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The purpose of this study was to test the hypothesis that increasing muscle contraction frequency, which alters the duty cycle and metabolic rate, would increase the contribution of the contractile phase to mean venous blood flow in isolated skeletal muscle during rhythmic contractions. Canine gastrocnemius muscle (n = 5) was isolated, and 3-min stimulation periods of isometric, tetanic contractions were elicited sequentially at rates of 0.25, 0.33, and 0.5 contractions/s. The O2 uptake, tension-time integral, and mean venous blood flow increased significantly (P < 0.05) with each contraction frequency. Venous blood flow during both the contractile (106 +/- 6, 139 +/- 8, and 145 +/- 8 ml x 100 g-1 x min-1) and noncontractile phases (64 +/- 3, 78 +/- 4, and 91 +/- 5 ml x 100 g-1 x min-1) increased with contraction frequency. Although developed force and duration of the contractile phase were never significantly different for a single contraction during the three contraction frequencies, the amount of blood expelled from the muscle during an individual contraction increased significantly with contraction frequency (0.24 +/- 0.03, 0.32 +/- 0.02, and 0.36 +/- 0.03 ml x N-1 x min-1, respectively). This increased blood expulsion per contraction, coupled with the decreased time in the noncontractile phase as contraction frequency increased, resulted in the contractile phase contribution to mean venous blood flow becoming significantly greater (21 +/- 4, 30 +/- 4, and 38 +/- 6%) as contraction frequency increased. These results demonstrate that the percent contribution of the muscle contractile phase to mean venous blood flow becomes significantly greater as contraction frequency (and thereby duty cycle and metabolic rate) increases and that this is in part due to increased blood expulsion per contraction.

The method described here allows the quantitative simultaneous determination of adenosine 5'-triphosphate, adenosine 5'-diphosphate, adenosine 5'-monophosphate, adenosine, guanosine 5'-triphosphate, guanosine 5'-diphosphate, guanosine, inosine 5'-monophosphate, inosine, uric acid, xanthine, hypoxanthine and beta-nicotinamide adenine dinucleotide by ion-pair high performance liquid chromatography. The chromatographic analysis requires 26 min per sample and allows the separation of the mentioned metabolites in a time as short as 16 min. Primary cultures of rat cerebellar granule cells were incubated in serum-free medium containing 25 mM KCl for 1.5-48 h and their acid extracts were injected onto column. Uric acid, inosine 5'-monophosphate, inosine, beta-nicotinamide adenine dinucleotide, adenosine, adenosine 5'-monophosphate, guanosine 5'-diphosphate, adenosine 5'-diphosphate, guanosine 5'-triphosphate and adenosine 5'-triphosphate were identified and quantified, while hypoxanthine, xanthine and guanosine were below the detection limit. This method makes use of a single-step sample pre-treatment procedure which allows a greater than 91% recovery of the compounds of interest and provides the assay of the metabolites of interest in little amounts of cell extracts. Therefore, this method is suitable to evaluate the energetic state in a variety of cell types, both under normal and dismetabolic conditions, such as after the induction of apoptosis or necrosis.

Objective: The postoperative course of cyanotic children is generally more complicated than that of acyanotic children. A possible reason is reoxygenation injury at the beginning of cardiopulmonary bypass. In this study we tested the hypothesis that reoxygenation of chronically hypoxic hearts is worse than that of normoxic hearts., Methods: Two groups of rats (n = 9 each) were exposed to either room air (fraction of inspired oxygen, 0.21%) or chronic hypoxia (fraction of inspired oxygen, 0.10%) for 2 weeks. Hearts were then isolated and perfused for 30 minutes with hypoxic buffer (oxygen saturation, 10%), followed by 30 minutes of reoxygenation (oxygen saturation, 100%)., Results: In hypoxic rats hematocrit values, hemoglobin concentrations, and red cells were higher (69% +/- 6% vs 40% +/- 6%, 219 +/- 14 vs 124 +/- 12 g/L, and 10.30 +/- 0.6 vs 6.32 +/- 0.5/microL/1000, respectively; P <.0001); the amount of ingested food was less (22.3 +/- 4.8 vs 30.7 +/- 3.9 g/d, P <.001), as was the amount of ingested water (21.0 +/- 3.1 vs 50.4 +/- 14.6 mL/d, P <.0001); and body weight was lower (182 +/- 14.2 vs 351 +/- 40.1 g, P <.0001), as was heart weight (1107 +/- 119 vs 1312 +/- 128 mg, P <.005). The heart weight/body weight ratio was higher (6.10 +/- 0.8 vs 3.74 +/- 0.1 mg/g, P <.0001). Systolic and diastolic functions, not different during the hypoxic baseline period, were more impaired in hypoxic than in normoxic hearts after the reoxygenation, whereas coronary resistance remained lower. During the hypoxic perfusion, the venous partial pressure of oxygen remained low in both groups, whereas during reoxygenation, partial pressure of oxygen was higher in hypoxic hearts, with a lower (P <.01) oxygen uptake. During hypoxic baseline adenosine triphosphate turnover, lactate production and lactate turnover were lower in hypoxic hearts (P <.005, P <.0001, and P <.0001, respectively)., Conclusions: Body and blood values are severely affected by chronic hypoxia, and the cardiac effects of uncontrolled reoxygenation after chronic hypoxia are more severe than after acute hypoxia.

Chronic hypoxia (CH) is believed to induce myocardial protection, but this is in contrast with clinical evidence. Here, we test the hypothesis that repeated brief reoxygenation episodes during prolonged CH improve myocardial tolerance to hypoxia-induced dysfunction. Male 5-week-old Sprague-Dawley rats (n = 7-9/group) were exposed for 2 weeks to CH (F(I)O(2) = 0.10), intermittent hypoxia (IH, same as CH, but 1 hr/day exposure to room air), or normoxia (N, F(I)O(2) = 0.21). Hearts were isolated, Langendorff perfused for 30 min with hypoxic medium (Krebs-Henseleit, PO(2) = 67 mmHg), and exposed to hyperoxia (PO(2) = 670 mm Hg). CH hearts displayed higher end-diastolic pressure, lower rate x pressure product, and higher vascular resistance than IH. During hypoxic perfusion, anaerobic mechanisms recruitment was similar in CH and IH hearts, but less than in N. Thus, despite differing only for 1 hr daily exposure to room air, CH and IH induced different responses in animal homeostasis, markers of oxidative stress, and myocardial tolerance to reoxygenation. We conclude that the protection in animals exposed to CH appears conferred by the hypoxic preconditioning due to the reoxygenation rather than by hypoxia per se.

