342 The Blood of Carnivorous Marsupials: Low Hemoglobin Oxygen Affinity J. F. Hallaml* R.A. B. Holland2 T.J. Dawson1 'School of Biological Science, University of New SouthWales, Sydney 2052, Australia; 2Schoolof Physiologyand Pharmacology, University of New South Wales, Sydney2052, Australia Accepted 10/10/94 Abstract We examined the blood offour dasyurid marsupials, all small, active insectivores and/or carnivores, to establish if there were any specializations in oxygen trans- port. All four species had hemoglobins with a low affinity for oxygen. This uwas demonstrated by high Pso's (the partialpressure of oxygen [Po2] at which the he- moglobin is half-saturated with oxygen) of the whole blood (means of 38.3-59. 8 mmHg) and of red cells suspended in a physiological buffer (means of 41. 6-49. O mmHg) at a CO2 tension of 43 mmHg. There uwas a strong correlation betuween increase in body mass over three orders of magnitude and increase in the oxy- gen affinity of the whole blood The effects of changing CO, tensions on the oxygen affinity of whole blood were measured as Alog Pso/Alog Pco2; mean values ranged from 0.25 to 0.373. Values for Alog Pso/ApH wuere measured for red-cell suspen- sions; means values ranged from -0.40 to -0. 481. The introduction of 2,3- diphosphoglycerate significantly decreased the oxygen affinity, of the hemoglobin ofDasyuroidesbyrnei. The blood ofD. byrnei and Dasyurus viverrinus had high hematocrits and high concentrations of hemoglobin. We conclude that hemoglo- bins with a low affinity for oxygen, an adaptation to an active lifestyle, are a characteristic of dasyurid marsupials generally. Introduction Highly specialized blood-oxygen transport systems are often found in small, active mammals such as bats (Maina and King 1984) and shrews (Bartels, Schmelzle, and Ulrich 1969; Gehr et al. 1980). Oxygen-carrying capacity of * To whom correspondence should beaddressed. Physiological Zoology 68(2):342-354. 1995. o 1995 byThe University of Chicago. All rights reserved. 0031-935X/95/6802-9417$02.00 This content downloaded on Sun, 17 Mar 2013 22:48:23 PM All use subject to JSTOR Terms and Conditions
Transcript
342
The Blood of Carnivorous Marsupials: Low Hemoglobin Oxygen Affinity
J. F. Hallaml* R. A. B. Holland2 T. J. Dawson1 'School of Biological Science, University of New South Wales, Sydney 2052, Australia; 2School of Physiology and Pharmacology, University of New South Wales, Sydney 2052, Australia
Accepted 10/10/94
Abstract We examined the blood offour dasyurid marsupials, all small, active insectivores and/or carnivores, to establish if there were any specializations in oxygen trans- port. All four species had hemoglobins with a low affinity for oxygen. This uwas demonstrated by high Pso's (the partialpressure of oxygen [Po2] at which the he- moglobin is half-saturated with oxygen) of the whole blood (means of 38.3-59. 8 mmHg) and of red cells suspended in a physiological buffer (means of 41. 6-49. O mmHg) at a CO2 tension of 43 mmHg. There uwas a strong correlation betuween increase in body mass over three orders of magnitude and increase in the oxy- gen affinity of the whole blood The effects of changing CO, tensions on the oxygen affinity of whole blood were measured as Alog Pso/Alog Pco2; mean values ranged from 0.25 to 0.373. Values for Alog Pso/ApH wuere measured for red-cell suspen- sions; means values ranged from -0.40 to -0. 481. The introduction of 2,3- diphosphoglycerate significantly decreased the oxygen affinity, of the hemoglobin ofDasyuroides byrnei. The blood ofD. byrnei and Dasyurus viverrinus had high hematocrits and high concentrations of hemoglobin. We conclude that hemoglo- bins with a low affinity for oxygen, an adaptation to an active lifestyle, are a characteristic of dasyurid marsupials generally.
