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Biological Research

versión impresa ISSN 0716-9760

Biol. Res. v.33 n.1 Santiago  2000 

Hemoglobin affinity for oxygen in three subspecies of toads (Bufo sp.) living at different altitudes


1 Clinicum Laboratorio Automatizado, Iquique, Chile
2 Laboratorio de Transporte de Oxígeno, Universidad Peruana Cayetano Heredia, Lima, Perú
3 Universidad Arturo Prat, Iquique, Chile


Blood oxygen affinity and red blood cell properties were measured in three subspecies of genus Bufo: Bufo spinulosus limensis, collected at sea level and at an average day temperature of 20ºC; Bufo spinulosus trifolium, from 3100 m, average day temperature of 15ºC; and Bufo spinulosus flavolineatus, from 4100 m, average day temperature of 10ºC. Electrophoresis of the hemoglobin showed the same component in each of the three subspecies. At 20ºC the blood oxygen affinities (P50) showed small differences between Bufo spinulosus limensis and Bufo spinulosus trifolium, whereas the value for Bufo spinulosus flavolineatus was markedly lower. At 10ºC, the ambient temperature of Bufo spinulosus flavolineatus, the P50 was extremely low compared with the other two subspecies at their corresponding ambient temperatures.

KEY WORDS: adaptations, altitude, amphibians, Bufo spinulosus limensis, Bufo spinulosus trifolium, Bufo spinulosus flavolineatus, hemoglobin affinity, hematocrits, oxygen binding, toads


In Peru the genus Bufo occupies an altitudinal gradient ranging from sea level to above 4000 m. The species Bufo spinulosus has three subspecies whose niches are at low, intermediate and high altitudes. These subspecies are Bufo spinulosus limensis (low altitude), Bufo spinulosus trifolium (intermediate altitude) and Bufo spinulosus flavolineatus (high altitude) (12). One of the most intensively studied environmental adaptations of the respiratory system in vertebrates is that of hypoxia, which occurs variously at high altitude, in embryonic development (e.g., in utero) and in aquatic, particularly fresh water, environments. These adaptations commonly involve changes in the properties of the red cells, in the oxygen-carrying capacity, or in the oxygen affinity of the hemoglobin. Intraspecific adaptations, which occur in the same animal under different conditions, generally involve changes in allosteric factors (i.e., the conditions under which the hemoglobin functions inside the red cells), whereas interspecific adaptations, which are evident when comparing different species, are attributable to changes in the properties of the hemoglobin molecules.

Most studies on hypoxic adaptations in red cell and hemoglobin properties refer to fish and mammals, and very little is known about lower ectothermic vertebrates. An earlier study showed that the Lake Titicaca frog Telmatobius culeus (2) living at 3800 m. had higher blood O2 affinity and smaller blood cells than sea level frogs. This study describes the adaptations in Bufo subspecies.


Anurans of the subspecies Bufo spinulosus limensis were collected from the coastal plains at approximately 400 km from our laboratory in Lima (sea level, average day temperature 20ºC). Bufo spinulosus trifolium were collected in the Central Peruvian Andes (about 3100 m, average day temperature 15ºC), and Bufo spinulosus flavolineatus also in the Central Andes (at 4100 m, average day temperature 10ºC). The animals were brought to our laboratory within a few hours. Blood was drawn in heparinized syringes from the hearts of previously-pithed animals. Approximately 3 ml of blood could be obtained from Bufo spinulosus limensis and 1 to 1.5 ml from the other subspecies. Blood from two to three animals were pooled in order to carry out the determinations. Red blood cell counts, hemoglobin concentration and hematocrits were measured by conventional techniques.

Electrophoresis of the hemoglobin was run on cellulose acetate strips in a buffer specific for hemoglobin electrophoresis, and the bands were stained with Ponceau S and read in a densitometer according to the Helena Laboratories (USA) instruction manual.

The blood-oxygen affinity (P50) was measured by the mixing technique (1,10) at 10ºC, 15ºC and 20ºC. The blood equilibration was carried out in a tonometer maintained at these temperatures using gas mixtures containing 4.5% CO2 and variable O2 and N2 concentrations. Blood O2 saturation varied between 30 and 80%. The pH meter was calibrated using high-precision phosphate buffers.