Lipids compete with glucose for utilization by the myocardium. Amino acids are an important energetic substrate in the heart but it is unknown whether they reduce glucose disposal. The molecular mechanisms by which lipids and amino acids impair insulin-mediated glucose disposal in the myocardium are unknown. We evaluated the effect of lipids and amino acids on the insulin stimulated glucose uptake in the isolated rat heart and explored the involved target proteins. The hearts were perfused with 16 mM glucose alone or with 6% lipid or 10% amino acid solutions at the rate of 15 ml/min. After 1 h of perfusion (basal period), insulin (240 nmol/l) was added and maintained for an additional hour. Both lipids and amino acids blocked the insulin effect on glucose uptake (P<0.01) and reduced the activity of the IRSs/PI 3-kinase/Akt/GSK3 axis leading to the activation of glucose transport and glycogen synthesis. Amino acids, but not lipids, increased the activity of the p70 S6 kinase leading to the stimulation of protein synthesis. Amino acids induce myocardial insulin resistance recruiting the same molecular mechanisms as lipids. Amino acids retain an insulin-like stimulatory effect on p70 S6 kinase, which is independent from the PI 3-Kinase downstream effectors.

Post-ischemic reperfusion leads to apoptosis-linked loss of myocytes in cultured cells and in vivo. We tested the hypothesis that apoptosis develops without reperfusion in Langendorff-perfused hearts exposed to either low-flow ischemia (LFI) or hypoxia (H). Rat hearts were perfused with amino-acid-enriched Krebs-Henseleit buffer and exposed for 6 h to LFI (flow=2 ml/min, PO(2)=500+/-50mmHg, mean+/-SD), H (10ml/min, 120+/-15mmHg), or control conditions (C, 10ml/min, 500+/-50mmHg). At selected times, DNA-fragmentation was measured by agarose-gel electrophoresis and in situ TUNEL assay. After 6 h, the ratio (TUNEL-positive)/(total nuclei) was 0.620+/-0.027, 0.615+/-0.005, 0.404+/-0.021 in LFI, H and C, respectively. The ratio was 0.813+/-0.021 in hearts exposed to 90 min global no-flow ischemia and reperfused (5 h). To assess the role of membrane-diffusible factors, separate experiments were performed recirculating the medium and exposing hearts to LFI or H as above. The degree of apoptosis was the same in both the recirculating and non-recirculating modes. Thus, apoptosis develops by similar extents and in a time-dependent fashion in crystalloid-perfused rat hearts during LFI or H at the same oxygen shortage (flow.PO(2)), even without the reperfusion., (Copyright 2002 S. Karger AG, Basel)

We tested the hypothesis that down-regulated hearts, as observed during low-flow ischemia, adapt better to low O2 supply than non-down-regulated, or hypoxic, hearts. To address the link between down-regulation and endogenous ischemic protection, we compared myocardial tolerance to ischemia and hypoxia of increasing duration. To that end, we exposed buffer-perfused rat hearts to either low-flow ischemia or hypoxia (same O2 shortage) for 20, 40 or 60 min (n = 8/group), followed by reperfusion or reoxygenation (20 min, full O2 supply). At the end of the O2 shortage, the rate-pressure product was less in ischemic than hypoxic hearts (p < 0.0001). The recovery of the rate-pressure product after reperfusion or reoxygenation was not different for t = 20 min, but was better in ischemic than hypoxic hearts for t = 40 and 60 min (p < 0.02 and p < 0.0002, respectively). The end-diastolic pressure remained unchanged during low-flow ischemia (0.024 +/- 0.013 mmHg x min(-1)), but increased significantly during hypoxia (0.334 +/- 0.079 mmHg x min(-1)). We conclude that, while the duration of hypoxia progressively impaired the rate-pressure product and the end-diastolic pressure, hearts were insensitive of the duration of low-flow ischemia, thereby providing evidence that myocardial down-regulation protects hearts from injury. Excessive ATP catabolism during ischemia in non-down-regulated hearts impaired myocardial recovery regardless of vascular, blood-related and neuro-hormonal factors. These observations support the view that protection is mediated by the maintenance of the ATP pool.

There is growing evidence that hypertriglyceridemia exacerbates ischemic injury. We tested the hypothesis that triglycerides impair myocardial recovery from low-flow ischemia in an ex vivo model and that such an effect is related to endothelin-1. Hyperglycemic (glucose concentration = 12 mmol/l) and hyperinsulinemic (insulin concentration = 1.2 micromol/l) isolated rat hearts were perfused with Krebs-Henseleit buffer (PO(2) = 670 mmHg, pH 7.4, 37 degrees C) added with increasing triglycerides (0, 1,000, 2,000, and 4,000 mg/dl, n = 6-9 rats/group). Hearts were exposed to 60 min of low-flow ischemia (10% of basal coronary flow), followed by 30 min of reperfusion. We found that increasing triglycerides impaired both the diastolic (P < 0.005) and systolic (P < 0.02) recovery. The release of endothelin-1 during reperfusion increased linearly with triglyceride concentration (P = 0.0009). Elevated triglycerides also increased the release of nitrite and nitrate (NO(x)), the end products of nitric oxide, up to 6 micromol/min. Trimetazidine (1 micromol) further increased NO(x) release, blunted endothelin-1 release, and protected myocardial function during recovery. We conclude that high triglyceride levels impair myocardial recovery after low-flow ischemia in association with endothelin-1 release. The endothelium-mediated effect of triglycerides on both contractile recovery and endothelin-1 release is prevented by 1 microM trimetazidine.

