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The respiratory properties of the blood of the bladdernose seal (Cystophora cristata)
Respiration Physiology (1969) 7, l-6; North-Holland Publishing Company, Amsterdam
TJ3E RESPIRATORY
PROPERTIES
THE BLADDERNOSE
OF THE BLOOD
OF
SEAL (Cystophora cristata)
GUNNAR CLAUSEN AND AMUNDERSLAND Institute of Physiology, University of Bergen, Norway
Abstract. The blood of about three months old Cystophora had an OS.capacity of 36 vol %. RBC was normal, 4.8 x 106/mms, but Hct and Hb content were very high, 63 % and 26.4 g /lOOml. When calculating the MCV as Hct/RBC x 10 and from the Price-Jones curve the mean values were 131 and 135 ps. The Cystophora red blood cell was 60% larger and had a 30% higher MCHC than that of humans. The TSOwas 24 mm Hg and the mean Bohr effect factor (A log Tsa/ApH) was -0.66, i.e. somewhat higher than in previously investigated mammalian divers. Relatively high Haldane effect, 8.4 vol % CO%,and buffering capacity (A[HCO;I/ApH), -36 meq/L, were found in the blood. It is generally concluded that the investigated characteristics of Cystophora blood differ from those of human blood in accordance with its higher Hb content. Buffering capacity COZ dissociation HbOz dissociation
In a sea lion (Eumethopsis
0s capacity T50 Red
cell volume
stelleri) an exceptionally high O2 capacity per volume red cells (0.68 cc O&c cells) was found by FL~RKIN and REDFIELD (1931). The Hct of the single sample available was only 29. IRVING et al. (1935) found 0.61 cc O&c cells in the blood from harbour seals (Phoca vitulina). They found a RBC of 6 x 106/mm3 and a normal Hct, and concluded that the red cells are smaller than those of human blood. IRVING et al. where not satisfied that their Hb data giving 1.78 cc 0,/g I-lb were correct. There is no doubt that seal blood does have exceptionally high O2 capacities. SCHOLANDER (1940) has shown this in the grey seal (Helichoerus grypus) and the present species. It is not clear, however, whether this high 0, capacity is associated with a high MCHC, a high Hct or with a special feature of seal Hb itself. Both the I-IbOz and the CO1 dissociation have been studied, but pH was not determined in any of the above mentioned papers. In the Cystophora the gas equilibrium of the blood was evaluated from alveolar gas concentrations and arterial gas contents during prolonged submersions (SCHOLANDER, 1940). These results, however, were influenced by progressive acidosis. Accepted for publication 7 December 1968.
2
GUNNARCLAUSEN ANDAMUNDERSLAND
In order to obtain a comparison to other diving mammals previously investigated or discussed by the present authors (CLAUSENand ERSLAND,1968), we have reinvestigated the respiratory properties of Cystophora blood in vitro by the same methods. Materials and methods The two seals available weighed about 50 kg and were about three months old (“blue backs”). The blood samples were obtained at one to two week intervals. Twenty to thirty ml of arterial blood was drawn into siliconed 10 ml syringes by puncturing a flipper artery. Heparin was used to prevent coagulation. The analyses began half an hour after sampling. The blood was stored in ice water during transport, later at + 4°C. Determination of O2 capacity and CO2 content, pH, Po2, Pco2, HbOz sat %, and Hb were made as described in a recent paper (CLAUSENand ERSLAND,1968). Hematocrit (Hct) was determined in triplicate or more, by 15 min centrifugation using a standard hematocrit centrifuge and capillary tubes (diam. 1.2-1.4, length 75 mm). (A stable Hct was obtained after less than 10 min