Introduction
Highly specialized blood-oxygen transport systems are often found in small, active mammals such as bats (Maina and King 1984) and shrews (Bartels, Schmelzle, and Ulrich 1969; Gehr et al. 1980). Oxygen-carrying capacity of
* To whom correspondence should be addressed.
Physiological Zoology 68(2):342-354. 1995. o 1995 by The University of Chicago. All rights reserved. 0031-935X/95/6802-9417$02.00
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the blood is increased; the blood has a high hematocrit and high circulating concentrations of hemoglobin. Furthermore, intrinsic differences in amino acid chains in the hemoglobin molecule or high levels of red-cell organic phosphates act to lower the oxygen affinity of the hemoglobin, thereby fa-
cilitating oxygen unloading in the active tissues (Bartels et al. 1979;Jtirgens, Bartels, and Bartels 1981).
The kowari, Das3yuroides byrnei, a small, carnivorous, dasyurid marsupial, is known to sustain resting levels of cold-induced energy metabolism that are 10-11 times its basal levels (Smith and Dawson 1985). These levels, which may be characteristic for dasyurid marsupials, are impressive, even
by the standard of levels for placental mammals (Dawson 1989). An extensive
study of the lung tissue and its pulmonary diffusion characteristics showed that this marsupial's level of oxygen consumption was not likely to be con- strained by the structural components of lung tissue (Hallam, Dawson, and Holland 1989). As with other marsupial mammals (Dawson and Needham 1981), D. byrnei was found to have a large respiratory tidal volume under basal metabolic conditions, and basal respiratory frequency was less than half of the value predicted for a comparable placental mammal (Hallam and Dawson 1993). Since they found that maximum increases in tidal volume and respiratory rates were similar to those achieved by placental mammals, Hallam and Dawson (1993) proposed that D. byrnei has an excellent re-
spiratory capacity to deal with the oxygen demands of high levels of energy metabolism.
The current investigation was undertaken to determine some of the char- acteristics of oxygen transport in the blood of D. byrnei and three other dasyurid marsupials.
Material and Methods
The animals used for this study were all adults. They were the narrow-nosed
planigale (Planigale tenuirostris, mean body mass 6 g), the stripe-faced dunnart (Sminthopsis macroura, mean body mass 23 g), the kowari (Das- yuroides byrnei, mean body mass 130 g), and the eastern quoll (Dasyurus vizverrin us, mean body mass 700 g). Five planigales (one male, four female) were wild caught at Fowlers Gap research station in western New South Wales; six dunnarts (three male, three female) were obtained from Mac-
quarie University in Sydney, New South Wales; six kowaris (three male, three female) were bred in captivity at the University of New South Wales from South Australian stock; and four quolls, all female, were from Taronga Zoological Park in Sydney, Australia.
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344 J. F. Hallam, R. A. B. Holland, and T. J. Dawson
Blood samples were taken from the lateral caudal vein of unanesthetized animals, except the D. viverrinus, which were lightly sedated with Diazepam (0.5 mg intramuscular) before samples were taken from the hind leg. The blood was taken quickly into a few microliters of heparin and immediately refrigerated. Handling of animals was kept to a minimum to avoid the prob- lem of metabolic acidosis.
Whole-blood characteristics for the two larger species (D. viverrinus and D. byrnei) were determined with an Ortho EH (800) hematology analyzer (Ortho Diagnostic Systems, Westwood, Mass.). Whole-blood samples were
centrifuged and washed three times in 0.85% NaCl solution. One part of washed red cells was hemolyzed in 100 parts of distilled, deionized water. The hemoglobin solutions were run against known samples of human or
sheep blood on polyacrylamide gel plates containing carrier ampholytes (Ampholine PAGplates for determination of HbA1c; LKB, Bromma, Sweden). The pH gradient of the plates was approximately 7.0-8.0, and samples were run at 10.00C. The isoelectric point, or pI (the point at which there is no net electric charge on the protein), was determined for the hemoglobin of each study animal.
