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Temperature and pH effects on the oxygen equilibrium curve of the thoroughbred horse
RESPIRATION PHYSIOLOGY ELSEVIER
Respiration Physiology 97 (1994) 293-300
Temperature and pH effects on the oxygen equilibrium curve of the thoroughbred horse K. Smale a'b a n d P.J. Butler b'* aDepartment of Comparative Phystology, Animal Health Trust, Newmarket, Suffolk, UK USchool of Biological Sciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK Accepted 21 March 1994
Abstract A new oxygen equilibrium curve is defined for the Thoroughbred horse under standard conditions of 37 °C, pH = 7.4 and Pco2 = 5.33 kPa. The "standard" Pso for the Thoroughbred is, at 2.83 + 0.04 (SE of mean) kPa, significantly lower than that found for the Hanoverian horse (3.17 + 0.03 kPa) by Clerbaux et al. (Can. J. Vet. Res. 50: 188-192, 1986), and lower than other values for horses in the literature. Using data from Butler et al. (J. Exp. Biol. 179:159-180, 1993), curves were also constructed, in vitro, under simulated conditions of intense exercise to examine the individual effects ofpH, temperature and Pc% on the standard curve. The fixed acid Bohr coefficient is similar to that in humans (-0.41) whereas the temperature coefficient is, at 0.019, lower than that for humans. The coefficients were shown to be saturation dependent. Keywords: Blood, 02 transport, horse, comparative; Bohr effect, blood, horse; Mammals, horse, Thoroughbred vs Hanoverian
1. Introduction Exercise-induced arterial hypoxaemia (EIH) has now been accepted as a normal response to exercise in the performance horse (Persson et aL, 1987; Littlejohn and Snow, 1988; Butler et al., 1993). It could be considered, therefore, that EIH might restrict the exercise potential of the Thoroughbred racehorse. However, the thoroughbred shows a marked increase in haemoglobin concentration with exercise - the equine spleen acting as a reservoir of erythrocytes which is released during exercise or under conditions of stress, via adrenergically induced contraction of the smooth muscle of the splenic capsule and trabeculae (Persson, 1967). Under intense exercise conditions, the oxygen carrying capacity has been shown to increase by more than 50% (Butler et aL, 1993). This could be a major adaptation to exercise in the Thoroughbred. * Corresponding author. Tel:(0)21 414 5470; Fax: (0)21 414 5925. 0034-5687/94/$7.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 3 4 - 5 6 8 7 ( 9 4 ) 0 0 0 3 7 - Z
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K. Smale and P.J. Butler / Respiration Physiology 97 (1994) 293-300
The relationship between Po: and oxygen content has been studied widely for human blood, resulting in several algorithms to describe the OEC. (Hill, 1910; Adair, 1925; Kelman, 1966; Severinghaus, 1979). Data regarding haemoglobin-oxygen binding in the horse are very limited and, as the relationship between the human and Thoroughbred OEC has not been established, these "human" nomograms cannot be used for blood from Thoroughbred horses with full confidence. In order to establish this relationship, the influence of temperature, pH and Pco2 on the shape and position of the OEC were determined for the blood of the Thoroughbred. Curves were constructed under "standard" conditions of 37 °C, pH 7.4 and Pco~ = 5.33 kPa, as well as under the conditions determined in horses at maximum exercise during a standard exercise test on a treadmill (Butler et al., 1993).