centrifugation). Erythrocyte concentration (RBC) was determined by counting in Biirker chamber and by celloscope. The mean volume of the erythrocytes (MCV) was determined by constructing the PriceJones curve, using the celloscope 101 produced by Ljungberg et Co., Stockholm. The celloscope measurements were made immediately after dilution of whole blood in buffered physiological saline. The celloscope was adjusted to give a MCV of 85 p3 of human erythrocytes. The mean cell diameter (MCD) was obtained from the MCV/ MCD relation table published by Ljungberg et Co. in the celloscope instruction manual: MCD ,~6 MCVp3
6.8
7.5
8.2
8.9
9.6
10.3
71.5
85.0
102.5
122.0
143.0
164.0
The counting accuracy of the celloscope is N 1%. Since the erythrocytes of the Cystophora blood had a normal discoidal shape and appeared to have a normal morphology when studied by microscope, the mean cell diameter was also calculated as Hct/RBC* 10. Results and discussion The results listed in table 1 show that the high O2 capacity of the blood (36 vol %) is due to a high content of Hb (26.4 g/100 ml) having a normal O2 capacity (1.36 ml 0,/g Hb) at N 300 mm Hg of Oz. The Hb is held by erythrocytes which are 60 % larger than those of human blood in a 30% higher concentration than in human erythrocytes. The shape of the erythrocyte studied in buffered physiological saline by microscope, appeared normal as compared to that of human blood. In fig. 1 the HbOz dissociation curve is presented. It was constructed from the data in table 2. Figure 2 demonstrates the Bohr effect. The Bohr factor (AlogT,,/A pH) was - 0.66 (- 0.71 and - 0.62 in seal 1 and 2 respectively). Tse at pH 7.4 was 24.2 mm Hg, i.e. about 5-10 mm Hg lower than the in viuo values of SCHOLANDER’S investigation on the same species. The difference in the position of his and the present curves should
BLOOD OF THB BLADDERNOSE SEAL TABLE
3
1
Characteristics of blood in three samples from each of the two specimens. Hb g/100 ml
Hct %
RBC million/n-ma
Oa cap. vol 0%
MCV ya
MCD
celloscope
Hct/RBC
10
26.4
63
4.8
36
135
131
9.3
(25.8-28.4)
(60-67)
(4.3-5.3)
(34-38)
(131-138)
(127-W)
(9.2-9.4)
Red cell concentration
(RBC)
Mean cell volume (MCV)
was determined by celloscope
Mean cell diameter (MCD)
in two samples only.
gives the mean value and range of both methods.
TABLE 2 COZ and Oa tensions, HbOa sat % and pH of the blood. SEAL
1
Pco,
N
PH
40mmHg Po*
Pco, sat%
N
PH
105mmHg Po,
Pco, sat %
ff
PH
21 mmHg Po,
sat%
7.415
15.1
30.1
7.155
25.6
30.1
7.550
15.5
38.0
7.425
21.7
43.0
7.13s
38.4
51.5
7.545
28.9
64.0
1.405
28.2
62.0
7.140
49.4
67.5
7.535
35.7
77.0
1.385
41.3
76.0
7.130
55.1
77.0
-
-
-
7.390
15.3
29.5
7.140
26.0
31.5
7.560
15.7
41.0
7.410
22.0
49.0
7.190
32.0
42.0
7.510
22.5
55.5
7.360
28.7
54.0
7.160
44.0
60.0
7.510
29.4
73.0
7.370
35.2
72.5
7.135
56.0
73.0
-
-
SEAL
Pco,
2 N
40mmHg
Pco,
N
105 mm Hg
Po,
sat %
PH
7.340
15.1
26.5
7.095
19.7
24.5
7.340
21.7
44.0
7.085
31.6
41.5
7.320
34.9
71.0
7.095
43.4
64.0
7.280
61.1
90.0
7.070
55.2
PH
7.375
-
PO*
15.2
30.0
7.140
19.9
sat %
Pco, -.
N
21 mm Hg PO*
sat %
7.460
15.5
35.5
7.455
29.0
65.5
-
-
-
16.5
-
-
-
24.5
7.495
15.6
35.0
PH
7.375
21.8
45.5
7.130
25.8
34.5
7.480
22.4
53.5
7.360
35.0
74.0
7.130
31.8
47.5
7.475
29.2
67.0
-
-
-
7.130
43.7
65.5
-
-
-
-
-
-
7.105
55.6
77.0
-
-
-
GUNNAR CLAUSEN AND AMUND ERSLAND
Hb sat. %
80 -
60 -
I
I
I
1
20
1
40
’
I
1
I
60
80
Po2 mm
Hg
Fig. 1. The HbOa dissociation curve at 40 mm Hg of COa and pH 7.4, constructed from the data in table 2. For comparison the HbOa-Pao, dissociation curves obtained during submersion (SCHOLANDER, 1940) have been entered.