The characteristics of the oxygen equilibrium curve (OEC) were deter- mined on a thin film of 2 pL of whole blood or red-blood-cell suspension with a Hem-O-Scan (Aminco Instrument, Silver Springs, Md.). Oxygen ten- sions were measured with an oxygen electrode, and fractional oxygen sat- uration of the hemoglobin was measured by dual-beam spectrophotometry at 560 and 576 nm. The sample- and gas-introduction systems of the machine were modified as described by Lapennas and Lutz (1982). The spectropho- tometer was taken to 100% saturation with injections of pure oxygen to ensure accurate calibration for OECs on the low-oxygen-affinity hemoglobins (Vorger 1987). The OECs were run at 36.00C, which is considered to be an
average dasyurid body temperature (Dawson and Hulbert 1970). Carbon dioxide tensions ranged between 7 and 143 mmHg. The OECs were deter- mined within 2 h on whole-blood samples. Red-cell suspensions were pre- pared by resuspending washed red cells in a saline-bicarbonate-phosphate buffer solution, which had an electrolyte composition similar to that of nor- mal mammalian plasma. The final concentration was approximately 5 mM
hemoglobin monomer (Hb1). Red-cell-suspension OECs were run within 4-6 h, except in the case of D. viverrinus, in which whole blood was
refrigerated and cell suspensions were made up and analyzed the follow-
ing day. The OECs were graphically plotted according to the logarithmic form of
the Hill equation
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where Yis the fractional saturation of the hemoglobin with oxygen, Po2 is the partial pressure of oxygen, and P50 is the partial pressure of oxygen at which the hemoglobin is half-saturated with oxygen. The slope of the plot,
nH, is the Hill coefficient and gives the degree of cooperation between subunits of the hemoglobin tetramer (Hb,). The Hill plots were found to be curvilinear, and the Ps0 was determined from quadratic equations, which
statistically gave a better fit to the data. A linear regression was fitted to a
selection of points in the midrange of the curves by the method of least
squares, and in this way a value for nH was obtained. A decrease in the pH of the blood results in a decrease in the affinity of
the hemoglobin for oxygen. The magnitude of this effect can be expressed by the Bohr factor, Alog P50/ApH. Since most of our blood samples were
too small to measure pH, we measured the effects of changing CO2 tensions on the P50 of whole blood (calculated as Alog P50/Alog Pco2). We determined the Bohr factors for the red-cell suspensions in the following manner. The
logarithmic relationship between CO2 tension and pH was experimentally determined for the cell-suspension buffer and was found to be highly sig- nificant (r2 = 0.996, P < 0.001). Using this relationship, we calculated the
pH of the red-cell suspensions at a known CO2 tension. Bohr factors were thus calculated as Alog P50/ApH. It is possible that the buffering capacity of the hemoglobin in the cell suspensions could have resulted in differences in pH from that measured for the suspension buffer alone. However, it was felt that this was the best estimate in the absence of sufficient blood samples to measure pH directly.
For D. byrnei, 2,3-diphosphoglycerate (2,3-DPG) concentrations were measured by ultraviolet spectrophotometry with a Sigma Diagnostics kit no. 35-UV (Sigma Chemical, St. Louis). A standard curve was constructed with a range of 2,3-DPG concentrations (r2 = 0.94, P< 0.001), and concentrations in the blood samples were calculated from A absorbance at 340 nm from
this. The moderating effect of 2,3-DPG on the oxygen affinity of hemoglobin was also evaluated. Red-blood-cell suspensions were prepared from whole- blood samples by the method outlined above ([Hb,] 6.7 mM). The pH was measured at 36.00C and the OECs run at 5.8% CO2 (41 mmHg) to determine the Ps0. In order to lower the organic phosphate levels in the red cells, whole-blood samples were mixed with an acid-citrate-dextrose solution (ACD; 5 vol blood:l vol ACD) and incubated at 37.00C for 17-19 h. A sus-
pension was prepared from the incubated cells as above, the pH was mea-
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346 J. F. Hallam, R. A. B. Holland, and T. J. Dawson
sured, and OECs were run under the same conditions as those of the un- stripped suspensions.
