Robert A.B. Holland

Rate at which CO replaces O2 from O2Hb in red cells of different species

Abstract The rate at which CO replaces O 2 from combination with O 2 Hb in cells and solution has been measured for different species. Red cells studied were those of the human adult and newborn rabbit, dog, cat, horse, goat and toad. At 37 °C the cell reaction rate was more affected by the rate in solution than by the size of the cells. At lower temperatures the cell and solution rates were closer together and at 25 °C the replacement reaction was as fast in the large cells of the toad as in some mammalian cells. The energy of activation (E) of the replacement reaction in solution was approximately 20 kcal. Mole -1 for the three hemoglobins in which it was measured. For the reaction in red cells, E was in the range predicted from E in solution. The rates of the replacement reaction were applied to measurements of D m and Vc by the method of Roughton and Forster. It was found that Vc can be measured without knowledge of the cell reaction rate provided that the solution rate at low CO tensions is known. Some workers have used the rate of the replacement reaction in man at 37 °C when making measurements on other species or at other temperatures. It has been shown that this can lead to errors in D m and Vc.

Oxygen carriage and carbonic anhydrase activity in the blood of a marsupial, the Tammar wallaby (Macropus eugenii), during early development.

Blood O2 transport and Hb type have been studied in pouch young and adult of a marsupial, the Tammar Wallaby. The O2-Hb equilibrium curves (at 35.5 degrees C and PCO2 = 34 Torr) had a high P50 in the first few days of life, up to 49 Torr. This fell to 32 Torr by 2 weeks of age. Also (delta log P50/delta PCO2) was low but it rose to adult levels by 2 weeks of age. The curves in these early pouch young showed a change in Hill coefficient (nH) at between 32 and 62% saturation, nH rising to more than 4.0 at higher O2 saturations. This indicates interaction between more than 4 Hb subunits. Model calculations showed that such curves could be produced by a mixture of 2 Hb components; one with a low P50 and low nH, and one with a high P50 and high nH. In this model the nH values were different from the nH values of either component. The temperature effect on P50 in early pouch young was higher than in adult Tammars and similar to that reported for adult eutherians. In the first 4 days all red cells were nucleated and four Hb types were present. Carbonic anhydrase activity in the blood before birth was about 30% of the adult levels. These levels remained until 2 days after birth, when a rapid rise in activity began, near-adult levels being reached at 5 days despite the animals being still very immature.

Oxygen affinity and 2,3-diphosphoglycerate in blood of Australian marsupials of differing body size.

Oxygen carriage and 2,3-diphosphoglycerate (2,3-DPG) levels have been measured in the blood of seven species of Australian marsupials ranging in size from 35 to 0.03 kg. They were Red and Grey Kangaroos, Wallaroo, Tammar Wallaby, Brush-tailed possum, Potoroo, and Brown Marsupial Mouse. Oxygen affinity decreased with decrease in adult body size, standard P50 (at 36 C) varying from 24.6 torr in the largest (Red Kangaroo) to 41.9 torr in the smallest (Brown Marsupial Mouse). The relationship between P50 and body size is similar to the relationship which has been described previously for eutherian mammals. The Bohr factor (--deltalog P50/deltapH) and value for Hill n were generally in the range found for other land-dwelling mammals. All species had 2,3-DPG in their erythrocytes acting as a regulator of oxygen affinity. The polymorphism at position beta 2 in hemoglobin of the Grey Kangaroo was shown to affect the respiratory properties of the molecule. When beta 2 = histidine, which has a positively charged side chain, erythrocyte 2,3-DPG was higher, and P50 was higher, than when beta 2 = glutamine which has a neutral side chain.