I
L
7.1
I
I
1
1
.2
.3
.4
.5
.6 PH Fig. 2. The TWJ-pHrelation demonstrating the Bohr effect. Squares: seal No. 1; circles: seal No. 2.
express the alveolar arterial tension difference of 0, (P&,-Pao,). This is especially interesting since SCHOLANDER’Sdata were obtained during submersion when the heart rate - and blood flow through the lungs - is about 10 % of normal owing to the profound bradycardia associated with submersion. SCHOLANDER’S curves are progressively displaced rightwards at falling Po2, because of the increasing Pco, and lactate
BLOOD OF THE BLADDERNOSE SEAL
3
5
40 PC0 mmM 2
Fig. 3. Characteristic COa dissociation curves of fully reduced and oxygenated blood (from seal No. 2).
TABLE 3 Blood pH and ~01% COs when Hb (26.6 go& is fully reduced and oxygenated Pco,
mm Hg 20.9
39.1 48.9 62.8
Hb
(Seal No.2).
HbOa
pH
COa
pH
COa
7.480 7.340 7.300 7.240
26.6 38.2 42.7 41.5
7.380 7.260 7.205 7.140
19.6 29.8 34.3 39.2
APH
ACOa
0.10 0.08 0.10 0.10
7.0 8.4 8.4 8.3
concentration in the blood. SCHOLANDER presents Pco, values corresponding to the HbO, dissociation curves. Using the present pH-log P,o, line and Bohr effect factor, T,, at pH 7.4 is 27.5 (26-30) mm Hg. Due to a slight lactate accumulation in the blood, that has not been accounted for in this calculation, the T,,-, must be somewhat lower than 27.5 mm Hg, i.e. the Pbl-Pao, is less than 4 mm Hg at TSo. A relatively high Haldane effect of 8.4 ~01% of CO, was found at 40 mm Hg of COz (fig. 3). The ACO, (Hb-HbO,) vol%/g Hb is 0.32, slightly lower than that of human blood (0.35). The buffering capacity of the blood (A[HCO;]/ApH) was - 36. meq/L at 26.6 g % Hb, i.e. higher than in human blood. The pH-log Po-,z lines that are shown in fig. 4, in comparison with that of human blood of equal Hb content, show that pH in re-
GUNNAR
CLAUSENAND
AMUND
ERSLAND
‘CO, mm Hg 120 -
20 -
t
I
I
I
I
I
I
20
1
.2
.3
x
.5
.6 pH
Fig.4.The pH-log PCO, lines made from the data in table 3 in comparison of equal Hb content (26.6 g%).
with that of human blood
duced blood, measured when making the CO2 dissociation curves, was 0.1 units higher than in oxygenated blood at constant Pco, (c$ table 3). Generally, the 0, capacity, Haldane effect and buffering capacity of C~,rtophoru blood appear to be modified as should be expected according to the high Hb content of the blood. A high Hb content, giving both high O2 capacity and buffering capacity, may be regarded as a valid adaptation to diving. The question of possible adaptations of the respiratory properties of mammalian blood to diving was discussed in a recent paper (CLAUSEN and ERSLAND, 1968). Acknowledgement
We wish to thank the Norwegian Fisheries Research Institute, Directorate Fisheries, which provided - and helped us with handling - the seals.
of
References CLAUSEN,G. and A. ER~LAND(1968). The respiratory properties of the blood of two diving rodents, the beaver and the water vole. Respir. Physiol. 5: 350-359. FLORKIN, M. and A. C. REDFIELD(1931). On the respiratory function of the blood of the sea lion. Biol. Bull. Woods Hole 61: 422-426. IRVING,L., 0. M. SOLANT,D. Y. SOLANTand K. G. FNJER (1935). Respiratory characteristics of the blood of the seal. J. Cell. Comp. Physiol. 6: 393-403. SCHOLANDER, P. F. (1940). Experimental investigations on the respiratory function in diving mammals and birds. Hvalrdd. Skr. 22: l-131.