The effects of the addition of 2,3-DPG to depleted hemoglobin solution were also examined. Stripped cells were hemolyzed with two volumes of doubly distilled water. Bis-tris buffer (0.1 M + 0.15 M NaCl adjusted to pH 7.4) was added to the hemolysate in an equal volume
([Hbl] 3.5
mM). The pH of the resulting solutions was measured and the OECs run to calculate the Ps0. The tris salt of 2,3-DPG was added to the hemoly- sate in a 2:1 ratio with the amount of Hb4 calculated to be present in the sample. The pH of the resultant solution was measured and the OECs were run. A paired t-test evaluated the significance of changes in P50 and pH.
Results
Isoelectric focusing showed that all four dasyurid species had a single major hemoglobin. These hemoglobins had higher pi values than does adult human hemoglobin. Planigale tenuirostris, Sminthopsis macroura, and Dasyurus viverrinus had hemoglobins with a pI of 6.9, while Das- yuroides byrnei hemoglobin had a pI of 7.3, all measured at 10.00C. Whole-blood characteristics for D. byrnei and D. viverrinus are shown in table 1.
Representative OECs for the four species are shown for whole blood in figure la and for red-blood-cell suspensions in figure lb. The curves
TABLE 1
Hematological parameters for Dasyuroides byrnei and Dasyurus viverrinus
Dasyuroides Dasyurus byrnei viverrinus
Body mass (g) .................... 133 + 6 706 19 Hematocrit ....................... .55 o .04 .48 o .03
Fig. 1. Representatizve OECs for the four dasyurid species at 360 C and a CO2 tension of 43 mmHg. a, Whole blood,; b, red-cell suspension.
for whole blood were found to follow the usual trend, with the smallest
animals having the most right-shifted curves. Washing and suspension of the red cells in buffer reduced the separation of the curves
(fig. 1 b). All four dasyurid species were found to have hemoglobins with a low
affinity for oxygen. This is demonstrated by the high mean P50's of whole blood and red-blood-cell suspensions shown in table 2. The P50's of the whole blood and red-blood-cell suspensions were significantly different in the case of P. tenuirostris, S. macroura, and D. viverrinus. Notable in the
two smallest dasyurids (P. tenuirostrisand S. macroura) is the significantly increased affinity of the hemoglobin for oxygen in the cell suspensions over that of whole blood.
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The relationship between P50 of the whole blood and body mass is shown in figure 2. The relationship between P50 of the red-cell suspensions and
body mass was not significant (P = 0.34). The nH values measured from the midsections of the OECs ranged from
1.91 to 2.97. A Tukey test showed no interspecific differences were significant. The change in P50 with changing CO2 tensions (Alog P50/Alog Pco2) for
whole blood, Bwb, and the Bohr factors calculated for the red-blood-cell
suspension, Brbc, are shown in table 2. In each species, the absolute value of Brbc was larger than Bwb. These differences were significant.
The mean 2,3-DPG level for D. byrnei was 6.2 + 0.7 tmol of 2,3-DPG/mL of red blood cells, which is intermediate in the range for mammals (Bunn, Seal, and Scott 1974). The mean ratio of 2,3-DPG to Hb4 was 1.3 + 0.2. When 2,3-DPG levels were depleted in the red blood cells by incubation, the mean
Ps0 fell significantly from 52 to 43 mmHg (P< 0.002); there was no significant change in the pH. When 2,3-DPG was added to the depleted hemoglobin solution in the ratio of 2:1 (2,3-DPG:Hb4), Ps0 increased from an initial mean of 38 mmHg to a mean value of 59 mmHg (P< 0.001). There was no significant change in the pH caused by the addition of 2,3-DPG.
