Respiratory functions of blood of the yak, llama, camel, Dybowski deer, and African elephant’

HEINZ BARTELS, PETER HILPERT, KLAUS BARBEY, KLAUS BETKE,2 KLAUS RIEGEL,2 ERNST MICHAEL LANG,3 AND JAMES METCALFE” Abteilung fiir Angewundte Physiologic am Physiologischen Institut, Tiibingen, West Germany, and Harvard Medical School, Boston, Massachusetts

BARTELS? HEINZ, PETER HILPERT, KLAUS BARBEY, KLAUS BETKE, KLAUS RIEGEL, ERNST MICHAEL LANG, AND JAMES METCALFE. Respiratory functions uf blood uf the yak, llama, camel, Dybowski deer, and African elephant. Am. J. Physiol. 205(2): 331--

336. I963* -Blood samples from a yak, llama, camel, deer, and African elephant were analyzed for oxygen capacity, Ccstandard bicarbonate” content, oxygen dissociation curve, and the magnitude of the Bohr and Haldane effects. These parameters of the respiratory function of the blood have been related to the morphology of the red cells, to the weights of the animals, and to the most important electrolytes in the erythro- cytes and in the plasma. The high affinity for oxygen described previously for llama blood is shared by its relative, the camel. Both these animals have a high concentration of hemoglobin within their erythrocytes. Blood from the African elephant showed the greatest affinity for oxygen among the subjects studied.

T o UNDERSTAND THE GAS TRANSPORT function of the blood, these data are required: the oxygen capacity, the oxygen dissociation curve, the buffering capacity, the carbon dioxide dissociation curve, the Bohr effect (the effect of changes in pH on the oxygen dissociation curve), and the Haldane effect (the effect of changes in oxygen saturation upon the carbon dioxide dissociation curve). The oxygen capacity and the oxygen dissociation curve of the blood are known for many mammals. It is not clear whether the established differences among animals are important for gas exchange. The high oxygen affinity of the blood of the llama suggests an adaptation to its high-altitude home. Blood of the rat, mouse, or cat shows a relatively low affinity for oxygen when com-

Received for publication 26 April 1962. I This study was aided in part by grants from the Josiah Macy,

*Jr., Foundation and the National Institutes of Health. 2 Present address: Universitgtskinderklinik, Tiibingen, West

Germany. 3 Present address : Base1 Zoologischer Garten, Basel, Switzer-

land. 4 Present address : University of Oregon Medical School, Port-

land, Ore.

pared to larger mammals, suggesting a relationship be- tween blood oxygen affinity and metabolic mass (I). A correlation also exists (2) between the shape of erythro- cytes and the oxygen affinity.

To further clarify these relationships we have studied blood from the yak (another high-altitude resident), the camel which has oval erythrocytes and is a relative of the llama, the deer whose erythrocytes become sickle- shaped, and the elephant which, among the mammals, has the largest red cells known. As the second largest living mammal, the elephant will also contribute to the suggested correlation between oxygen affinity and oxygen consumption.

The rare opportunity of studying the blood of these animals led us to carry out some measurements which have no direct bearing on the respiratory function of the blood but which may be important for comparative physiology.

METHODS

General. About 50 ml of venous blood were drawn with- out anesthesia from each animal. The blood for gas analysis was mixed with o. I % sodium fluoride, o. 2 % po- tassium oxal &ate, and powdered heparin (Vetren) . It was kept in ice water for a maximum of 4.5 hr before analysis. Blood for elect *olyte analysis was prevented from clotting with calcium h.eparinate and centrifuged within 45 min of sampling. Blood counts, blood smears, and hemato- crits were prepared within 20 min of blood withdrawal.

