Response of the glycolysis of human erythrocytes to the transition from the oxygenated to the deoxygenated state at constant intracellular pH

The time course of the rate of the glycolysis of human erythrocytes and of some metabolites were determined before and after rapid deoxygenation at constant intracellular pH. For this purpose stripped deoxygenated haemoglobin was used as a rapid oxgen acceptor.Deoxygenation causes an increase of the glycolytic rate by 26%. Glucose 6-phosphate is decreased while the adenine nucleotides and 2,3-bisphosphoglycerate remain constant. Fructose 1,6-bisphosphate and the triose phosphates decrease transiently before rising.The data can be explained by increased binding of phosphocompounds to deoxygenated as compared with oxygenated haemoglobin. Thereby the control enzymes hexokinase and phosphofructokinase are influenced. It is concluded that under physiological conditions changes in the oxygenation state of haemoglobin per se alter the glycolytic rate.     

Response of the glycolysis of human erythrocytes to the transition from the oxygenated to the deoxygenated state at constant intracellular pH.

The time course of the rate of the glycolysis of human erythrocytes and of some metabolites were determined before and after rapid deoxygenation at constant intracellular pH. For this purpose stripped deoxygenated haemoglobin was used as a rapid oxygen acceptor. Deoxygenation causes an increase of the glycolytic rate by 26%. Glucose 6-phosphate is decreased while the adenine nucleotides and 2,3-bisphosphoglycerate remain constant. Fructose 1,6-bisphosphate and the triose phosphates decrease transiently before rising. The data can be explained by increased binding of phosphocompounds to deoxygenated as compared with oxygenated haemoglobin. Thereby the control enzymes hexokinase and phosphofructokinase are influenced. It is concluded that under physiological conditions changes in the oxygenation state of haemoglobin per se alter the glycolytic rate.     

Monitoring blood loss with near infrared spectroscopy

Experimental research has shown correlation between near infrared spectroscopy (NIRS) and blood loss, but these findings have not been validated in man. Ten blood donors were monitored before, during and for 10 min after blood collection (470 ml) with NIRS. A Somanetics INVOS 4100 oximeter monitored regional haemoglobin saturation in the cerebral cortex (cSO(2)-left frontal area) and from the left calf (pSO(2)). A Critikon 2001 Cerebral Redox Model monitored total (tHb), oxygenated (O(2)Hb) and deoxygenated (HHb) haemoglobin from the right calf. The oxygenation index [HbD]=[O(2)Hb]-[HHb] was derived from the data. cSO(2) (P<0.001), pSO(2) (P<0.001) and HbD (P=0.001) decreased during blood collection. Maximum changes occurred 10 minutes after collection for cSO(2), with a mean fall (95% C.I.) of 2.5 (-0.06-4.86)%, at the end of blood collection for pSO(2), with a mean fall (95% C.I.) of 3 (0.74-5.26)% and after 8% of blood volume loss for HbD, with a mean fall (95% C.I.) of 7.2 (2.25-12.16). Cerebral and peripheral oxygenation did not recover after blood collection. There was good correlation between NIRS parameters and blood loss. NIRS is a potentially useful technique for monitoring blood loss in humans. Further research is needed to define its role in clinical practice.     

Effect of transient hypoxia on oxygenation of the developing rat brain: relationships among haemoglobin saturation, autoregulation of blood flow and mitochondrial redox state

Multi-wavelength, differential spectroscopy was used to examine the effects of transient hypoxia on oxygen delivery and intracellular utilization in the brain of developing rats. The in vivo redox status of cytochrome a,a3 was compared simultaneously with changes in relative haemoglobin saturation and blood volume in the cerebral cortex during lowered FiO2. During hypoxia, neonates maintained their intracellular cytochrome a,a3 redox state as well as did adults, but did so through unusual characteristics, including: (1) maintenance of haemoglobin oxygenation at lower FiO2; (2) regulation of cerebral blood volume at blood pressures below the point at which autoregulation would fail in the adult; and (3) the capacity to tolerate a greater reduction of cytochrome a,a3 relative to haemoglobin desaturation at lowered FiO2. These data suggest that mechanisms which protect the neonate from hypoxic insult involve preservation of oxygen delivery, increased respiratory compensation for metabolic acidosis, and maintenance of cellular energy requirements predominantly through anaerobic metabolism.     