Discussion
The four dasyurid marsupials used for this study are all active insectivores and/or carnivores. Low oxygen affinity of the hemoglobin has often been
* P. tenuirostris
70 * S. macroura
I 65 0 D.byrnei E E 60 A D. viverrinus
o 55 - o I
S50 - 0
S45 - r-
40 - o 0 C 35 -
3 0 . . . . , ,, . . . . , ,**1 I . . . . . . . i
1 10 100 1000
Body mass (g)
Fig 2. The relationship between Pso of the whole blood and body mass for the four dasyurid species at 360 C and a CO, tension of 43 mmHg. The line is described by the equation Pso = 69. 53 - 10 61 log Mb (r2 = . 73, P < 0001).
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350 J. F. Hallam, R. A. B. Holland, and T. J. Dawson
suggested as an adaptation to an active lifestyle, as it enables the blood to unload oxygen at the tissues when oxygen partial pressures are still relatively high (Schmidt-Nielsen and Larimer 1958). High Ps50's have similarly been found in small, energetic, placental mammals such as shrews and bats (Bar- tels et al. 1979; Jtirgens, Bartels, and Barrels 1981). Another strategy for coping with the requirements of a high metabolic rate is to have an increase in the blood's capacity to carry oxygen. The blood of Dasyuroides byrnei can carry 24 mL of oxygen in 100 mL of blood (Hallam, Dawson, and Holland 1989) compared with the usual 20 mL 02/100 mL blood oxygen-carrying capacity of many placental bloods. Note that, like the blood of D. byrnei, the blood of Dasyurus viverrinus has a high hematocrit and high concen- trations of circulating hemoglobin.
The strong correlation between the P50 of the whole blood and body mass (fig. 2) reflects that previously demonstrated for placental mammals. However, dasyurid P50's are about 20% higher than would be predicted on the basis of body mass (Schmidt-Nielsen and Larimer 1958). The rea- son for such extreme elevation in P50 remains unexplained. Blood with
very low oxygen affinity requires high oxygen tensions to be fully satu- rated. Indeed, at the normal partial pressure of 100 mmHg oxygen in alveolar air, Planigale tenuirostris and Sminthopsis macroura would only be able to load about 80% of their hemoglobin with oxygen (see fig. 1 a). However, in small animals, the reduction in alveolar diameter leading to a greater surface area to volume ratio in the lungs, coupled with higher respiratory frequencies, has been proposed to result in better alveolar ventilation (Steen 1971; Weibel 1972). Furthermore, D. byrnei and P. tenuirostris have both been shown to have a large tidal volume by com-
parison to similar-sized placental mammals (Hallam and Dawson 1993; Chappell and Dawson 1994). Higher ventilation perfusion ratios in the alveoli would cause an increase in alveolar oxygen tension and alleviate the problem of loading oxygen onto a low-affinity hemoglobin. Higher ventilation perfusion ratios in the alveoli would also lead to a decrease in alveolar CO2 tension. If the dasyurids in this study had arterial Pco2'S of less than 40 mmHg, as has been found in some placental mammals (Lahiri 1975), then this might explain why the P50's reported here (Pco2 = 43 mmHg) are so high.
Unfortunately, the small size of the blood samples precluded measure- ment of the pH of the whole blood, and this would have been of particular interest in the two smallest species. Note that Nicol (1982) found a P50 of 38 mmHg for the whole blood of D. viverrinus at 370C and a pH of 7.4. When corrected to 36.00C, this gives a P50 of 36.0 mmHg, which is close to the value found in this study. For the two smallest dasyurids, the large
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and significant increase in oxygen affinity when the red cells were sus-
pended in buffer at a Pco2 of 43 mmHg (pH assumed to be 7.4) suggests that the bicarbonate content of the plasma may be less than that of our
"physiological" buffer. Such a difference in bicarbonate content would cause the whole blood to be more acid than the cell suspension when both were equilibrated with the same Pco2.