Blood gas analysis. Equilibration, analysis, and calcula- tion of gas content in the blood samples and construction of the oxygen dissociation curves were done as described in detail by Bartels and Harms (3). Because of the rapid sedimentation rate of elephant red blood cells, samples of this blood were hemolyzed anaerobically immediately after equilibration. Hemolysis was accomplished by freez- ing (in a bath of dry ice and acetone) of the blood in polyvinyl chloride tubing which had previously been rinsed for I hr or more with pure CO2. After thawing the hemolysate was transferred directly to pipettes for im-

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332 BARTELS ET AL.

mediate introduction into the Van Slyke apparatus. Analysis for oxygen content of similar hemolysates gave results within 0.1 ~01% (randomly distributed) of analyses of the same human or goat blood analyzed without hemolysis. Analyses for CO2 content and pH were done without hemolysis, as speedily as possible. The equilibrations were all made at 37 C although the body temperatures of some species (the deer and the yak) may be as much as 2 C higher. With the facilities avail- able, analyses at body temperatures were not possible. If one uses the temperature correction factor for human blood (4), the T50 value is increased by approximately 0.5 mm Hg for the African elephant and the llama, I -7

mm Hg for the camel, and about 2.6 mm Hg for the yak and the deer. The human temperature factor may not be correct, however, since a smaller value has been found for goat blood (unpublished results).

For measurement of the Bohr effect, blood samples were equilibrated with such gas mixtures that at oxygen saturations of approximately 50%, the pH varied from

-0 20 40 60 80 mmHg FIG. I. Oxygen dissociation curves of the yak, llama, camel

deer, and African elephant at 37 C and a blood pH of 7.40.

TABLE I. General information about the animah studied

7.0 to 7.7. From the ~0, values for 50% saturation (T50) and the measured pH values, the Bohr effect was deter- mined and expressed as A log pOz/A pH (see Table 3).

The Haldane effect (expressed as the difference in carbon dioxide capacity at a pCOZ of 40 mm Hg be- tween fully oxygenated blood and blood coritaining no oxygen) was calculated using a carbon dioxide dissocia- tion curve at full oxygen saturation drawn with a nomo- gram which Henderson published in an appendix to the German edition (5) of his book. This nomogram was constructed from data obtained on human blood. The carbon dioxide contents of the samples which were 50 % or less saturated with oxygen at a pC02 or 40 mm Hg were sketched in. The increase of carbon dioxide con- tent resulting from decreasing oxygen saturation was read off and the Haldane effect for complete desatura- tion calculated.

@H Meusurement. Duplicate electrometric measure- ments were made on each blood sample used for the determination of the Bohr effect. The pH of the red cells was measured as described elsewhere (6).

Morphology of the blood. Number of erythrocytes (RBC) was counted in duplicate in the Neubauer chamber after dilution with physiological saline (7). Hematocrit (Vc) determinations were done in duplicate using a microhematocrit centrifuge. Hemoglobin (Hb) concen- tration was calculated from triplicate measurements of the oxygen capacity. Erythrocyte surface (MS) was measured by planimetry of the magnified red cell image.

Values for mean cell volume (MCV), erythrocyte diameter (MD) (in the camel and yak, two additional diameters are given since we are dealing with elliptical erythrocytes), erythrocyte thickness (MT), and erythro- cyte surface area (MSA) were obtained by methods described elsewhere (8).

Ehtrolytes. Plasma and an hemolysate of packed red cells were analyzed for potassium and sodium with a flame photometel (without phosphorus correction), for magnesium according to Orange and Rhein (g), and for chloride (I 0). Total phosphorus was determined by a method modified from Fiske and Subbarow (I I ). Blood water was measured by drying at 80 C to constant weight. Plasma proteins were determined according to Kingsley

( 1 12 .