TJ3E RESPIRATORY
PROPERTIES
THE BLADDERNOSE
OF THE BLOOD
OF
SEAL (Cystophora cristata)
GUNNAR CLAUSEN AND AMUNDERSLAND Institute of Physiology, University of Bergen, Norway
Abstract. The blood of about three months old Cystophora had an OS.capacity of 36 vol %. RBC was normal, 4.8 x 106/mms, but Hct and Hb content were very high, 63 % and 26.4 g /lOOml. When calculating the MCV as Hct/RBC x 10 and from the Price-Jones curve the mean values were 131 and 135 ps. The Cystophora red blood cell was 60% larger and had a 30% higher MCHC than that of humans. The TSOwas 24 mm Hg and the mean Bohr effect factor (A log Tsa/ApH) was -0.66, i.e. somewhat higher than in previously investigated mammalian divers. Relatively high Haldane effect, 8.4 vol % CO%,and buffering capacity (A[HCO;I/ApH), -36 meq/L, were found in the blood. It is generally concluded that the investigated characteristics of Cystophora blood differ from those of human blood in accordance with its higher Hb content. Buffering capacity COZ dissociation HbOz dissociation
In a sea lion (Eumethopsis
0s capacity T50 Red
cell volume
stelleri) an exceptionally high O2 capacity per volume red cells (0.68 cc O&c cells) was found by FL~RKIN and REDFIELD (1931). The Hct of the single sample available was only 29. IRVING et al. (1935) found 0.61 cc O&c cells in the blood from harbour seals (Phoca vitulina). They found a RBC of 6 x 106/mm3 and a normal Hct, and concluded that the red cells are smaller than those of human blood. IRVING et al. where not satisfied that their Hb data giving 1.78 cc 0,/g I-lb were correct. There is no doubt that seal blood does have exceptionally high O2 capacities. SCHOLANDER (1940) has shown this in the grey seal (Helichoerus grypus) and the present species. It is not clear, however, whether this high 0, capacity is associated with a high MCHC, a high Hct or with a special feature of seal Hb itself. Both the I-IbOz and the CO1 dissociation have been studied, but pH was not determined in any of the above mentioned papers. In the Cystophora the gas equilibrium of the blood was evaluated from alveolar gas concentrations and arterial gas contents during prolonged submersions (SCHOLANDER, 1940). These results, however, were influenced by progressive acidosis. Accepted for publication 7 December 1968.
2
GUNNARCLAUSEN ANDAMUNDERSLAND
In order to obtain a comparison to other diving mammals previously investigated or discussed by the present authors (CLAUSENand ERSLAND,1968), we have reinvestigated the respiratory properties of Cystophora blood in vitro by the same methods. Materials and methods The two seals available weighed about 50 kg and were about three months old (“blue backs”). The blood samples were obtained at one to two week intervals. Twenty to thirty ml of arterial blood was drawn into siliconed 10 ml syringes by puncturing a flipper artery. Heparin was used to prevent coagulation. The analyses began half an hour after sampling. The blood was stored in ice water during transport, later at + 4°C. Determination of O2 capacity and CO2 content, pH, Po2, Pco2, HbOz sat %, and Hb were made as described in a recent paper (CLAUSENand ERSLAND,1968). Hematocrit (Hct) was determined in triplicate or more, by 15 min centrifugation using a standard hematocrit centrifuge and capillary tubes (diam. 1.2-1.4, length 75 mm). (A stable Hct was obtained after less than 10 min centrifugation). Erythrocyte concentration (RBC) was determined by counting in Biirker chamber and by celloscope. The mean volume of the erythrocytes (MCV) was determined by constructing the PriceJones curve, using the celloscope 101 produced by Ljungberg et Co., Stockholm. The celloscope measurements were made immediately after dilution of whole blood in buffered physiological saline. The celloscope was adjusted to give a MCV of 85 p3 of human erythrocytes. The mean cell diameter (MCD) was obtained from the MCV/ MCD relation table published by Ljungberg et Co. in the celloscope instruction manual: MCD ,~6 MCVp3
