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M Samaja

Publications and source records attributed to M Samaja.

At least 55 records · Page 3Linked to original sources
PubMed1991

Dual role of hypoxanthine in the reoxygenation of hypoxic isolated rat hearts.

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.

Animals↗
PubMed1990

Human red cell age, oxygen affinity and oxygen transport.

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].

Adult↗
PubMed1990

Red cell aging and active calcium transport.

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.

Adult↗
PubMed1990

Impairment of the post-anoxic recovery of isolated rat hearts by intravascular hypoxanthine and xanthine.

Hypoxanthine is the final product of the catabolism of ATP in the stored red cell. Upon transfusion, this purine may be uptaken by the endothelial cell and oxidized in a post-ischemic or post-anoxic environment with production of oxygen-derived free radicals. We have tested this hypothesis with a isolated perfused rat heart model monitoring the recovery of the heart function from 20 min anoxia in the presence of 0.1 mM hypoxanthine or xanthine. Addition of 0.1 mM guanine minimized the fraction of hypoxanthine to be salvaged. The presence of hypoxanthine in the vascular space impaired the recovery of the end-diastolic pressure, left ventricular developed pressure, contraction rate, and coronary perfusion pressure. We conclude that intravascular hypoxanthine is oxidized by the endothelial cell xanthine oxidase contributing to the post-anoxic reoxygenation injury. Since the injury led by equimolar xanthine was nearly half of that observed for hypoxanthine, this injury appears to be correlated to the stoichiometry of the oxygen-derived free radical generating reaction.

Animals↗
PubMed1989

The effect of in vitro and in vivo cellular aging on the active calcium transport in human inside-out red cell membrane vesicles.

Modelling of the in vivo and in vitro aging processes in the human red cell has stressed the following features of the active calcium uptake by inside-out vesicles: 1) it is higher in the outdated, in vitro aged, than in the fresh red cell (p less than 0.0005), and in the densest, in vivo aged fraction than in the lightest, young fraction (p = 0.08); 2) it increases following stimulation by excess calmodulin to values that are not significantly different; 3) it decreases to the same value in the absence of endogenous calmodulin and inhibitor, with and without exogenous calmodulin; 4) it is the target of a non-competitive inhibition, that is stronger in the fresh than in the outdated red cell. We conclude that the aging process does not involve neither membrane Ca-ATPase nor calmodulin, but rather the interaction of the calcium pump with the inhibitor of Ca-ATPase.

Biological Transport, Active↗
PubMed1989

Different hematologic responses to hypoxia in Sherpas and Quechua Indians.

Previous studies of the erythropoietic response to hypoxia in high-altitude natives suggest that the hematocrit and hemoglobin values in Himalayan natives (Sherpas) are lower than expected for the altitude, perhaps because of a genetic adaptation. However, differences in sampling techniques and experimental methods make comparisons difficult. Our studies were carried out to compare the erythropoietic response with the same altitude in age-matched natives of the Himalayas and Andes by the same experimental techniques. Healthy male subjects were selected in Ollagüe, Chile (n = 29, 27.3 +/- 5.9 yr) and in Khunde, Nepal (n = 30, 24.7 +/- 3.8 yr). Both of these villages are located at 3,700 m above sea level. Hematologic measurements confirmed lower hematocrit values in Nepal (48.4 +/- 4.5%) than in Chile (52.2 +/- 4.6%) (P less than 0.003). When subjects were matched for hematocrit, erythropoietin concentrations in Chile were higher than in Nepal (P less than 0.01). Detailed measurements of blood O2 affinity in Nepal showed no differences in shape or position of the O2 equilibrium curve between Sherpas and Western sojourners. Our results indicate that although Quechua Indians have higher hematocrits than Sherpas living at the same altitude, nevertheless they may be functionally anemic.

Adult↗
PubMed1988

Prediction of the oxygenation of human organs at varying blood oxygen carrying properties.

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.

Adult↗
PubMed1987

The dissociation of carbon monoxide from the alpha and the beta subunits of human carbonmonoxy hemoglobin.

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.