Normal cell homeostasis relies on the ordered flow of nutrients and substrates through metabolic pathways. Any perturbation of this flow eventually leads to dysfunction, impairment of defense mechanisms, loss of viability and death. High altitude and pathological hypoxia represent a serious and frequent cause for the loss of cell viability. Although organisms customarily respond by triggering adaptive or maladaptive mechanisms, all forms of life eventually succumb to hypoxia if it is severe enough, irrespectively of the primary cause. This paper reviews one of the mechanisms by which organisms respond to hypoxia: erythropoiesis. Although such response is not always beneficial, the discovery of the biochemical mechanisms underlying erythropoiesis has triggered an active field of research that is actually applying lessons learned in the mountains to a more clinical environment.

The aim is to determine if a single measurement of blood 2,3-diphosphoglycerate combined with gas analysis (pH, PCO2, PO2 and saturation) can identify the cause of an altered blood-oxygen affinity: the presence of an abnormal haemoglobin or a red cell disorder. The population (n=94) was divided into healthy controls (A, n=14), carriers of red cell disorders (B, n=72) and carriers of high oxygen affinity haemoglobins (C, n=8). Those variables were measured both in samples equilibrated at selected PCO2 and PO2 and in venous blood. In the univariable approach applied to equilibrated samples, we correctly identified C subjects in 93.6% or 96.8% of the cases depending on the selected variable, the standard P50 (PO2 at which 50% of haemoglobin is oxygenated) or a composite variable calculated from the above measurements. After introducing the haemoglobin concentration as a further discriminating variable, the A and B subjects were correctly identified in 91.9% or 94.2% of the cases, respectively. These figures become 93.0% or 86.1%, and 93.7% or 94.9% of the cases when using direct readings from venous blood, thereby avoiding the blood equilibration step. This test is feasible also in blood samples stored at 4 degrees C for 48 h, or at room temperature for 8 h.

Metabolic events during ischaemia are probably important in determining post-ischaemic myocardial recovery. The aim of this study was to assess the effects of the beta-blocker atenolol and the high energy demand in an ischaemia-reperfusion model free of neurohormonal and vascular factors. We exposed Langendorff-perfused isolated rat hearts to low-flow ischaemia (30 min) and reflow (20 min). Three groups of hearts were used: control hearts (n =11), hearts that were perfused with 2.5 micrograms l-1atenolol (n =9), and hearts electrically paced during ischaemia to distinguish the effect of heart rate from that of the drug (n =9). The hearts were freeze-clamped at the end of reflow to determine high-energy phosphates and their metabolites. During ischaemia, the pressure-rate product was 2.3+/-0.2, 5.2+/-1.1, and 3.3+/-0.3 mmHg 10(3)min in the control, atenolol and paced hearts, respectively. In addition, the ATP turnover rate, calculated from venous (lactate), oxygen uptake and flow, was higher in atenolol (11.2+/-1.7 micromol min-1) and paced (8.1+/-0.8 micromol min-1) hearts than in control (6.2+/-0.8 micromol min-1). At the end of reflow, the pressurexrate product recovered 75.1+/-6.4% of baseline in control vs 54.1+/-9.1 and 48.8+/-4.4% in atenolol and paced hearts (P<0.05). In addition, the tissue content of ATP was higher in the control hearts (15.8+/-1. 0 micromol g(dw)(-1)) than in atenolol (10.5+/-2.6 micromol g(dw)(-1)) and paced (10.9+/-1.3 micromol g(dw)(-1)) hearts. Thus, by suppressing the protective effects of down-regulation, both atenolol and pacing apparently depress myocardial recovery in this model., (Copyright 1999 Academic Press.)

It is still unclear if performance recovery in postischemic hearts is related to their tissue level of high-energy phosphates before reflow. To test the existence of this link, we monitored performance, metabolism and histological damage in isolated, crystalloid-perfused rat hearts during 20 min of low-flow ischemia (90% coronary flow reduction) and reflow. To prevent interference from different ischemia times and perfusing media compositions, the ischemic ATP level was varied by changing energy demand (electrical pacing at 330 min(-1)). Under full coronary flow conditions, work output, as well as ATP and phosphocreatine contents were the same in control, spontaneously contracting (n = 23) and paced (n = 21) hearts. During low-flow ischemia, the higher work output (p < 0.0001) in paced hearts decreased their tissue content of ATP, phosphocreatine and total adenylates and purines (p < 0.05), as opposed to maintained values in control hearts. During reflow, the recovery of mechanical performance and O2 uptake was 94 +/- 5% and 110 +/- 9% (p = NS vs. baseline) in controls, vs. 71 +/- 5% and 74 +/- 6% in paced hearts (p < 0.004 vs. baseline). The levels of ATP and total adenylates and purines remained constant in control, but were markedly depressed (p < 0.05 vs. baseline) in paced hearts. Phosphocreatine+creatine was the same in both groups. These data, together with the observed lack of creatine kinase leakage and of structural damage, indicate that myocardial recovery during reflow reflects the tissue level of ATP, phosphocreatine and total adenylates and purines during ischemia, regardless of physical cell damage.

The effects of both high blood H+ concentration ([H+]) and high blood lactate concentration ([lactate]) under ischemia-reperfusion conditions are receiving attention, but little is known about their effects in nonischemic hearts. Isolated rat hearts were Langendorff perfused at constant flow with media at two pH values (7.4 and 7.0) and two [lactate] (0 and 20 mM) in various sequences (n = 6/group). Coronary flow and arterial O2 content were kept constant at levels that allowed hearts to function without O2 supply limitation. We measured contractility, O2 uptake, diastolic pressure, and at the end of the protocol, tissue [lactate] and pH. Perfusion with high [lactate] raised tissue [lactate] from 5.5 +/- 0.1 to 17.5 +/- 2.6 micromol/heart (P < 0.0001), whereas decreasing the pH of the medium decreased tissue pH from 6.94 +/- 0.02 to 6.81 +/- 0.06 (P = 0.002). Heart rate was not affected by high [lactate] but was reversibly depressed by high [H+] (P = 0.004). Developed pressure declined by 20% in response to high [lactate], high [H+], and high [lactate] + high [H+] (P = 0.002). After the high-[lactate] challenge was withdrawn, pressure continued to decline. In contrast, withdrawing the high [H+] challenge allowed partial recovery. The behavior of diastolic pressure mirrored that of developed pressure. Although unaffected by high [lactate], the O2 uptake was reversibly depressed by high [H+]. This suggests higher O2 cost per contraction in the presence of high [lactate]. We conclude that for similar acute contractility depression, high [lactate] induces irreversible damage, likely at some point in the pathway of O2 utilization. In contrast, the effect of high [H+] appears reversible. These differential behaviors may have implications for heart function during heavy exercise and ischemia-reperfusion events.