Figure 3 shows the relationship between P50 of hemoglobin with a pH of 7.4 at 36.0oC and the logarithm of body mass for a range of marsupial mammals. The line shown is described by the equation P50 = 35.21 - 5.14 log Mb, where Mb is body mass in kilograms (r2 = 0.60, P< 0.001). Bland and Holland (1977) derived a logarithmic relationship between
Ps0 and body mass for seven marsupial species ranging from 30 g to 35
kg Mb. Their line does not differ significantly from the relationship shown in figure 3.
The value of the Bohr effect was lower than expected in all four dasyurid species. Although Bland and Holland (1977) found no significant correlation between the Bohr factor and body mass for a range of marsupials, the Bohr factor is generally found to be higher in small mammals (Riggs 1960; Lahiri
1975). This is a mechanism to aid loading of oxygen onto the lower-oxygen- affinity hemoglobins in the lung.
Bland and Holland (1977) showed that 2,3-DPG decreased the oxygen affinity of the hemoglobin in four species of macropod marsupials. The results of this study clearly demonstrate the moderating effect of 2,3-DPG on the hemoglobin of D. byrnei. Bunn, Seal, and Scott (1974) conducted a study of the blood of 71 mammalian species representing 14 orders.
They found that most mammals had high levels of red-cell 2,3-DPG and that the phosphate-free hemoglobin solutions had intrinsically high ox-
ygen affinity that was significantly affected by the addition of 2,3-DPG. In contrast, all the members of the families Bovidae and Felidae examined in their study had low red-cell 2,3-DPG and intrinsically low oxygen- affinity hemoglobins that showed little reactivity to addition of 2,3-DPG. Notably, the P50 of the phosphate-depleted hemoglobin solution for D.
byrnei is a great deal higher than any previously reported for stripped hemoglobin (Bunn, Seal, and Scott 1974), indicating that D. byrnei has a hemoglobin with an intrinsically low oxygen affinity. Earlier studies
covering eight marsupial species resulted in similar data to those for D.
byrneiin that all species showed levels of red-cell 2,3-DPG with the ratio of 2,3-DPG to Hb4 > 1 (Bland and Holland 1977; Dodgson and Holland 1982; Isaacks et al. 1984).
It is clear from this study that hemoglobins with a low affinity for oxygen are indeed a characteristic of the dasyurid marsupials. There is also a
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352 J. F. Hallam, R. A. B. Holland, and T. J. Dawson
* This study
60 -
0O Dasyurids
O Other marsupials 55 -
O Macropodids 50
S50 -A Opossum E* E 45
so *
o 0 -o o IA
4
35 Bartels
,
o Io 30 - 0
I1.
25 0a
0.001 0.01 0.1 1 10 100
Body mass (kg)
Fig. 3. The relationship between Pso of whole blood at a pH of 7. 4 and
body mass for a range of marsupial mammals at 360 C. The line is de- scribed by the equation Pso = 35.21 - 5.14 * log Mb (r2 = 0.60, P < 0.001). The Pso values for animals in this study taken from the red-
blood-cell suspensions at a pH of 7. 4. Where necessary, values have been corrected from 37.00 C by dividing by 1.056 (Severinghaus 1971). In-
cluded for comparison is the line given by Bartels (1964)for placental mammals. Also plotted are the data for the Tasmanian devil (carnivo-
supial) (Holland et al. 1988);fat-tailed dunnart (insectivorous dasyurid marsupial), brushtail possum, koala, and red kangaroo (herbivorous di-
protodont marsupials) (Lutz, personal communication).
strong correlation between oxygen affinity of the hemoglobin and body mass over three degrees of magnitude for these four dasyurid species. Since dasyurids are very active animals, this lends support to the theory that low-affinity hemoglobins are an adaptation to an active lifestyle (Schmidt-Nielsen and Larimer 1958). For even if these animals are shown to have lower in vivo Pco2'S, as seems probable, the low Bohr effects in these bloods mean that corrections for pH would not depress the P50 to "normal" values.
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We thank Dr. Garry Reddacliff of Taronga Zoological Park, Sydney, for the blood samples from Dasyurus viverrinus. This study was funded by Aus- tralian Research Council grants to T.J.D. and R.A.B.H.
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