Kinetics of combination of O2 and CO with human hemoglobin F in cells and in solution

Kinetic constants for the combination of oxygen and carbon monoxide with reduced human fetal hemoglobin in cells and solution were measured using a double beam stopped-flow apparatus. At 37 °C the mean velocity constant for the uptake of oxygen by reduced fetal red cells was 83 mM-1. sec-1 and for the uptake of CO was 68 mM-1.sec-1, these being little different from values previously found for adult cells. For combination of CO with reduced fetal hemoglobin solution the velocity constant was 370 mM-1.sec-1 at 37 °C and the energy of activation was 9.2 kcal/mole. The results indicate that the chemical differences between human fetal and adult hemoglobin do not affect the rate of combination with carbon monoxide, and that the differences between fetal and adult red cells confer no advantage with respect to velocity of gas uptake. Carbon monoxide Reaction kinetics Fetal blood Red blood cells Hemoglobin Red cell membrane Oxygen

Rate of O2 dissociation from O2Hb and relative combination rate of CO and O2 in mammals at 37 °C

Abstract The rate at which CO displaced oxygen from combination with oxyhemoglobin solution has been measured at different oxygen tensions at 37 °C. The hemoglobins studied were human adult and fetal, horse, goat, dog, cat and rabbit. From these results we have determined (i) the velocity constant for the dissociation of oxygen from fully saturated hemoglobin ( k 4), (ii) the velocity constant for replacement of oxygen from fully saturated hemoglobin at low tensions of CO( m ′ ∞ ), and (iii) the ratio of the velocity constants for the combination of CO and O 2 with three-parts saturated hemoglobin (′ l 4 /′ k 4 ). The values found for k 4 and ( m ′ ∞ ) (each in units of sec -1 ) and for the ratio (′ l 4 /′ k 4 ) were: human adult, 222, 16.9, and 0.30; human new-born, 300, 16.0, and 0.21; horse, 208, 13.3, and 0.26; goat, 214, 14.2, and 0.27; dog, 338, 21.1, and 0.25; cat, 286,17.4, and 0.24. The velocity of the displacement reaction varied among rabbits despite their having electrophoretically similar hemoglobins, and was slower than in the other species. The value of k 4 for dog Hb, human Hb F, and cat Hb was significantly higher than for the hemoglobins of the human adult, horse, goat and rabbit. Goat hemoglobins A and B, whose α-chains differ, showed the same rate for the replacement reaction.

Gas exchange in the lung of a dasyurid marsupial: morphometric estimation of diffusion capacity and blood oxygen uptake kinetics

Placental and marsupial mammals differ in the balance of their cardio-respiratory systems. To determine if differences also occur in lung structure, a morphometric study of the lungs of a dasyurid marsupial, Dasyuroides byrnei, was undertaken. The total lung volume was large, but a comparatively smaller proportion was devoted to gas exchange tissue. Eighty-seven percent of lung parenchyma was air space. The volume of capillary blood was lower than would be predicted on a body mass basis, but the hematocrit was high (55%). The rate of oxygen uptake by the blood of D. byrnei was not different from that of recent determinations on human blood. However, a large oxygen capacity resulted in a high theta O2. Morphometric data were used, with theta O2 calculated for D. byrnei, to estimate the pulmonary diffusion capacity. Mass specific DLO2 was high by comparison to those reported for placentals. The erythrocytes contributed 70% of the diffusion resistance to oxygen transfer, with the large theta O2 counteracting the low Vc. We conclude that, similarly to placentals, lung structure is unlikely to limit oxygen transport in this animal, although the structural emphasis appears to be geared towards maximising alveolar ventilation.

Respiratory Properties of the Neonatal Blood of the Common Brushtail Possum (Trichosurus vulpecula)

The respiratory properties of the blood of a newborn marsupial, the common brushtail possum (Trichosurus vulpecula of the family Phalangeridae), have been examined. The O₂ equilibrium curves (OECs) under standard conditions (36° C and partial pressure of CO₂ [Pco₂] = 42 mmHg) were right shifted, being to the right of the maternal curves. The partial pressure of O₂ (Po₂) at which blood is 50% saturated with oxygen (P50) under these conditions was 42 mmHg in the neonatal blood. All the neonatal OECs had a Hill plot bent at about 50% saturation, and the mean value of the Hill coefficient (nH) was 5.96 for the upper part of the curve, indicating aggregation of Hb tetramers or interaction between adjacent tetramers. Apart from previous findings in another marsupial (Macropus eugenii), the first of these properties had not been shown before in any species, the second had not been shown before in any normal mammalian blood. At higher and lower Pco₂ (72 and 21 mmHg) the neonatal OECs were of the same shape and were also to the right of the maternal OECs under equivalent conditions. The Bohr effect of the neonatal blood was lower than that of adult blood; the neonatal red blood cells were nearly all nucleated and contained four Hb types, each different from adult Hbs. In these respects it was characteristic of embryonic blood and similar to the blood of the prenatal and neonatal tammar wallaby of the family Macropodidae