The objective of this study was to test the hypothesis that the beneficial effect of trimetazidine during reflow of ischemic hearts is mediated by energy sparing and ATP pool preservation during ischemia. Isolated rat hearts (controls and rats treated with 10(-6) M trimetazidine, n = 17 per group) underwent the following protocol: baseline perfusion at normal coronary flow (20 minutes), low-flow ischemia at 10% flow (60 minutes), and reflow (20 minutes). We measured contractile function, O2 uptake, lactate release, venous pH and PCO2, and the tissue content of high-energy phosphates and their metabolites. During baseline, trimetazidine induced higher venous pH and lower PCO2 without influencing performance and metabolism. During low-flow ischemia, trimetazidine reduced myocardial performance (P = 0.04) and ATP turnover (P = 0.02). During reflow, trimetazidine improved performance (91 +/- 6% versus. 55 +/- 6% of baseline), prevented the development of diastolic contracture and coronary resistance, and reduced myocardial depletion of adenine nucleotides and purines. ATP turnover during low-flow ischemia was inversely related to recovery of the rate-pressure product (P = 0.002), end-diastolic pressure (P = 0.007), and perfusion pressure (P = 0.05). We conclude that trimetazidine-induced protection of ischemic-reperfused hearts is also mediated by energy sparing during ischemia, which presumably preserves the ATP pool during reflow.

The mechanism(s) limiting muscle O2 uptake (VO2) kinetics was investigated in isolated canine gastrocnemius muscles (n = 7) during transitions from rest to 3 min of electrically stimulated isometric tetanic contractions (200-ms trains, 50 Hz; 1 contraction/2 s; 60-70% of peak V(O2)). Two conditions were mainly compared: 1) spontaneous adjustment of blood flow (Q) [control, spontaneous Q (C Spont)]; and 2) pump-perfused Q, adjusted approximately 15 s before contractions at a constant level corresponding to the steady-state value during contractions in C Spont [faster adjustment of O2 delivery (Fast O2 Delivery)]. During Fast O2 Delivery, 1-2 ml/min of 10(-2) M adenosine were infused intra-arterially to prevent inordinate pressure increases with the elevated Q. The purpose of the study was to determine whether a faster adjustment of O2 delivery would affect V(O2) kinetics. Q was measured continuously; arterial (Ca(O2)) and popliteal venous (Cv(O2)) O2 contents were determined at rest and at 5- to 7-s intervals during contractions; O2 delivery was calculated as Q x Ca(O2), and V(O2) was calculated as Q x arteriovenous O2 content difference. Times to reach 63% of the difference between baseline and steady-state VO2 during contractions were 23.8 +/- 2.0 (SE) s in C Spont and 21.8 +/- 0.9 s in Fast O2 Delivery (not significant). In the present experimental model, elimination of any delay in O2 delivery during the rest-to-contraction transition did not affect muscle V(O2) kinetics, which suggests that this kinetics was mainly set by an intrinsic inertia of oxidative metabolism.

The purpose of this study was to examine the bioenergetics and regulation of O2 uptake (VO2) and force production in contracting muscle when blood flow was moderately reduced during a steady-state contractile period. Canine gastrocnemius muscle (n = 5) was isolated, and 3-min stimulation periods of isometric, tetanic contractions were elicited sequentially at rates of 0.25, 0.33, and 0.5 contractions/s (Hz) immediately followed by a reduction of blood flow [ischemic (I) condition] to 46 +/- 3% of the value obtained at 0.5 Hz with normal blood flow. The VO2 of the contracting muscle was significantly (P < 0.05) reduced during the I condition [6.5 +/- 0.8 (SE) ml . 100 g-1 . min-1] compared with the same stimulation frequency with normal flow (11.2 +/- 1.5 ml . 100 g-1 . min-1), as was the tension-time index (79 +/- 12 vs. 123 +/- 22 N . g-1 . min-1, respectively). The ratio of VO2 to tension-time index remained constant throughout all contraction periods. Muscle phosphocreatine concentration, ATP concentration, and lactate efflux were not significantly different during the I condition compared with the 0. 5-Hz condition with normal blood flow. However, at comparable rates of VO2 and tension-time index, muscle phosphocreatine concentration and ATP concentration were significantly less during the I condition compared with normal-flow conditions. These results demonstrate that, in this highly oxidative muscle, the normal balance of O2 supply to force output was maintained during moderate ischemia by downregulation of force production. In addition, 1) the minimal disruption in intracellular homeostasis after the initiation of ischemia was likely a result of steady-state metabolic conditions having already been activated, and 2) the difference in intracellular conditions at comparable rates of VO2 and tension-time index between the normal flow and I condition may have been due to altered intracellular O2 tension.

As a model of human hypoxia, exposure to high altitude causes a number of ventilatory, circulatory and hemopoietic adaptations. A review of the literature on blood gas transport responses to hypoxia indicates that they are influenced not only by altitude, but also by factors related to acclimatization. In addition, it appears that the need to oxygenate tissues conflicts with the need to maintain H+ homeostasis. Thus, the final situation represents a compromise between the respiratory adjustment aimed at increasing blood alkalosis in order to optimize the oxygen transport system, and the metabolic readjustment aimed at reestablishing normal blood pH. There are factors like red cell 2,3-diphosphoglycerate, a compound that decreases the hemoglobin affinity for oxygen, that can influence that balance by affecting arterial oxygen saturation through mechanisms independent of respiration.