Oxygen binding by Helix pomatia alpha-haemocyanin studied by X-ray-absorption spectroscopy.

The X-ray absorption spectra of haemocyanin from Helix pomatia were obtained by using X-rays from synchrotron radiation. Cu K-edges were recorded at four conditions, namely fully oxygenated, 85% oxygenated, 12% oxygenated and fully deoxygenated. The percentage oxygenation calculated from the edge-shift of the partially oxygenated samples did not agree with the percentage oxygenation as determined by u.v. measurements. Two intermediates in the oxygenation process are presented to explain the observed dissimilarities.     

Ligand-induced spectral changes in cytochrome c oxidase and their possible significance.

1. The spectral shifts induced on the binding of H2S to ferric cytochrome aa3 are similar to those induced by cyanide, reflecting a possible high- to low-spin state change in the a3 haem. Opposite shifts are seen with either formate or low azide concentrations, while high azide concentrations reverse the change induced at lower concentrations. The unusually high Soret band in the half-reduced sulphide-inhibited species (a2+a33+H2S) results from the superposition of cytochrome a2+ and cytochrome a33+H2S peaks. 2. The difference spectra in the visible region for cytochrome a2+ minus cytochrome a3+ obtained with four inhibitors (cytochrome a2+ a3+I minus minus a3+a33+I)are similar, except that azide and sulphide induce blue shifts of the alpha-peak. The trough in the Soret region for the azide complex is much deeper than that for the other complexes, suggesting changes in the cytochrome a33+HN3 centre on reduction of cytochrome a. 3. The "oxygenated" and "high-energy" forms of cytochrome aa3 both involve spectral changes at the a3 haem similar to the changes induced by cyanide and sulphide. The spectrum of partially reduced cytochrome aa3 in the presence of reductant and oxygen indicates the steady-state occurrence of appreciable levels of low-spin (oxygenated) cytochrome aa3. These may be important for energy conservation during the action of cytochrome aa3 in the intact mitochondrial membrane.     

The detection of cytochrome oxidase heme iron and copper absorption in the blood-perfused and blood-free brain in normoxia and hypoxia.

The resolution of cytochrome and hemoglobin changes in in vivo rat and cat brains has required studies over wide wavelength ranges (580-1100 nm) with a novel spectroscopic technique using blood-free and blood-perfused brains. Tissue oxygen was varied from physiological levels to 0 and hematocrits were varied from normal to less than 1%. The experimental results were subjected to a multicomponent analysis using the Beer-Lambert law. At normal hematocrits, the oxygen saturation of hemoglobin in the brain was found to be 30-50% in rats and cats, indicating that the optical method responded primarily to the saturation of the venous ends of the capillary beds. With low hematocrits, both brains showed the absorption band of reduced cytochrome c, the iron component of cytochrome aa3, plus the absorption band of the oxidized copper component. In cat brains, the background absorption changed at all wavelengths. Thus, no isosbestic points were observed in the spectra. In rat brains, however, they were readily observed. The "overtones" of water absorption in the NIR region were found to be significant in the difference spectra of the cat brain, but not in the rat brain. Parallel absorbance changes in the heme and copper components of cytochrome aa3 were obtained in rat and cat brains during the normoxic-hypoxic transition. The ratio of the iron absorbance at 605 nm to the copper absorbance at 830 nm is much smaller in both brains than the in vitro value due to the shorter path length of photon migration at the shorter wavelengths.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo optical/near-infrared spectroscopy and imaging of metalloproteins

