase above 4 mM x L(-1) during the second. Although exercise intensity was much lower in hypoxia than in normoxia, [NH3]b was always higher at simulated altitude. Thus, for the same workload, [NH3]b in hypoxia was significantly higher (p<0.05) than in normoxia. Our data suggest that there is a close relationship between [NH3]b and [la-]b in normoxia and hypoxia during graded intensity exercises. The accumulation of ammonia in blood is independent of that of lactate during constant intense exercise. Hypoxia increases the concentration of ammonia in blood during exercise.     

Increased affinity to substrate in sarcolemmal ATPases from hearts acclimatized to high altitude hypoxia

It has been well documented that acclimatization to chronic high altitude hypoxia involves a complex of adaptation changes which are capable of protecting the myocardium in diverse situations such as in acute hypoxia, coronary occlusion-induced ischaemia or isoprenaline-induced calcium overload. Since many of the former changes concern membrane functions, namely those of the sarcolemma, the activities and kinetic properties of sarcolemmal Mg2+-, Ca2+- and (Na+ + K+)-ATPase were investigated in right heart ventricles of rats acclimatized to intermittent high altitude hypoxia simulated in a barochamber. In the course of the experiment, the ventricles were subjected to a special anoxic test in vitro. The high altitude induced increase in cardiac tolerance to anoxia was not accompanied by any preservation of the sarcolemmal ATPase activities. On the contrary, membrane preparations obtained from the right ventricles of hearts acclimatized to high altitude exhibited significantly lower ATPase activities in comparison to non-acclimatized controls. The significant diminution in Km values of ATPases established in acclimatized hearts points to an increase in the affinity of their active sites to ATP. The latter effect is in agreement with the lowered rate of both the decrease in ATPase activities and the reduction of contractility in acclimatized hearts during the anoxic test, as well as with the considerably improved postanoxic reparability of contractions as compared to the controls. It is being concluded that the sarcolemmal changes at the level of ATPases involved in ionic transport processes represent an integral part of the adaptation complex to chronic high altitude hypoxia.     

Increased tissue PO2 and decreased O2 delivery and consumption after 80% exchange transfusion with polymerized hemoglobin

The O2-carrying blood substitute based on polymerized bovine hemoglobin (PBH) was used to determine efficacy in maintaining tissue Po2 after an 80% isovolemic blood exchange leading to a hematocrit of 19% [5.4 g Hb/dl from red blood cells (RBCs) and 6.3 g Hb/dl from PBH]. Effects were studied in terms of O2 delivery, O2 extraction, and tissue Po2 at the microcirculatory level at 1, 12, and 24 h after exchange transfusion in awake hamsters prepared with a window chamber model. At 1 h after exchange, arteriolar and venular diameters were decreased compared with baseline. Arteriolar diameter did not fully recover at 12 h after exchange, but venular diameter returned to normal. At 24 h after exchange, arteriolar and venular diameters were not different from baseline. Combining diameter and flow velocity data allowed us to calculate arteriolar and venular flows. At 1 h after exchange, arteriolar and venular flow was reduced compared with baseline. Arteriolar flow was lower at 12 h after exchange and recovered after 24 h. The number of capillaries with RBC passage [functional capillary density (FCD)] at 1 h after exchange with PBH was significantly lower than baseline. FCD remained decreased at 12 h; at 24 h after exchange transfusion, FCD was fully recovered. Tissue Po2 was maximal at 1 h after exchange and decreased progressively at 12 and 24 h after exchange. O2 release to the tissue was minimal at 1 h and increased at 12 and 24 h after exchange. These results suggest the impairment of tissue O2 metabolism after introduction of PBH into the circulation, which is mitigated as PBH concentration declines.     

Urate produced during hypoxia protects heart proteins from peroxynitrite-mediated protein nitration

Tyrosine nitration is a common modification to proteins in vivo, but the reactive nitrogen species responsible for nitration are often studied in vitro using just the amino acid tyrosine in simple phosphate solutions. To investigate which reactive nitrogen species could nitrate proteins in a complex biological system, we exposed rat heart and brain homogenates to peroxynitrite, nitric oxide under aerobic conditions, and other putative nitrating agents. Peroxynitrite was by far the most efficient nitrating agent when alternative targets were available to compete with tyrosine. Curiously, proteins in heart homogenates were substantially more resistant to nitration than brain homogenates. Ultrafiltration to remove low molecular weight compounds made the heart proteins equally susceptible as the brain proteins to nitration. Endogenous ascorbate and free thiols had little effect on nitration by peroxynitrite in either heart or brain. However, accumulation of urate formed by the oxidation of hypoxanthine by xanthine dehydrogenase and oxidase in heart appeared to be the major inhibitor of nitration. Heart homogenates treated with uricase, which converts urate to allantoin, showed equivalent nitration as in brain homogenates. Urate, as assayed by HPLC, was 58 +/- 8 microM in heart but only 4 +/- 2 microM in brain homogenates. Although xanthine dehydrogenase conversion to a free radical-producing oxidase can serve as an important source of superoxide and hydrogen peroxide during ischemia/reperfusion, our results suggest that urate formation by xanthine dehydrogenase may provide a significant antioxidant defense against peroxynitrite and related nitric oxide-derived oxidants.