In this study, we assessed one particular aspect of the arrhythmogenic phenomena that occur during reperfusion secondary to thrombolysis, that is the therein involved metabolic mechanisms. The employed experimental model (isolated Langendorff-perfused rat heart) allowed us to distinguish which factor involved during ischemia, low coronary flow or low oxygen tension, is primarily involved during arrhythmogenesis. This was made possible by comparing two settings characterized by the same oxygen supply, but with different coronary flows and PO2 values, i.e., ischemia and hypoxemia. As expected, the contractile dysfunction was higher during reoxygenation at the end of hypoxemia than during reperfusion at the end of ischemia (p < 0.05). However, the incidence of arrhythmias was similar in both cases. Therefore, whereas the contractile dysfunction appears to be more sensitive to coronary flow, the incidence of arrhythmias appears to be more sensitive to the total oxygen supply to the heart. This implies that the mechanisms underlying the development of contractile dysfunction and arrhythmogenesis follow different paths.

We tested the hypothesis that residual oxygen supply during acute low-flow ischaemia or hypoxemia is a major regulator of myocardial performance, metabolism and recovery. Rat hearts were exposed for 20 min to either ischemia (coronary flow reduced to 10% of baseline), hypoxemia (oxygen content reduced to 10% baseline) or a "mixed" condition (combined ischaemia and hypoxemia). The oxygen supply (coronary flow x oxygen content) was matched in all groups (n = 16 per group). Hypoxemic hearts had the highest performance (systolic and developed pressures, +/- dP/dtmax and oxygen uptake) and content of IMP and AMP. Ischaemic hearts had the highest content of ATP, phosphocreatine, adenine nucleotides and purines. As flow and/or oxygenation were restored, post-ischemic hearts showed better functional and metabolic recovery than post-hypoxemic ones. "Mixed" hearts were more similar to hypoxemic ones during oxygen shortage but to ischemic ones during recovery. We conclude that as oxygenation is critically limiting, coronary flow is relatively more important than oxygen supply in determining myocardial function, metabolism and recovery, most likely secondary to changes in the metabolism of diffusible substances.

Aim of this study was to assess the role of O2, lactate and energy demand in the regulation of myocardial work during severe dysoxia. For this purpose, we measured function and metabolism in isolated Langendorff-perfused rat hearts exposed to either ischemia or hypoxemia (matched for the O2 supply, 10% of baseline) with/out electrical stimulation. When hearts could adjust their HR, hypoxemia demanded more energy than ischemia (p < 0.05) despite same O2 supply. Venous PO2 was 12 +/- 2 or 139 +/- 20 mmHg (p < 0.0001), respectively, but VO2 was the same. After 10 min at HR = 300 min-1, myocardial performance increased in ischemic but not in hypoxemic hearts. PvO2 and VO2 were not affected by pacing. In contrast, both venous [lactate] and lactate production rate increased, but in ischemic hearts only. We conclude that ischemic hearts were downregulated while hypoxemic hearts were not. Likely, depressed washout of lactate during ischemia could offset the effects of O2 in severely dysoxic hearts. Anaerobic glycolysis provided the energy necessary to meet increased energy demand in ischemic hearts, but could not exploit this action in hypoxemic hearts probably because in these hearts it was already working near maximum.

The xanthine/xanthine oxidase reaction produces reproducible amounts of oxygen-derived free radicals that oxidize human oxyhemoglobin (Hb). We monitored the kinetics of the oxidation of stripped Hb (sHb), purified HbA0 and alpha-alpha cross-linked Hb (HbXL99 alpha) at [Hb] in the 5 to 150 microM (heme) range. For increasing [Hb], the oxidation halftime (t1/2) increased for all Hbs, but t1/2 was always less for HbXL99 alpha than for HbA0 and sHb. Such feature was attributed to the lower affinity for O2 of HbXL99 alpha and may represent a serious problem for use of this Hb as blood substitute.

Thyroid hormones may influence the active transport of Ca2+ across the cell membrane. To test the physiologic relevance of this mechanism, we used inside-out human red cell membrane vesicles as a model of the cell membrane Ca2+ pump. We monitored by spectrophotometric methods the kinetics of the uptake of Ca2+ in the presence of 10(-5)-10(-10) M thyroid hormones or their analogues. Vesicles freed of calmodulin and protein inhibitor(s) of the Ca2+ pump were also obtained. The results are as follows: (1) Thyroxine inhibits the active Ca2+ uptake; (2) this effect antagonizes that of soluble calmodulin; and (3) triiodothyronine and other analogues of the thyroid hormones are less active than thyroxine. We conclude that the thyroid hormones may influence cell Ca2+ homeostasis by direct action on the Ca2+ pump.

Aim of this study was to assess the effect of propionyl-L-carnitine (PLC), a naturally occurring derivative of L-carnitine, in cardiac hypertrophy induced by pressure overload in rats. The abdominal aorta was banded and the rats received one daily administration of PLC (50 mg/kg) or saline for four days. The hearts were excised 24 h after the last administration and were perfused retrogradely with oxygenated Krebs-Henseleit buffer containing 1.2 mM palmitate bound to 3% (w/v) albumin, 2.5 microM PLC and 25 microM L-carnitine. A saline-filled balloon was inserted into the left ventricle and the heart contractility was measured at three volumes of the balloon, corresponding to zero diastolic pressure and to increased volumes (110 and 220 microliters) over the zero volume. At the end of the perfusion, the hearts were freeze-clamped, weighed and analyzed for adenine nucleotide and phosphocreatine (PCr) content by HPLC methods. No differences in the myocardial performance were found at zero diastolic pressure. In contrast, at high intraventricular volume, the maximal rate of ventricular relaxation was increased in PLC-treated with respect to saline-treated controls (p < 0.05). In addition, the increase of the end-diastolic pressure at increasing balloon volume was more marked in controls than in the PLC-treated hearts (p < 0.02). These data correlate well with the measured higher level of total adenine nucleotides (p < 0.05) and ATP (p < 0.02) in the PLC-treated hearts, while PCr was the same in both groups. Parallel experiments performed in the absence of palmitate in the perfusing media failed to show any effect of PLC.(ABSTRACT TRUNCATED AT 250 WORDS)