A number of medical applications of near-infrared spectroscopy are growing closer to clinical acceptance, and new techniques involving both spectroscopy and imaging are evolving rapidly. In vivo spectroscopy and, more recently, imaging techniques are largely based upon optical electronic transitions involving the metal centers of hemoglobin (blood), myoglobin (muscle) and cytochrome aa3 (mitochondria). The wide variety of near-IR based applications includes heart and stroke research, monitoring cerebral oxygenation of premature babies, and 'functional activation' (response of brain to mental tasks). All of these applications are founded upon changes in hemoglobin O2 saturation; these changes are monitored by following trends in the near-infrared absorptions of deoxyhemoglobin (760 nm) and oxyhemoglobin (920 nm). The same absorptions provide a basis for imaging regional variations in blood oxygenation. This report presents and discusses examples, both from the literature and from our recent work, of near-infrared spectroscopy and imaging in medical applications.     

Near infrared monitoring of human skeletal muscle oxygenation during forearm ischemia

Changes in tissue oxygenation of forearm muscles were measured by near infrared (NIR) spectrophotometry in 10 healthy adults during tourniquet ischemia and venous outflow restriction. Muscle O2 stores were depleted rapidly by forearm ischemia manifest by a progressive decrease in tissue oxyhemoglobin and oxymyoglobin over 4-5 min. Muscle ischemia significantly decreased the oxidation level of cytochrome aa3, to below resting base line after only 1.5 min, and the enzyme became fully reduced after 6.5 min. After 8 min of ischemia, tourniquet release was accompanied by a transient increase in muscle blood volume due to influx of oxyhemoglobin. The cytochrome aa3 oxidation level increased above resting base line within 1 min after tourniquet release. Transcutaneous PO2 measurements recorded simultaneously from the same forearm correlated poorly with the kinetics of O2 availability and cytochrome oxidation in the underlying muscle tissue; this was not unexpected because overlying skin did not contribute significantly to NIR muscle signals. Venous outflow restriction without inflow obstruction increased muscle deoxyhemoglobin and tissue blood volume but did not change muscle O2 stores or cytochrome aa3 oxidation level. The ability of the NIR technique to detect dynamic trends in tissue oxygenation reveals that muscle O2 is rapidly consumed during tourniquet ischemia and rapidly restored by hyperemic responses after brief ischemia.     

Effect of oxygen binding on the dielectric properties of hemoglobin

The dielectric properties of horse hemoglobin have been investigated in the frequency range for 100 kcps to 15 Mcps at varying degrees of oxygenation. A linear dependence of the specific increment on the degree of oxygenation was found under a variety of experimental conditions, the increment of oxygenated hemoglobin being about 10% larger than that of deoxygenated hemoglobin. A similar difference was obtained with human adult and fetal hemoglobin. No variation of the dielectric parameters as reported by Takashima and Lumry could be detected.     

The functional significance of the haemoglobin in a marine nematode, Enoplus brevis (Bastian).

An animal chamber and a simple microspectrophotometer for investigating the in vivo oxygenation of the haemoglobin of E. brevis are described. The in vivo absorption peaks of this haemoglobin occur at similar wavelengths to those of other nematodes. Mean values, given with their corresponding standard errors, occur at 577.6 plus or minus 0.6 nm, 543.6 plus or minus 0.5 nm and 421.7 plus or minus 1.9 nm for oxyhaemoglobin, and 555.2 plus or minus 0.9 nm and 432.2 plus or minus 1.3 nm for the deoxygenated pigment. The percentage of oxyhaemoglobin in the pharynx of E. brevis decreased at external oxygen tensions of less than 20 Torr, and the pigment was completely deoxygenated at 5 Torr. Stimulation of individuals in aerated sea water for 1-2 min caused a partial deoxygenation of the haemoglobin; the pigment reloaded soon after this period of increased activity had ended. The functional significance of the haemoglobin of E. brevis is disucssed.     