Blood O2 transport in newborn and adult of a very small marsupial (Sminthopsis crassicaudata)

Blood O2 transport and hemoglobin types have been studied in a Dasyurid marsupial (Sminthopsis crassicaudata) at the neonatal (10-20 mg) and adult (16 g) stages, and in part of the transition period. In neonates the blood was embryonic in type with erythrocytes nucleated and containing two Hb types both different from adult Hb. The oxygen equilibrium curves (OECs) at day 1 had a P50 of 38 mmHg at 36 degrees C and PCO2 = 43 mmHg. This is lower than in other neonatal marsupials, but higher than in fetal or neonatal eutherian mammals. Adult P50 under the same conditions were higher (59 mmHg), the normal relationship in viviparous animals. Hill plots of neonatal OECs showed a sharp upward bend at about 50% saturation. As in other embryonic and neonatal marsupials, in the upper part of the plot nH was greater than 4. This indicates aggregation of Hb tetramers. The Bohr effect of neonatal blood at higher PCO2 values (43-71 mmHg PCO2) was zero. The special features of neonatal blood had largely disappeared by day 6.

Blood O2 transport and Hb types in the embryonic Tammar Wallaby (Marsupialia, Macropus eugenii).

Blood oxygen transport and hemoglobin type have been studied in the developing Tammar Wallaby from 20 days gestation, just after the circulation first forms, until 28 days gestation, 2 days after birth. The oxygen equilibrium curves (OECs) of the embryonic whole blood had high P50 values, mean being 44.6 mmHg at 36 degrees C, PCO2 = 40 mmHg. In contrast to other viviparous species studied, the embryonic OECs were well to the right of the maternal OEC. There was no significant change in P50 throughout the age range studied. The curves had high Hill coefficients above about 50% saturation (mean = 5.65), indicating cooperativity between hemoglobin tetramers. The Bohr effect (measured as delta log P50/delta log PCO2) was low, about half that of the adult. The early embryos (days 20 and 21) had only 2 or 3 Hb types, with the other embryonic Hbs appearing by 22 days of gestation. The proportion of the four embryonic Hbs changed during development although one type was always predominant (> 45%). This is the first serial study of blood O2 carriage and Hb type in developing marsupial embryos. The finding of a right-shifted embryonic OEC throughout intrauterine development suggests that, contrary to current belief, a right-shifted curve may be physiologically advantageous to a developing embryo.

Blood oxygen carriage in the marsupial, tammar wallaby (Macropus eugenii), at prenatal and neonatal stages

The measurements reported here are the first to be made on oxygen carriage of a prenatal marsupial. The oxygen equilibrium curves (OECs) of tammar wallaby blood 1-2 days before the due date of birth showed a high P50 (mean = 44 Torr at 36 degrees C at a PCO2 of 34 Torr), more than 1.5 times that of the mother. This was confirmed by measurements in red cell suspensions at controlled pH. The finding of a higher P50 than in adult is in contrast to the general finding in eutherian (placental) mammals. Also they showed interaction between O2 and CO2 carriage (expressed as delta log P50/delta log PCO2 between 34 and 64 Torr PCO2) about half the magnitude of that in adults. At high PCO2 this effect reversed in the lower part but not in the upper part of the OEC. The Hill plot of the OECs showed a bend in the middle range of saturation: in nearly all cases the Hill coefficient (nH) was greater than 4.0 above about 50% saturation suggesting aggregation of haemoglobin tetramers. These results are similar to those previously reported for neonatal tammars and confirmed by further measurements in this study. The prenatals all had four haemoglobin types, identical with those found in the neonates.