The events associated to myocardial ischemia result from 2 overlapping phenomena due to reduced blood flow (ischemia) and reduced O2 supply (hypoxemia). To distinguish these effects, 2 groups of isolated rat hearts were perfused through the aorta (Langendorff's method) with Krebs-Henseleit buffer, and were exposed for 20 min to hypoxemia or ischemia, matched in terms of the O2 supply (10% of baseline), with continuous monitoring of cardiac contractility, O2 uptake and lactate production. The developed pressure and the O2 uptake were similar in hypoxemic and ischemic hearts; heart rate, end-diastolic pressure and lactate production rate were higher in hypoxemia than in ischemia; the recovery from hypoxemia was less than that from ischemia, despite the same O2 supplies; treatment with superoxide dismutase and catalase, scavengers of the O2 derived free radicals, during hypoxemia, allowed hypoxemic hearts to recover as ischemic hearts. Therefore, the main determinant of the reperfusion injury is to be attributed to the low O2 supply rather than to the low coronary flow; part of the injury is due to free radicals; a substantial portion is mediated by the energy demand during the stress which was higher in hypoxemia than in ischemia.

We have studied the relationship between the in vivo aging process of the human red cell (RBC) and its main function, the transport of O2 from the lungs to the tissues. This study included several approaches. First, we observed that the affinity for O2 in young RBCs was lower than in old RBCs (p less than 0.0005) due to different intracellular concentration of 2,3-diphosphoglycerate, main effector of hemoglobin. Second, we explored whether there are some subgroups of the healthy human population with altered RBC age distribution: females in the age range 25-35 exhibited significantly younger RBCs (p less than 0.0005) and lower RBC-O2 affinity (p less than 0.01) than other groups. Correspondingly, the RBC-O2 affinity in female blood was significantly lower (p less than 0.002) than in male blood. Third, we correlated by two independent methods the lowered RBC-O2 affinity to a more efficient O2 delivery to the tissues by two independent methods: 1) calculating the size of the cardiac output increase required to sustain the tissue oxygenation after an increase of the RBC affinity for O2; and 2) monitoring the enhanced cardiac function in isolated rat hearts perfused with RBCs at low O2 affinity. Finally, comparing some hematologic findings relevant for the O2 transport in two healthy populations with different RBC age distributions, such as age-matched females and males, it appeared that the low RBC-O2 affinity in females is an adaptive response to their lower [Hb].(ABSTRACT TRUNCATED AT 250 WORDS)

In the reoxygenated hypoxic heart, hypoxanthine is either oxidized by xanthine oxidase with production of toxic oxygen species or salvaged for the ATP pool by hypoxanthine-guanine phosphoribosyl transferase. To characterize the repartition of hypoxanthine between the two pathways, we have subjected rat hearts to 20 min hypoxia and monitored the recovery (ventricular, end-diastolic and coronary pressures, and the contraction rate) during the reoxygenation (30 min) in the presence of either hypoxanthine or guanine alone, or both. The rate-pressure product recovered 78% of the pre-hypoxia values in hearts reoxygenated with 100 microM hypoxanthine and 80% in hearts reoxygenated with 100 microM guanine, in contrast to 49% in the presence of both hypoxanthine and guanine (100 microM each). Thus, it is likely that hypoxanthine is salvaged when present alone and is oxidized generating the reperfusion injury when the salvage is prevented by guanine that competes with hypoxanthine from the same site of hypoxanthine-guanine phosphoribosyl transferase. The functional impairment was slower when hypoxanthine was replaced by xanthine, and was eliminated by superoxide dismutase and catalase, indicating that the injury is caused by toxic oxygen species generated from hypoxanthine and xanthine oxidase. These data suggest that the salvage pathway may be critical in preventing the reperfusion injury in hypoxic hearts.

The [2,3-DPG]/[Hb] ratio and the P50 were found to be lower in the 10% denser (old) than in the 10% lighter (young) red blood cell (RBC) fractions (0.57 +/- 0.13 vs 0.96 +/- 0.13 and 23.02 +/- 0.85 vs 27.47 +/- 1.05 Torr, respectively, mean +/- SD, P less than 0.0005 for both, n = 6). The RBC aging processes appear thus to affect the RBC oxygen affinity. However, the [2,3-DPG] changes do not fully explain the drop of not fully explain the drop of P50 as measured at constant [H+], [CO2] and [HbCO]. It is therefore postulated that an additional factor is involved in the regulation of the oxygen affinity in the ageing RBC. The RBC density in 59 normal individuals matched for age (infants, adult, and aged) and for sex was found to be younger in adult females than in all other groups (P less than 0.0005), including an age-matched group of pregnant women. Correspondingly, the [2,3-DPG]/[Hb] ratio and the P50 are higher in adult females than in adult males (0.92 +/- 0.10 vs 0.82 +/- 0.09, P less than 0.009, and 29.03 +/- 1.07 vs 27.72 +/- 0.82 Torr, P less than 0.002, respectively). These data are evaluated in terms of the efficiency of the oxygen transport calculating the circulatory load required to transport a given amount of oxygen to the tissues. The results indicate that the lower oxygen affinity (due to the younger RBC population) in adult females partially compensates for their lower [Hb].

The authors have investigated the relationships between the active calcium transport across the human red blood cell (RBC) membrane and the RBC aging processes in vivo and in vitro. For the study of this biological system, the authors have determined the active calcium uptake by inside-out membrane vesicles obtained from selected RBC populations. This model provided an optimal way to assess the biochemical and functional responses of the human cell to the oxidative stimulus triggered by the cellular aging processes. The activity of the calcium pump is indeed strictly correlated to the oxidative damage suffered by the RBC, being higher in the aged RBC. It appears that the main controller of the active calcium transport is the age-dependent protein inhibitor of the calcium pump.