Seizure-associated pulmonary edema and cerebral oxygenation in the rat

Cerebral partial pressure of O2 (PO2), relative changes in the ratio of reduced/oxidized cytochrome aa3, blood flow, and the arteriovenous difference in O2 content were measured during seizures with and without pulmonary edema. Seizures were induced with bicuculline (0.2-1.2 mg/kg iv) in rats anesthetized with 70% N2O and paralyzed with curare. Briefer seizures were accompanied by increased cerebral PO2 and increased oxidation of cytochrome aa3. Lung water content and arterial O2 partial pressure (PaO2) remained normal. Longer duration seizures were also accompanied initially by increases in cerebral oxygenation. Within minutes, however, PaO2 fell from a mean of 118 to 51 mmHg, and lung water content increased from 76.2 to 83.6%. Cerebral PO2 fell but most often rose back to or above control levels, while cytochrome aa3 became markedly reduced. Simultaneously, cerebral blood flow increased more than 300% above preseizure values and O2 delivery increased more than O2 consumption. The reductive shift of cytochrome aa3 was greater than that produced by lowering PaO2 to equivalent values in seizure-free rats. The reductive shift of cytochrome aa3, despite increased O2 delivery, may be indicative of derangements in cerebral O2 diffusion or energy metabolism.     

Inhibition of adrenergic proton extrusion in rainbow trout red cells by nitrite-induced methaemoglobinaemia

Summary The effects of nitrite-induced methaemoglobinaemia on adrenergic proton extrusion from rainbow trout red blood cells were studied using the pH-stat method. In control conditions adrenergic proton extrusion was completely inhibited by amiloride and was greater in deoxygenated than in oxygenated erythrocytes. Nitrite-induced methaemoglobinaemia was associated with a pronounced reduction in the catecholamine-stimulated proton efflux from both deoxygenated and oxygenated erythrocytes. In deoxygenated erythrocytes the initial proton efflux upon catecholamine stimulation decreased by 60–70%, while the percentage of methaemoglobin in the red cells increased from the control level of 1–3% to 20%. In oxygenated erythrocytes the decrease was 30% at the same methaemoglobin percentage range. It is suggested that the pronounced influence of nitriteinduced methaemoglobinaemia on adrenergic proton efflux results from an inhibition of the red cell sodium/proton exchanger by the R-like haemoglobin conformations.Abbreviations DIDS 4,4-diisothiocyanostilbene-2,2-disulfonic acid - DMO 5,5-dimethyloxazolidine-2,4-dione - RBC red blood cell     

Regional transcranial oximetry with near infrared spectroscopy (NIRS) in comparison with measuring oxygen saturation in the jugular bulb in infants and children for monitoring cerebral oxygenation]

Using a dual channel near infrared (NIR) in vivo optical spectroscopy (INVOS) system (INVOS 3100A, Somanetics Corp. Troy, MI, USA) we investigated the relationship between jugular venous oxygen saturation (SjvO2) and regional cerebral oxygen saturation (rSO2) in 30 infants and children (mean age 4.5 years) with congenital heart disease undergoing cardiac catheterisation. The NIRS-SomaSensor (emitter and dual receiver probe) was applied at a standardised right fronto-temporal location (over the right frontal cortex) on the infant's head and covered with an adhesive flexible bandage. Using NIR light (730 and 810 nm) and two source-detector spacings (3 and 4 cm from the transmitter), percentage values of rSO2 were calculated from detected haemoglobin saturations. Simultaneously, jugular venous oxygen saturation (SjvO2) monitoring was performed via a catheter placed in the right internal jugular vein with its tip positioned in the jugular bulb, as verified by fluoroscopy. To compare the reliability of NIRS measurement characteristics, jugular venous blood was analysed for SjvO2 as a reference measure of global cerebral oxygenation, by co-oximetry (OSM3-Hemoximeter, Radiometer Copenhagen, Denmark). Other measured variables included pulse oximetry, arterial blood pressure, and venous and arterial oxygen saturations. Over a jugular venous oxygen saturation range of 31-83%, a significant positive linear correlation was found between rSO2 (NIRS measurement) and SjvO2 (jugular bulb oximetry) (r = 0.93, p < 0.001). No significant correlation was observed between rSO2 values and arterial blood saturation or pulse oximetry. The quantitative correlation between rSO2 (haemoglobin oxygenation in a small hemi-elliptical area of the brain) and reference SjvO2 measurement (method for monitoring global cerebral oxygenation) suggests that NIRS measurement with subtraction algorithm should identify predominantly intracranial saturation in the pediatric age group, and will tend to reflect global oxygenation under physiological conditions. Transcranial oximetry using dual receiving channel NIRS offers a noninvasive, real-time, reliable and practicable means of monitoring cerebral haemoglobin oxygenation changes infants and children with cyanotic and noncyanotic congenital heart disease.     