Starch block electrujhoresis. The hemolysate of washed erythrocytes was prepared with carbon tetrachloride and

ZooIogical Name Age, Yr

Sex Birthplace

Body Temperature, C

Estkated kg” our Literature

Measurements (27)

Yak Llama Camel Dybowski deer African elephant

Bos grunniens Llama glamu Camelus hacfrianus Pseudaxis hortulorum Loxodonta africana

--

2

I l 5 IO

I.5 10.5

Q Q Q

8 CT

Base1 Zoo Base1 Zoo Rotterdam Baael Zoo Tanganyika

25O 38*5-39*5 200 37-38 300 38-39 37.5

35 38-5-39-5 38.2 2,300 37-38 36.2

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RESPIRATORY FUNCTIONS OF BLOOD 333

TABLE 2. Gas pressures and contents, 02 saturation, and $7 of blood (calculated from gas analyses and measured with glass electrode), and in hemolysates of red cells (measured with glass electrode)

26590 45-4 21.52 46.0 100 15.8 44.0 5-13 49-8 24-5 23.7 46-3 7.68 52-3 36.8 25.6 44.6 IO. 19 49-3 48-9 27-5 43*6 II .22 47-3 53-8

265-o 45*4 21.69 45.6 100 18.2 24.4 7403 40.6 33.4 23*7 46.3 7.74 52.6 36.8

3943 126.5 10.30 74-4 48.8

265-o 45.4 21.67 45-9 100 18.2 24.4 7-05 40.6 33-5 23.7 46.3 7-78 52.6 37 -0 39.3 126.5 10.18 74-6 48-3

Llama

202.2‘ 40.0 25 l 97 202.2 40.0 25.76

16.4 43.1 8.51 20.9 41.4 10.32 22.0 40.4 12.52 26.6 45.2 14.23 32.0 46.6 17.52 41.9 36.6 20.99

52.4 48.1

49-4 49*4 49-I 43-2

100

I.00

3345 40.6

49-3 56.1 69.0 82.7

Camel

265-o 45.3 21.18 48.2 100

7-3 42-9 2.56 57*6 12.5

15.5 43-9 6.34 55.9 30-9 27-o 43*5 12.31 54-5 60.1 26.1 44.5 13-45 55-6 65-7

265-o 45.3 22 .oo 55*6 100

18.2 24.3 g-20 47*o 43.2 23.6 46.2 10.42 58.5 48.9 39.2 126.1 13.05 80.8 61 .I 28.6 50.6 12.86 58.7 60.3

265*0 45.3 21.87 54.8 100 18.2 24.3 9.08 46.6 42.9 23.6 46.2 10.40 58.2 49.3 39.2 126. I 12*77 79.9 60.1 61.1 39.7 18.69 49-8 88.0

265.0 45*4 22.70 39.8 100 8.1 42-9 I .51 44.9 8-4

15.8 43*9 3*27 44*2 14.8 16.6 44-6 8.58 43*3 38.8

27.5 43-6 8.76 43*o 39-6 265.0 45-3 23*‘9 39-4 100

18.3 24-4 5.29 33-9 23 -4 23-7 46.3 5-94 46*7 26.3

39*3 126.5 7.92 67*4 34.9 28.6 50*3 8.43 46.1 37*4

265 -0 45.3 23.17 39.4 100 18.3 24.4 5-34 3396 23*7 23-7 46.3 5-99 46.4 26.5 39*3 126.5 8.04 67-4 35*4 61.2 39.8 16.05 37.8 71-7

Yak

Deer

7-375 7-376 7*37o 7-361

7.559 7-379 7,048

7 *561 7 a379 7 -048

7-419 7 0402 7 -427 7-376 7.361 7.418

7 -448 7-427 7 -422 7.425

7 a633 7 *433 7 -091 7*39”

7.628 7.429 7 -085 7 -439

7 *550 7*335 7 -045

7 -552 7 -339 7 l o31

7-3’6 22.4 7.172 27.8 6.948 40.0

7*336 22.3 7.166 27.7 6-94s 40.2

7.581 7.218 19.3: 7.385 7.111 23.9 7*o75 6.917 34.7

7.581 7.218 19.7 7.390 7.120 23.8 7.075 G-923 35*I

7 0340 79326 I i 1

7.486 7 -327 7.002 7 -285

7 -482 7*325 7.001 7*313

7.441 7.230 26.2 7.266 7.106 32.5 6.985 6.900 47.7

7+443 7.230 25-I 7.266 7.106 32.4 G-990 6.897 47.3

TABLE 2 + (Continued)

African Elephant

100

40.8 64.2 78.4 96.8

The oxygen pressure necessary for half saturation of the hemoglobin (T& at the corresponding pH is also given.

separated at a pH of 8.6 using 520 v and 36 ma for 16 hr at 5 C.