We describe a new polarographic method to measure the haemoglobin oxygen saturation in whole blood, employing up to 10 microliters of sample in a standard case. The measurement is done in an anaerobic stainless-steel cuvette (1 ml) recording three oxygen tension values: (1) that of an air-equilibrated buffer before the addition of the sample; (ii) that after the addition of the sample; and (iii) that after the addition of an oxidant. The haemoglobin oxygen saturation is then calculated from the three oxygen tension values, the volume of the reagents, and the solubility coefficient of oxygen. This method is simple, inexpensive and accurate, and correlates well with other standard methods.

We compare three separation techniques for preparative purposes, i.e. ion-exchange chromatography on CM-cellulose, conventional isoelectric focusing in polyacrylamide gel slabs and immobilized pH gradients. The biological system used to test the three methods is a solution containing four hemoglobin (Hb) valence intermediates, i.e. metHb, oxyHb, (alpha + beta O2)2 and (alpha O2 beta +)2. The delta pI between the two valence intermediates is 0.04 pH units. Immobilized pH gradients give the best performance in terms of resolving power, total amount of protein which can be loaded and retention of biological activity by the protein (the latter assessed by determination of CO dissociation rates).

A computer program is described, relating blood flow with venous PO2 for any given set of the following parameters: oxygen uptake, respiratory quotient, the 2,3-DPG/Hb molar concentration ratio (G), arterial PO2, PCO2, and pH. Two compartments (total body and one leg) and two conditions (rest and maximal exercise) are considered. Calculations are performed at five altitudes (0, 3850, 5400, 6300 and 8848 m), for which the above variables are known. The results indicate that an increased G value has a negative effect on the oxygen delivery to tissues at very high altitudes (greater than 5400 m), irrespectively of the work load, since larger blood flows (delta Q on the summit of Mt. Everest is +4 to +71/min, and +1 to +2.5 l/min, for whole body and one leg, respectively) are required for a given oxygen uptake. For submaximal work at altitudes ranging from sea level up to 5400 m, as well as for moderate work at 5400 m, high G values improve the oxygen delivery to tissues.

Investigations into the properties of haemoglobin often require the isolation of the valence intermediates (alpha o2 beta+)2 and (alpha+ beta o2)2. Chromatofocusing with an anion-exchange gel (Mono PTM; Pharmacia, particle size 10 micron) in an HR5/20 column at various temperatures (10-25 degrees C) provides an excellent method for this task. A linearly decreasing pH gradient (8 to 7, generated by Polybuffer 96, Pharmacia) eluted sequentially the species methaemoglobin, (alpha o2 beta+)2, (alpha+ beta o2)2 and oxygenated haemoglobin. Calibration graphs help in quantitative analyses. This method is simpler and less time consuming and provides a similar or even better resolution than the traditional ion-exchange or isoelectric focusing methods.

The oxygen affinity in the blood of adult Dorset sheep with HbB was studied by determining several oxygen equilibrium curves under different conditions of pH and PCO2 by a method that allowed strict control of pH, PCO2, and HCO3- concentration over the entire curve. Nomograms and equations were derived that allowed the estimation of the oxygen saturation in a sample of blood in the range 0 to 100%, either from pH, PCO2 and PO2, or from PO2 and known P50. The advantage of this approach is that no assumptions about the shape of the oxygen equilibrium curve need be made (i.e. the Hill parameter, n, is not required to be constant), since the entire curve was measured.

We have measured the intrinsic CO dissociation rates from the subunits of the human hemoglobin tetramers (alpha CO beta NO)2 and (alpha NO beta CO)2 using microperoxidase and a stopped-flow spectrophotometer. The dissociation of NO is negligible. The rate constants for the and the subunits are similar (0.014 s-1 vs. 0.011 s-1, respectively, at pH 7, 20 C; and 0.016 s-1 for both in the presence of inositol hexaphosphate), indicating that they are equivalent in the first step of the CO dissociation. Therefore, the chain unequality observed in the third and fourth steps (Samaja, M., Rovida, E., Niggeler, M., Perrella, M., and Rossi-Bernardi, L. (1987). J. Biol. Chem.: 262, 4528-4533) are not due to the intrinsic properties of the subunits, but to the conformational state of the molecule.

A new spectrophotometric cuvette holder to be used for subzero temperature is described. The device is easily adaptable to a commercial spectrophotometer and it was checked down to --40 degrees C. Satisfactory mixing of the reactants contained in the cuvette at low temperatures is attained using a special stirrer and suitable solution volumes. The rate of carbonmonoxyhemoglobin oxidation by K3Fe(CN)6 at different subzero temperatures has been studied using this apparatus; the results are in agreement with the extrapolated data at room temperature.

A simplified model is described to estimate the oxygen delivery to tissues as a function of oxygen uptake, gas exchange ratio, 2,3-DPG/Hb concentration ratio, arterial and venous PO2, PCO2 and pH. Due to the complexity of the oxygen delivery system, the aim of this model is to predict relative changes of the oxygen delivery to tissues induced by changes of the other variables, rather than to yield absolute values. In this work, the importance of the observed shifts of the hemoglobin oxygen equilibrium curve at altitude is evaluated in terms of the efficiency of the oxygen transport system. It will be shown that a rightward shift of the oxygen equilibrium curve is beneficial up to 5400 m.a.s.l., while for higher altitudes such shifts lead to less efficient oxygen delivery to tissues.

The oxygenation of some human organs (brain, heart, kidneys, and legs) is correlated to the oxygen carrying characteristics of blood by predicting the PVO2 vs Q relationship at a given metabolic level of the organ. The effect of moderate shifts of the oxygen equilibrium curve [approximately equal to 2 Torr (0.27 kPa), comparable to the shifts caused by non-massive transfusions of blood with altered 2,3-diphosphoglycerate (DPG) concentration] is evaluated in terms of the efficiency of organ oxygenation. The results indicate the following. (1) An increase of the P50 from 27.9 to 30.0 Torr (3.71 to 3.99 kPa), that is the consequence of an increase of the [DPG]/[Hb] ratio from 0.8 to 1.04 M/M, is advantageous for all organs, because the normal metabolic level can be maintained with a considerable reduction of Q (approximately 10%). (2) This reduction is similar to that caused by an increase of [Hb] from 160 to 182 g/L, but without increasing the blood viscosity and the vascular resistance. (3) This advantage is different for the various organs, as a function of their blood supply and metabolic level characteristics. These features were also observed at any PaO2 in the range 60-300 Torr (7.98-39.9 kPa), and when simulating acidemia or alkalemia, as well as a pH gradient across the organ.