Changes in muscle oxygenation during weight-lifting exercise

The quantitative analysis of haemoglobin oxygenation of contracting human muscle during weight-lifting exercise was studied noninvasively and directly using near-infrared spectroscopy. This method was developed as a three-wavelength method which confirmed the volume changes in oxygenated haemoglobin (oxy-Hb), deoxygenated haemoglobin (deoxy-Hb) and blood volume (total-Hb; Oxy-Hb + deoxy-Hb). Nine healthy adult men with various levels of training experience took part in the study. Ten repetition maximum (10 RM) one-arm curl exercise was performed by all the subjects. Results showed that at the beginning of the 10-RM exercise, rapid increases of deoxy-Hb and decreases of oxy-Hb were observed. In addition, total-Hb gradually increased during exercise. These results corresponded to the condition of arm blood flow experimentally restricted using a tourniquet in contact with the shoulder joint, and they showed the restriction of venous blood flow and an anoxic state occurring in the dynamically contracted muscle. In three sets of lifting exercise with short rest periods, these tendencies were accelerated in each set, while total-Hb volume did not return to the resting state after the third set for more than 90 s. These results would suggest that a training regimen emphasizing a moderately high load and a high number of repetitions, and a serial set with short rest periods such as usually performed by bodybuilders, caused a relatively long-term anoxic state in the muscle.     

Role of hemoglobin oxygenation in the modulation of red blood cell mechanical properties by nitric oxide

It has been previously demonstrated that both externally generated and internally synthesized nitric oxide (NO) can affect red blood cell (RBC) deformability. Further studies have shown that the RBC has active NO synthesizing mechanisms and that these mechanisms may play role in maintaining normal RBC mechanical properties. However, hemoglobin within the RBC is known to be a potent scavenger of NO; oxy-hemoglobin scavenges NO faster than deoxy-hemoglobin via the dioxygenation reaction to nitrate. The present study aimed at investigating the role of hemoglobin oxygenation in the modulation of RBC rheologic behavior by NO. Human blood was obtained from healthy volunteers, anticoagulated with sodium heparin (15 IU/mL), and the hematocrit was adjusted to 0.4 L/L by adding or removing autologous plasma. Several two mL aliquots of blood were equilibrated at room temperature (22 ± 2 °C) with moisturized air or 100% nitrogen by a membrane gas exchanger, The NO donor sodium nitroprusside (SNP), at a concentration range of 10^?7–10^?4 M, was added to the equilibrated aliquots which were maintained under the same conditions for an additional 60 min. The effect of the non-specific NOS inhibitor l-NAME was also tested at a concentration of 10^?3 M. RBC deformability was measured using an ektacytometer with an environment corresponding to that used for the prior incubation (i.e., oxygenated or deoxygenated). Our results indicate an improvement of RBC deformability with the NO donor SNP that was much more pronounced in the deoxygenated aliquots. SNP also had a more pronounced effect on RBC aggregation for deoxygenated RBC. Conversely, l-NAME had no effect on deoxygenated blood but resulted in impaired deformability, with no change in aggregation for oxygenated blood. These findings can be explained by a differential behavior of hemoglobin under oxygenated and deoxygenated conditions; the influence of oxygen partial pressure on NOS activity may also play a role. It is therefore critical to consider the oxygenation state of intracellular hemoglobin while studying the role of NO as a regulator of RBC mechanical properties.     