Oxidation of hemoglobin with potassium ferricyanide. This method (I 3) measures a process closely related to the uptake of oxygen because conversion of the iron in hemoglobin to the ferric state depends upon the disso- ciation of 02 (or CO) from hemoglobin (I 4).

Activity of glucose 6-phusphate dehydrugenase. A method modified from Motulsky (15) was used.

OBSERVATIONS AND DISCUSSION

Data on the respiratory function of the blood can be interpreted with full validity only when the gas pressures and contents in arterial and mixed venous blood are known. It remains to be seen if such values can be ob- tained in these animals without narcosis.

Oxygen dissuciatiun curues. In Tables 2 and 3 and Fig. I, the most important data are presented. Among the mammals which live at high altitude, only the llama glama and llama vicuna have previously been studied (16, I 7). The high oxygen affinity of the blood in these animals has been likened to that of fetal blood (I 7) and clearly aids in the uptake of oxygen by blood in the lungs at high altitude. It has not been demonstrated, however, whether this increased oxygen affinity is an adaptation to high altitude or if these animals, because of previous high blood oxygen affinity, have been able to survive and succeed biologically in places of great height. The blood of the camel, which belongs to the same family as the llama, I-as a half-saturation pressure of 24. I mm Hg, relatively close to that measured in the llama. In contrast, the’ yak, an animal which lives in altitudes equally high as the llama, has a lower blood oxygen affinity, resembling that ol” humans (a TG0 value of 26 mm Hg) and its relative, the domestic cow. This association of evidence suggests that the shape of the oxygen disso- ciation curve of llama blood is a family characteristic which enables the species to live in high places rather than an adaptation to high-altitude living. When the shape of the curves is considered (Fig. I), the steepness of the upper part of the yak blood curve is a feature which would encourage oxygen uptake in the lungs under lowered oxygen partial pressure.

The lowest oxygen affinity (expressed as the Tso value)

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334 BARTELS ET AL.

TABLE 3. 02 pressure necessary for half saturation at a blood PH of 7.4 (TAO, pH, 7.4) and an erythrocyte pH of 7.2 (T50, PH. 7.2), oxygen cu#~ucity, standard bicarbonate (CO% content in uol% in fully oxygenated blood at a PC& of 40 mm Hg and 37 C), the Bohr e$ect expressed per unit change of blood pH and of erythrucyte PH, the ” effectiue” Bohr e$ect (additional oxygen released at the same T 60 value by acidifying the blood from PH 7.4 to 7.31, the Haldane e$ect, and PH of the red cell hemolysate at a blood pH of 7.4

T60 PHb 7.49

m m Hg

T60 PHC

Bohr Bohr Effect, Effect

A log PO:! A log pO2

AP&, AP&

Standard Bicarbonate,

vol % “Effective”

Bohr Effect PHC at PHI

7.4 7.2,

mm Hg

Yak 26.3 26.9 20.81 40.6 0.50 0.68 x.98 8.0 7.2I Llama 23-3 22.9 25 l 23 4245 0.42 0.76 I .70 6-4 7.20

Camel 24.1 20.3 20.86 46.2 o-49 0.84 I .48 9-8 7.11 Deer 27,6 274 22.20 34*3 0.56 0.78 2.46 7-8 7.2I

African elephant 22.4 =9*7 16.45 44*4 0.40 0.58 1 l 39 6-7 7.12

TABLE 4. Morphology of the blood of animals studied

RBC, mil-

lions/ mm3

Hct, MCV, MCH, % P3 PW

15.6 7-99 47-3 58.2 19.5 18.8 16* 79 41 .o 24.2 II .2

15.8 II .oa 35-2 31.9 14.3 16.7 I3*58 45-o 33-x 12.3 12.4 3.20 38.2 119.2 39.3

02 Ca- paci ty, vol %

--

20.9

25.2 21 .I

22.4 16.5

MS, MSA, MT P2 P2 P MD, lit MD (28)

.