The hemoglobinemia S is a genetic defect due to a replacement of a single amino acid in the beta-chain of the human hemoglobin, leading to the most characteristic case of primary blood hyperviscosity. The difference in beta-chain of normal HbA compared to that of HbS is represented by the replacement of glutamic acid, normally found in the 6th position, with valine. The sickling of HbS-containing erythrocytes is due to the polymerization of deoxygenated HbS tetramers with formation of linear structures and to the parallel distribution of these fibers in the red blood cell. The full deoxygenation of HbS blood induces the characteristic morphological changes of red blood cell and increases the blood viscosity from 40 to 120% if compared to the viscosity of the same oxygenated blood. Any change in the viscosity of normal blood following deoxygenation was observed. Almost all the clinical symptoms found in patients carrying HbS can be directly or indirectly correlated to the increased blood viscosity following deoxygenation.

The acid-base equilibrium in the blood of sheep is different from that of human blood mainly because of a lower concentration of 2,3-DPG. A nomogram relating pH, pCO2, total CO2 content and base excess has been developed.

To investigate the mechanism of allosteric switching in human hemoglobin, we have studied the dissociation of the ligand (CO) from several intermediate ligation states by a stopped-flow kinetic technique that utilizes competitive binding of CO by microperoxidase. The hemoglobin species investigated include Hb(CO)4, the diliganded symmetrical species (alpha beta-CO)2 and (alpha-CO beta)2, and the di- and monoliganded asymmetrical species (alpha-CO beta-CO)(alpha beta), (alpha-CO beta)(alpha beta-CO), (alpha beta-CO) (alpha beta), and (alpha-CO beta)(alpha beta). They were obtained by rapid reduction with dithionite of the corresponding valence intermediates that in turn were obtained by chromatography or by hybridization. The nature and concentration of the intermediates were determined by isoelectric focusing at -25 degrees C. The study was performed at varying hemoglobin concentrations (0.1, 0.02, and 0.001 mM [heme]), pH (6.0, 7.0, 8.0), with and without inositol hexaphosphate. The results indicate that: (a) hemoglobin concentration in the 0.1-0.02 mM range does not significantly affect the kinetic rates; (b) the alpha chains dissociate CO faster than the beta chains; (c) the symmetrical diliganded intermediates show cooperativity with respect to ligand dissociation that disappears in the presence of inositol hexaphosphate; (d) the monoliganded intermediates dissociate CO faster than the diliganded intermediates; (e) the asymmetrical diliganded intermediates are functionally different from the symmetrical species.

Oxygen equilibrium curves of fresh, normal human blood have been measured by new methods which allow the control of pH, pCO2, and 2,3-diphosphoglycerate and which yield higher accuracy at the extremes of saturation than was possible previously. The curve determined by these techniques lies slightly to the right of the standard curve of Roughton et al. (Roughton, F.J.W., Deland, E.C., Kernohan, J.C., and Severinghaus, J.W. (1972) in Oxygen Affinity of Hemoglobin and Red Cell Acid Base Status (Astrup, P., and Rørth, M., eds) pp. 73-83, Academic Press, New York). The greatest difference is at low oxygen saturation, probably owing to the fact that the latter data were obtained under conditions which would lead to depletion of cellular 2,3-diphosphoglycerate. The range of p50 (oxygen pressure at half-saturation) values for four normal subjects was 28.3 mm Hg to 29.0 mm Hg. Adair's stepwise oxygenation scheme has been used to analyze the curves with the result that a1 = 0.1514 X 10(-1) (+/- 10%) mm-1; a2 = 0.9723 X 10(-3) (+/- 8%) mm-2; a3 = 0.1703 X 10(-3) (+/- 50%) mm-3; a4 = 0.1671 X 10(-5) (+/- 2%) mm-4 for the best of four data sets. Because these constants are very sensitive to changes in the shape of the oxygenation curve, this analysis is much more useful than p50 measurements in the investigation of the various allosteric effectors of the function of hemoglobin within the red cell.

We determined normal human blood p50 at various pH values (range 7.0 to 7.6) as a function of the proportion of carboxyhemoglobin (COHb) in total hemoglobin, from 0 to 23%. The d(log p50)/d[COHb] coefficient is 0.00848, independent of pH and 2,3-diphosphoglycerate. The derived equation allows the calculation of p50 as a function of COHb with an approximation of +/- 0.54 mmHg (about 72 Pa), and can be combined with other calculations (Clin Chem 27:1856-1861, 1981; Clin Chem 29:110-114, 1983) to predict p50 under any condition of pH within the range 7.0-7.6, ratio of [2,3-diphosphoglycerate] to [total hemoglobin] (range 0.3-2.5), pCO2 (range 20-90 mmHg), temperature (range 19-43 degrees C), and COHb (range 0-23%).

We report here the development of a new method that allows continuous determination of the oxygen dissociation curve for microsamples (600 mul) of whole blood under conditions of pH, pCO2, methemoglobin concentration, and 2,3-diphosphoglycerate content closely approaching those found in the circulatory system. The method consists of gradually oxygenating a blood sample by adding H2O2 in the presence of catalase (EC 1.11.1.6), to produce the reaction H2O2 leads to H2O + 1/2 O2. Because the total oxygen content of blood can be derived from the known rate of H202 addition and the pO2 is determined in the liquid phase by an oxygen electrode, the two functions (total O2 content) and (% oxygen saturation) vs. pO2 are simple to calculate. pCO2 and pH are controlled by adding base simultaneously with the gradual oxygenation of blood. The method described thus avoids the direct measurement of oxygen saturation of whole blood.