The pH values at the equilibrium temperatures (eq) were calculated from those at measurement temperatures (meas) using the equation given by Rosenthal (8):
pHeq = pH meas + 0.0147 (Teq _ Tmeas)

The Bohr factor was obtained by changing the blood pH as follows. Plasma prepared from blood aliquots was either acidified with lactic acid or alkalinized with sodium hydroxide and reconstituted with the corresponding red blood cells. The blood samples from Bufo spinulosus limensis and Bufo spinulosus trifolium were equilibrated at 20ºC and 15ºC and the blood from Bufo spinulosus flavolineatus at 20ºC and 10ºC and at different pH values in order to calculate the Bohr factor for each temperature (14). This factor allowed the calculation of the standard blood pH at the three temperatures. In order to calculate the Hill's coefficient (n), a method of successive approximations was used (10). A value from the Bohr equation for each subspecies was calculated from data in separate experiments.

The van't Hoff equation was used to calculate the overall heat of oxygenation DH' from the P50 values. For Bufo spinulosus limensis values at 20ºC and 15ºC were used taking 20ºC as the ambient temperature. For Bufo spinulosus trifolium the values at 15ºC and 20ºC were used taking 15ºC as the ambient temperature. For Bufo spinulosus flavolineatus the values at 10ºC and 20ºC were used taking 10ºC as the ambient temperature. The statistical significance was determined by Student's t test with software Systat 6.0 for Windows.


Tables I and II summarize the hematological data. There is no statistical difference between the values of Bufo spinulosus limensis and Bufo spinulosus trifolium. The values of the hematocrit, hemoglobin, and the quantity of RBC in Bufo spinulosus flavolineatus are much lower.

The electrophoresis experiments showed the same migration distances in the hemoglobins of each of the three subspecies and the presence of two bands in some of the runs (Fig. 4). No further studies were carried out to define the hemoglobin properties.

Table II shows the functional properties of the hemoglobin in blood, revealing small differences in the P50 between Bufo spinulosus limensis and Bufo spinulosus trifolium, whereas the value for Bufo spinulosus flavolineatus is lower. The values at the ambient temperature where the animals were collected did not differ significantly between Bufo spinulosus limensis and Bufo spinulosus trifolium. In the case of Bufo spinulosus flavolineatus, at 10ºC the P50 value is very low. Figure 1 shows a plot of log P50 vs. pH. Figure 2 shows hemoglobin oxygen dissociation curves constructed with the values of P50 and Hill's coefficient at 20º C. The curves for Bufo spinulosus limensis and Bufo spinulosus trifolium are very close to each other, whereas that for the Bufo spinulosus flavolineatus is shifted to the left. Figure 3 is a plot of blood O2 content vs. blood PO2 at the ambient temperature of each subspecies. This shows the low O2 content and very low P50 of Bufo spinulosus flavolineatus and a moderate displacement to the left of Bufo spinulosus trifolium compared to Bufo spinulosus limensis with no reduction of the blood O2 content in Bufo spinulosus limensis and Bufo spinulosus trifolium.

Fig. 1: Bohr effect in three subspecies of Bufo spinulosus. Bufo spinulosus flavolineatus shows the largest Bohr factor.

Fig. 2: Blooddissociations curve for the three subspecies at pH of 7.84 and 20º C. The curve of the Bufo spinulosus flavolineatus is displaced to the left.

Fig. 3: Hemoglobin O2 content vs partial oxygen pressure for each subspeceis at the respective ecological temperature. Bufo Spinulosus limensis (20º C), Bufo spinulosus trifolium (15º C)Bufo spinulosus flavolineatus (10º C). The hemoglobin content of Bufo spinulosus flavolineatus is lower than in the two other subspecies and the P50 is very low.

Fig. 4: Electrophoresis of the three subspecies of Bufo Spinulosus. The experiment shows the same migrations distances in the hemoglobins of each of the three subspecies and the presence of two bands in some of the runs.