I*99 6.12 5-4-6-25 I .05 5443; 7-45 x 4*21 7-56 x 4*o4 I-33 S-52; 7-46 x 4-10 8~3 X 4-32 I .6g 5.00 3*57-6*45 I .83 9.11 9-25

44J 29.2 97.3 61.6 23.2 6443 60.0 24.0 80.0 49-7 x9.6 65.8 43-2 65.2 182.7

Yak Llama Camel Deer African

elephant

Abbreviations are given in text, under Morphology of the blood.

TABLE 5. Electrolyte concentrations in the plasma and the erythrocytes

mEq/li ter mEq/liter Hz0

K % 520

91 l 32 69.48

91.88

56*3o

91.69 67.30

91 l 97 65*7o

Mg P K Cl Na

I5744 22.8

167.0 44-o

171-7 39-8

150.5 I4-4

Cl -

3*3

I .8 4.1

I .6 2-5

4*5 95.0 22.6 60.1

2.0

7-4

2.7 ro4. I 37.3 49-O

4*5 95-O 25.2 61.1

2.9 82. I 3542 37-I

Ca

148.0 4.9 3*9 107.0

Protein

15.3

=4*9

13.4

I7*4

=4*9

Na Mg

5.9 172.4 109.0 32.8 4*7

104. I 86.5

5*8 181.8 2.0 x13*4 70.0 78.2 7-3 87.0

4-9 187.2 I .8 103.6

135-5 59-I 3-7 go.8

6.1 I63*5 2.1 89*3 166.7 21 l g II .2 56-5

5-4 75-7

5.3 39-4

4-5 91 .I

5*6 1°9-5

5.2

Yak Plasma Red cells

Camel Plasma Red cells

Deer Plasma Red cells

African Plasma elephant Red cells

Llama Plasma

was found in the deer. Here there appears to be a parallel to the diminished oxygen affinity of human sickle erythro- cytes (I 8). The animal which we studied had sickle cells (I o % in the blood smear), as has been described for several varieties of deer. It must be pointed out, how- ever, that sickling in these animals is not related to low oxygen tension as in human drepanocytosis, but to alka- losis of the blood (I 9).

The highest oxygen affinity was shown by blood from the African elephant.

The suggested correlation between metabolic mass and oxygen affinity (I) is supported by our findings that the T50 value is lowest in the African elephant (2,300 kg)

and highest in the deer (35 kg). Although our other subjects were of intermediate weight and had inter- mediate Tco values, there was no regular correlation among them between T 50 and body weight. However, the general rule of increasing oxygen affinity with in- creasing body size finds general support from this work.

Oxygen capacity. We found a high oxygen capacity in the llama in agreement with Hall and co-workers (16) in llamas at sea level. In the single animal which they studied at high altitude they found a decrease of oxygen capacity with increasing altitude. Some confirmation of this remarkable finding has been given by Meschia et al. (I cl), who found an oxygen capacity of only about I 3.5

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RESPIRATORY FUNCTIONS OF BLOOD

vol % in a llama at Morococha (altitude 15,000 ft). However, their animal was pregnant.

The oxygen capacities of yak, camel, and deer blood lie in the area of 20-22 ~01%. Only the African elephant has an appreciably lower value with I 6 ~01%. Despite the reservation mentioned above, we believe this finding to be valid since three values showed only small varia- tion. Lang and Undritz (20) showed that the circus elephant frequently has an anemia which can be cor- rected by the administration of iron. The animal which we studied had been treated in this way, so the low blood oxygen capacity can be regarded as physiological.

“Standdrd bicarbonate” content of the blood. This value was about the same for all the animals except the deer. The lower value seen in the deer could have resulted from the great difficulty experienced in obtaining blood. The great motor activity before the puncture was ac- complished would probably lower the carbon dioxide binding capacity (and increase the oxygen capacity).