6.8
7.5
8.2
8.9
9.6
10.3
71.5
85.0
102.5
122.0
143.0
164.0
The counting accuracy of the celloscope is N 1%. Since the erythrocytes of the Cystophora blood had a normal discoidal shape and appeared to have a normal morphology when studied by microscope, the mean cell diameter was also calculated as Hct/RBC* 10. Results and discussion The results listed in table 1 show that the high O2 capacity of the blood (36 vol %) is due to a high content of Hb (26.4 g/100 ml) having a normal O2 capacity (1.36 ml 0,/g Hb) at N 300 mm Hg of Oz. The Hb is held by erythrocytes which are 60 % larger than those of human blood in a 30% higher concentration than in human erythrocytes. The shape of the erythrocyte studied in buffered physiological saline by microscope, appeared normal as compared to that of human blood. In fig. 1 the HbOz dissociation curve is presented. It was constructed from the data in table 2. Figure 2 demonstrates the Bohr effect. The Bohr factor (AlogT,,/A pH) was - 0.66 (- 0.71 and - 0.62 in seal 1 and 2 respectively). Tse at pH 7.4 was 24.2 mm Hg, i.e. about 5-10 mm Hg lower than the in viuo values of SCHOLANDER’S investigation on the same species. The difference in the position of his and the present curves should
BLOOD OF THB BLADDERNOSE SEAL TABLE
3
1
Characteristics of blood in three samples from each of the two specimens. Hb g/100 ml
Hct %
RBC million/n-ma
Oa cap. vol 0%
MCV ya
MCD
celloscope
Hct/RBC
10
26.4
63
4.8
36
135
131
9.3
(25.8-28.4)
(60-67)
(4.3-5.3)
(34-38)
(131-138)
(127-W)
(9.2-9.4)
Red cell concentration
(RBC)
Mean cell volume (MCV)
was determined by celloscope
Mean cell diameter (MCD)
in two samples only.
gives the mean value and range of both methods.
TABLE 2 COZ and Oa tensions, HbOa sat % and pH of the blood. SEAL
1
Pco,
N
PH
40mmHg Po*
Pco, sat%
N
PH
105mmHg Po,
Pco, sat %
ff
PH
21 mmHg Po,
sat%
7.415
15.1
30.1
7.155
25.6
30.1
7.550
15.5
38.0
7.425
21.7
43.0
7.13s
38.4
51.5
7.545
28.9
64.0
1.405
28.2
62.0
7.140
49.4
67.5
7.535
35.7
77.0
1.385
41.3
76.0
7.130
55.1
77.0
-
-
-
7.390
15.3
29.5
7.140
26.0
31.5
7.560
15.7
41.0
7.410
22.0
49.0
7.190
32.0
42.0
7.510
22.5
55.5
7.360
28.7
54.0
7.160
44.0
60.0
7.510
29.4
73.0
7.370
35.2
72.5
7.135
56.0
73.0
-
-
SEAL
Pco,
2 N
40mmHg
Pco,
N
105 mm Hg
Po,
sat %
PH
7.340
15.1
26.5
7.095
19.7
24.5
7.340
21.7
44.0
7.085
31.6
41.5
7.320
34.9
71.0
7.095
43.4
64.0
7.280
61.1
90.0
7.070
55.2
PH
7.375
-
PO*
15.2
30.0
7.140
19.9
sat %
Pco, -.
N
21 mm Hg PO*
sat %
7.460
15.5
35.5
7.455
29.0
65.5
-
-
-
16.5
-
-
-
24.5
7.495
15.6
35.0
PH
7.375
21.8
45.5
7.130
25.8
34.5
7.480
22.4
53.5
7.360
35.0
74.0
7.130
31.8
47.5
7.475
29.2
67.0
-
-
-
7.130
43.7
65.5
-
-
-
-
-
-
7.105
55.6
77.0
-
-
-
GUNNAR CLAUSEN AND AMUND ERSLAND
Hb sat. %
80 -
60 -
I
I
I
1
20
1
40
’
I
1
I
60
80
Po2 mm
Hg
Fig. 1. The HbOa dissociation curve at 40 mm Hg of COa and pH 7.4, constructed from the data in table 2. For comparison the HbOa-Pao, dissociation curves obtained during submersion (SCHOLANDER, 1940) have been entered.