These results show that at 20ºC Bufo spinulosus flavolineatus, whose natural habitat is close to 4000 m and whose ambient temperature averages 10ºC, has a low hemoglobin content and a high hemoglobin affinity when compared to Bufo spinulosus trifolium, whose average ambient altitude is close to 3000 m and whose ambient average temperature is 15ºC. Bufo spinulosus limensis (sea level) and Bufo spinulosus trifolium had almost identical curves. If the curves were obtained at the ambient temperatures, the Bufo spinulosus flavolineatus showed a very low O2 content and high hemoglobin affinity compared to the other two subspecies. In Bufo spinulosus flavolineatus, the high DH' indicates a high sensitivity of the hemoglobin affinity to changes in ambient temperature, and the high Bohr factor also indicates a high sensitivity of hemoglobin affinity to changes in blood pH.


The genus Bufo is widespread in Andean countries. Bufo spinulosus has three subspecies whose niches are located in the lower Andean slopes, the intermediate valleys (3000 m) and the highest plains (above 4000 m). The intermediate altitude subspecies Bufo spinulosus trifolium and the sea level subspecies Bufo spinulosus limensis do not differ in their hematological values. The high altitude Bufo spinulosus flavolineatus has a much lower hemoglobin concentration, but the red cells characteristics are similar to the other two subspecies. The RBCs appear to be smaller than those in the other two subspecies, unlike Ruiz's (9) findings, which indicated the opposite. It is known that the genus Bufo has a higher hemoglobin concentration than other amphibians. Hutchinson and Szarki (3) attribute this to the high vascularization and terrestrial habit. However, Bufo spinulosus flavolineatus has very low hemoglobin concentration.

In the three groups, the hemoglobin electrophoretic study shows individuals whose electrophoretic pattern present two clearly separate hemoglobin bands. There is no difference in the hemoglobin affinity between Bufo spinulosus trifolium and Bufo spinulosus limensis at 20ºC, whereas that in the Bufo spinulosus flavolineatus is higher. When the affinities were studied at the ambient temperatures where the animals live, Bufo spinulosus trifolium had oxygen dissociation curves that shifted moderately to the left of Bufo spinulosus limensis, and Bufo spinulosus flavolineatus showed a very marked shift, indicating a very high hemoglobin affinity.

High altitude native mammals and birds tend to have high blood oxygen affinities and not to show polycythemia at high altitude (5). It would be interesting to observe whether these responses to ambient hypoxia and cold conditions are present in ectothermic animals. In theory, cold conditions should help to increase the tolerance to high altitude because it decreases the metabolic rate. In the case of Bufo spinulosus flavolineatus,whose day temperature under rocks at 4100 m is about 10ºC and about 6ºC or less at night in the ponds where they rest half immersed, the high affinity indicates selection pressure towards hypoxia despite a possible reduction in metabolic rate. Packard and Stiverson (7) did not find a relationship between altitude and hemoglobin concentration in the chorus frog (Pseudacris triseriata), but the altitude range in their study (1500 m and 3000 m) was small.

The hemoglobin affinity is a genetic mark apparently selected by the hypoxic environment in vertebrates. In mammals and birds the affinity is a rather fixed property that does not increase in hypoxia and does not disappear in normoxia (6). In fish, on the other hand, blood oxygen increases markedly under hypoxia (13).

From a comparative point of view, there are some similarities between ectotherms and endotherms. It has been found that there is some correlation between hemoglobin affinity and altitude in subspecies of mice occupying an altitudinal gradient (11). It has also been shown that within the same mammalian species living along a vertical gradient in the Andes, individuals living in the highest range have hemoglobins with higher affinity than individuals living in the lowest range. These differences were greater in the case of the Andean fox, where two separate species occupy the two extremes of the altitude range (4).

This work reinforces the concept that polycythemia is not selected as an adaptive parameter to the hypoxic environment. On the contrary, high-affinity hemoglobin seems to be strongly selected in a variety of animals living at high altitude.


We thank Dr. Roy E. Weber for his help in clarifying our thinking on this subject and for reviewing the manuscript.

Corresponding Author: Hrvoj Ostojic P. - Clinicum Laboratorio Automatizado, Casilla 169, Iquique, Chile. Telephone: (56-57) 420599, Fax: (56-57) 427683, e-mail:

Received: August 13, 1999. Accepted in revised form: May 6, 2000


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