T/ze Bohr e$ect. This is of about the same size in the camel and the yak, smallest in the African elephant, and largest in the deer. The correlation between the magni- tude of the Bohr effect and body weight which Riggs (2 I ) found in hemoglobin solutions does not appear in our results (P = 0.5). Furthermore, a quantitative estima- tion of the Bohr effect with regard to its efficiency in the delivery of oxygen to the tissue requires, in addition to knowledge of the Bohr effect, an understanding of its interaction with the oxygen capacity and the oxygen dissociation curve (5). One sees (Table 3), for example, that with practically identical Bohr effects in the camel and the yak, the blood of the yak can deliver consider- ably more oxygen by acidification of o. I pH unit, largely because of a steeper oxygen dissociation curve. The blood of the deer has functionally the highest potential because of both a high oxygen capacity and a moderately steep oxygen dissociation curve. Therefore, the single value A log pOZ/A pH tells us relatively little. In general, one can say that with the same Bohr effect, its efficiency in promoting oxygen delivery increases with increasing oxygen capacity and increasing steepness of the oxygen dissociation curve.

Tize Haldane effect. The values obtained in the llama, yak, deer, and African elephant lie near values which have been found in humans (approximately 6 ~01%). Camel blood contains IO ~01% more CO2 at a pCO2 of 4o mm Hg when it is completely reduced than when it is completely saturated with oxygen.

Morphology of the red blood cells. See Table 4. The num- ber of erythrocytes varied between 3.2 (African elephant) and 16.8 (llama) million/mm3. The erythrocyte volume varied in inverse ratio to the number of erythrocytes: I 20 p3 in the African elephant and 25 p3 in the llama. In previously studied mammalian erythrocytes the mean corpuscular hemoglobin concentration is remarkably constant at values between 2g and 35% (22). In the family Camelidae (the camel and the llama), we found 50 % higher concentrations. (Wintrobe (23) gives a hemoglobin concentration of 4o % in llama erythrocytes

0 I 2 3 4

FIG. 2. Speed of oxidation of hemoglobin with potassium ferri- cyanide at pH 7.0 and room temperature. The ordinate is the logarithm of the ratio CO/C, (the amount of oxyhemoglobin at the beginning of the experiment divided by the amount of oxyhemo- globin at the time represented).

on the basis of calorimetric measurements.) The oxygen binding capacity can also be related to the erythrocyte volume (Oz/Vc ratio) (24). Again, 50 % more oxygen can be taken up per unit volume of cells in the Camelidae than in other mammals. The belief that we are dealing with a higher hemoglobin concentration in the erythro- cytes is further supported by the lower cell water content (Table 5) in these animals. These findings are especially remarkable since Perutz (25) has given 34 % as the high- est possible hemoglobin concentration in the erythro- cytes.

The thickness of the red cells is greatest in the yak and least in the Tylopoda. Our observations give no support to the finding (26) that with increasing thickness of the red cells there is increasing oxygen affinity.

(22). A connection between the oxygen affinity and the intra- or from our

extracellular studies.

electrolytes cannot be established

Starch block electrophoresis. Hemoglobin travels with in- creasing speed in the following order: camel, yak, deer, and African elephant. Llama hemoglobin travels slowly at a speed similar to camel hemoglobin, but a strict simultaneous comparison of its migration speed with the other species was not carried out. Only in the yak does the pigment separate into two equally large components.

Oxidation speed. See Fig. 2. The speed is greatest for camel and yak blood and least in the blood from the African elephant. In the deer and camel the oxidation behaves, as in the human, like a monomolecular reac- tion. Yak, llama, and African elephant hemoglobin each

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336 BARTELS ET AL.

behave like a mixture of two hemoglobins with different reaction speeds.

Activity of glucose 6-phos$ate dehydrogenase. In compari- son to human erythrocytes which are taken as IOO %,

the activity in the erythrocytes of the llama is greater than 200 %, camel 165 %, African elephant 55 %, deer 40 %, and yak o %. It is noteworthy that the yak erythro- cytes are the thickest, while erythrocytes from the llama

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