I
L
7.1
I
I
1
1
.2
.3
.4
.5
.6 PH Fig. 2. The TWJ-pHrelation demonstrating the Bohr effect. Squares: seal No. 1; circles: seal No. 2.
express the alveolar arterial tension difference of 0, (P&,-Pao,). This is especially interesting since SCHOLANDER’Sdata were obtained during submersion when the heart rate - and blood flow through the lungs - is about 10 % of normal owing to the profound bradycardia associated with submersion. SCHOLANDER’S curves are progressively displaced rightwards at falling Po2, because of the increasing Pco, and lactate
BLOOD OF THE BLADDERNOSE SEAL
3
5
40 PC0 mmM 2
Fig. 3. Characteristic COa dissociation curves of fully reduced and oxygenated blood (from seal No. 2).
TABLE 3 Blood pH and ~01% COs when Hb (26.6 go& is fully reduced and oxygenated Pco,
mm Hg 20.9
39.1 48.9 62.8
Hb
(Seal No.2).
HbOa
pH
COa
pH
COa
7.480 7.340 7.300 7.240
26.6 38.2 42.7 41.5
7.380 7.260 7.205 7.140
19.6 29.8 34.3 39.2
APH
ACOa
0.10 0.08 0.10 0.10
7.0 8.4 8.4 8.3
concentration in the blood. SCHOLANDER presents Pco, values corresponding to the HbO, dissociation curves. Using the present pH-log P,o, line and Bohr effect factor, T,, at pH 7.4 is 27.5 (26-30) mm Hg. Due to a slight lactate accumulation in the blood, that has not been accounted for in this calculation, the T,,-, must be somewhat lower than 27.5 mm Hg, i.e. the Pbl-Pao, is less than 4 mm Hg at TSo. A relatively high Haldane effect of 8.4 ~01% of CO, was found at 40 mm Hg of COz (fig. 3). The ACO, (Hb-HbO,) vol%/g Hb is 0.32, slightly lower than that of human blood (0.35). The buffering capacity of the blood (A[HCO;]/ApH) was - 36. meq/L at 26.6 g % Hb, i.e. higher than in human blood. The pH-log Po-,z lines that are shown in fig. 4, in comparison with that of human blood of equal Hb content, show that pH in re-
GUNNAR
CLAUSENAND
AMUND
ERSLAND
‘CO, mm Hg 120 -
20 -
t
I
I
I
I
I
I
20
1
.2
.3
x
.5
.6 pH
Fig.4.The pH-log PCO, lines made from the data in table 3 in comparison of equal Hb content (26.6 g%).
with that of human blood
duced blood, measured when making the CO2 dissociation curves, was 0.1 units higher than in oxygenated blood at constant Pco, (c$ table 3). Generally, the 0, capacity, Haldane effect and buffering capacity of C~,rtophoru blood appear to be modified as should be expected according to the high Hb content of the blood. A high Hb content, giving both high O2 capacity and buffering capacity, may be regarded as a valid adaptation to diving. The question of possible adaptations of the respiratory properties of mammalian blood to diving was discussed in a recent paper (CLAUSEN and ERSLAND, 1968). Acknowledgement
We wish to thank the Norwegian Fisheries Research Institute, Directorate Fisheries, which provided - and helped us with handling - the seals.
of
References CLAUSEN,G. and A. ER~LAND(1968). The respiratory properties of the blood of two diving rodents, the beaver and the water vole. Respir. Physiol. 5: 350-359. FLORKIN, M. and A. C. REDFIELD(1931). On the respiratory function of the blood of the sea lion. Biol. Bull. Woods Hole 61: 422-426. IRVING,L., 0. M. SOLANT,D. Y. SOLANTand K. G. FNJER (1935). Respiratory characteristics of the blood of the seal. J. Cell. Comp. Physiol. 6: 393-403. SCHOLANDER, P. F. (1940). Experimental investigations on the respiratory function in diving mammals and birds. Hvalrdd. Skr. 22: l-131.