Allosteric Regulation↗
PubMed1987

Separation of the valence intermediates of human haemoglobin by high-performance chromatofocusing.

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.

Chromatography, Ion Exchange↗
PubMed1987

The dissociation of carbon monoxide from hemoglobin intermediate.

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.

Carbon Monoxide↗
PubMed1987

Purification of human hemoglobin valence intermediates by preparative immobilized pH gradients.

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).

Carboxyhemoglobin↗
PubMed1986

The intermediate compounds between human hemoglobin and carbon monoxide at equilibrium and during approach to equilibrium.

The procedure of Perrella et al. (Perrella, M., Benazzi, L., Cremonesi, L., Vesely, S., Viggiano, G., and Rossi-Bernardi, L. (1983) J. Biol. Chem. 258, 4511-4517) for trapping the intermediate compounds between human hemoglobin and carbon monoxide was validated by quantitatively determining during the approach to equilibrium all the species present in a solution containing large amounts of intermediates. An accurate estimate of the intermediate compounds at 50% carbon monoxide saturation in 0.1 M KCl, pH 7, at 22 degrees C, allowed the calculation, according to Adair's scheme, of the four equilibrium constants. At 50% ligand saturation, the pool of intermediate species was about 12% of the total. A slightly greater concentration of tri-liganded than mono-liganded species was found. Carbon monoxide bound to beta chains in slightly greater excess with respect to alpha chains in both the mono- and tri-liganded species. The symmetrical bi-liganded intermediates, alpha 2 beta CO2 and alpha 2CO beta 2, were absent. The nature of the bi-liganded intermediate found to be present in detectable amounts by our technique has yet to be clarified: it could be either the asymmetrical species (alpha beta) (alpha CO beta CO) and (alpha beta CO) (alpha CO beta) or both of them. Such a finding on the functional heterogeneity among the four possible bi-liganded intermediates is consistent with hypotheses of the existence of more than two quaternary structures in the course of ligand binding to hemoglobin.

Carbon Monoxide↗
PubMed1986

The role of 2,3-DPG in the oxygen transport at altitude.

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.

2,3-Diphosphoglycerate↗
PubMed1985

Simulation of oxygen delivery to tissues: the role of the hemoglobin oxygen equilibrium curve at altitude.

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.

Altitude↗
PubMed1984

Red cell function at extreme altitude on Mount Everest.

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

2,3-Diphosphoglycerate↗
PubMed1984

Blood O2 affinity and maximal O2 consumption in elite bicycle racers.

The PO2 at which hemoglobin is half-saturated with O2 (P50) at 37 degrees C, PCO2 = 42 Torr, measured pH and 2,3-diphosphoglycerate-to-hemoglobin concentration ratio ( [2,3-DPG]/[Hb]) values, Hill's coefficient (n) at rest, and maximal O2 consumption (VO2max) were determined in 11 world-class professional bicycle racers off-season (control, C), after 3 mo of 3 h daily training (preseason, PrS), and after additional 6 mo of competitions (competitive season, CoS). The results indicate that the P50 observed in trained athletes was the same as that of a comparable group of sedentary subjects (Sed) under the same conditions of pH, PCO2, and [2,3-DPG]/[Hb] and was similar to that obtained after "normalization" in respect to pH and the [2,3-DPG]/[Hb]; [2,3-DPG]/[Hb] increased as a function of training from 0.72 to 0.95 (P less than 0.001); the slope of the central portion of the O2 equilibrium curve (OEC) was nearly unaffected by endurance training as indicated by the n value (NCoS = 2.70 +/- 0.08; nSed = 2.65 +/- 0.08); and VO2max increased in the course of training 7 and 9% (P less than 0.001), respectively, when expressed in absolute units or per kilogram body weight. The VO2max predicted on the basis of a computer simulation does not increase significantly as a consequence of the measured rise in [2,3-DPG]. Therefore, the observed increase of VO2max cannot be explained with adaptive changes of the OEC. The present results differ from previous findings reported in other types of athletes.

2,3-Diphosphoglycerate↗
PubMed1984

Carboxyhemoglobin and oxygen affinity of human blood.

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%).

2,3-Diphosphoglycerate↗