Oxygen binding and aggregation of bullfrog hemoglobin

The hemoglobin of the bullfrog, Rana catesbeiana, forms aggregates larger than tetramers in two ways. The first, which results from intermolecular disulfide bonds, can be prevented by treatment with iodoacetamide. The second way results from the association of the deoxygenated forms of the two major components, B and C, to form reversibly an aggregate which is believed to be a trimer, BC2. The sedimentation velocity data show that the stoichiometry of the aggregate cannot be 1:1. The electrophoretic pattern of the deoxygenated B/C mixture suggests that the association is not indefinite. No significant aggregation of the separate deoxygenated tetramers of the components nor of the oxygenated components or mixture occurs. Gel chromatography of the oxygenated forms of components B and C and of mixtures indicates that the B and C tetramers both form dimers upon dilution with a dissociation constant of 2-3 micron. The oxygen-binding data indicate that the B/C aggregate has a much lower oxygen affinity than its constituent tetramers. Dissociation of the low affinity B/C aggregate to higher affinity B and C tetramers with increasing oxygenation gives rise to enhanced cooperativity as measured by the Hill coefficient which is maximal near 75-80% oxygenation and is as high as 4.1 at a heme concentration of 15 mM.     

Noninvasive estimation of the oxygen state of experimental tumor by diffuse optical spectroscopy

The potentialities of diffuse optical spectroscopy for the noninvasive estimation of the oxygen state of experimental tumors have been demonstrated. The distribution of total, oxygenated and deoxygenated hemoglobin, as well as the level oxygen saturation of blood have been shown using two tumor models differing in the histological structure and functional characteristics. The results obtained by the optical method have been verified by immunohistochemical examination of tissue specimens with the exogenous hypoxia marker pimonidazole.     

Noninvasive estimation of the oxygen status of experimental tumors by diffuse optical spectroscopy

The potentialities of diffuse optical spectroscopy for noninvasive estimation of the oxygen status of experimental tumors have been demonstrated. The distribution of total, oxygenated, and deoxygenated hemoglobin, as well as the level of oxygen saturation of blood have been assessed using two tumor models differing in the histological structure and functional characteristics. The results obtained by the optical method have been verified by immunohistochemical examination of tissue specimens with an exogenous hypoxia marker pimonidazole.     

Oxygen absorption by skin exposed to oxygen supersaturated water

The present study tests the hypothesis that skin on the plantar surface of the foot absorbs oxygen (O(2)) when immersed in water that has a high dissolved O(2) content. Healthy male and female subjects (24.2 ± 1.4 years) soaked each foot in tap water (1.7 ± 0.1 mg O(2)·L(-1); 30.7 ± 0.3 °C) or O(2)-infused water (50.2 ± 1.7 mg O(2)·L(-1); 32.1 ± 0.5 °C) for up to 30 min in 50 different experiments. Transcutaneous oximetry and near infrared spectroscopy were used to evaluate changes in skin PO(2), oxygenated haemoglobin, and cytochrome oxidase aa(3) that resulted from treatment. Compared with the tap water condition, tissue oxygenation index was 3.5% ± 1.3% higher in feet treated for 30 min with O(2)-infused water. This effect persisted after treatment, as skin PO(2) was higher in feet treated with O(2)-infused water at 2 min (237 ± 9 vs. 112 ± 5 mm HG) and 15 min (131 ± 1 vs. 87 ± 4 mm HG) post-treatment. When blood flow to the foot was occluded for 5 min, feet resting in O(2)-infused water maintained a 3-fold higher O(2) consumption rate than feet treated with tap water (9.1 ± 1.4 vs. 3.0 ± 1.0 μL·100 g(-1)·min(-1)). We estimate that skin absorbs 4.5 mL of O(2)·m(-2)·min(-1) from O(2)-infused water. Thus, skin absorbs appreciable amounts of O(2) from O(2)-infused water. This finding may prove useful and assist development of treatments targeting skin diseases with ischemic origin.