2. M e t h o d s
The pool of experimental animals consisted of 14 Thoroughbred horses (three fillies and 11 geldings, weighing approximately 450 kg), aged between four and 17 years. Blood samples were taken from the jugular vein by needle [21-gauge, Monoject, Sherwood Medical, Crawley, UK) into heparinised (heparin sodium injection BP (mucous), 5000 units in 1 ml, Evans Medical, Sussex, UK)] polypropylene syringes and used immediately for the construction of equilibrium curves. Mixing pumps, (2M301/1-f, WOsthoff, Bochum, Germany) were used to create gas mixtures of oxygen and carbon dioxide with high purity nitrogen (Air products, Ipswich, UK] as the balancing gas. The required gas mixture was passed through a spinning tonometer (IL237, Instrumentation Laboratory, Milan) at a rate of approximately 300 ml.min -~ where it was humidified before equilibrating with the spinning blood. Blood samples were spun for at least fifteen minutes at each gas mixture which ensured equilibration, although a longer time was required for initial deoxygenation and for a larger sample volume. Packed cell volume (PCV) was measured by the micro-haematocrit method. Haemoglobin was converted to cyanmethaemoglobin and measured spectrophotometrically using a haemoglobinometer (Coulter Electronics Ltd, Reading, UK). Haemoglobin concentration in plasma before and after tonometry was below 0.008 raM, confirming that the action of the tonometer did not cause haemolysis. For the analysis of 2,3-bisphosphoglycerate (2,3-BPG), venous blood (1 ml) was added to 3 ml cold trichloroacetic acid (8~o w/v), shaken and kept on ice until the sample was spun at 4000 rpm for 4 min. The pellet was then discarded and the supernatant stored at -20 °C. Analysis was performed using a Sigma kit (procedure number 665, Sigma UK). In 28 samples, 2,3-BPG concentration was measured before and after tonometry and showed no significant change (P<0.05). Mean 2,3-BPG concentration was 14.7 + 0.2 gmol (g Hb) -l. Oxygen content was measured by the method of Tucker (1967), and pH measurements were performed by a blood gas analyser thermostatted at 37 °C (ABL330, Radiometer, Copenhagen, Denmark). The ABL 330 has an inbuilt temperature corAnimals and blood collection
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rection facility which is sufficiently accurate for pH of horse blood (Butler et al., 1991). Po2 and Pco2 were calculated from the composition of the equilibrating gas, barometric pressure, water vapour pressure etc. A single OEC was constructed by completely desaturating the blood and then slowly resaturating it by increasing the oxygen concentration in the gas mixture. Samples were taken at points along the resaturation curve, and curves were originally run in both directions to ensure identical results. For each curve, temperature, pH, P¢o2 and haemoglobin concentration were recorded. Nine levels of oxygen were used for each curve up to a maximum of 25 ~o. The latter gave the same amount of oxygen combined to haemoglobin (CHbo:) as 30 ~o 02, thus 100 ~o saturation of haemoglobin with oxygen was measured using a gas mixture containing 25 ~o 02. Samples were withdrawn from the tonometer, using a syringe which had been flushed with the circulating gas mixture, and introduced into the blood gas analyser and the Tucker apparatus. Total 02 content is the sum of the oxygen dissolved in the plasma (% x Po2, where % is the solubility of O2 in the plasma) and that combined with the haemoglobin: Co = CHbo + ( ~ x Po ). Dissolved oxygen [% x Po2, where ~p = 0.000031 ml O2.m1-1 mmHg-1 (Aclamsand I~Iahn, 1979)] was subtracted from the total 02 content. Percent saturation was then calculated as: CHbo2/(CHb o . . . . x 100, where CHbo2ma x is the maximum amount of oxygen that can combine chemically with haemoglobin. The effect of temperature, p H and P C o 2 on the standard O E C Standard arterial conditions were taken as 37 °C, pH = 7.4 and P¢o~ = 5.33 kPa. The effects of temperature, pH, and Pco2 were examined within the physiological ranges observed during exercise (Butler et al., 1991; Butler et al., 1993): 37-41°C, pH 7.01-7.4 and P¢o: 6.3-16.9 kPa. Curves were drawn by hand to determine the temperature coefficient (TC), the fixed acid Bohr coefficient (BC) and the effect of CO 2 (CO2 effect) on the OEC. The effect of temperature was examined by altering the temperature of the waterbaths surrounding the tonometer. Three temperatures were used: 37, 39 and 41 °C. The temperature coefficient was calculated as blog Ps0/bT, at constant Pco, (mean pH was taken over the OEC). The fixed acid Bohr coefficient was determined by adding 0.1 mol.L -I HC1 to the blood sample until the required pH was achieved in the tonometer, and calculated as Slog Ps0/cSpH. The CO 2 effect was determined by increasing the P¢o2 in the gas mixture but no attempt was made to keep the pH constant. However, the subtraction of the fixed acid coefficient from the CO2 effect (calculated as Slog Ps0/~SpH) gives the net CO z coefficient. The effect of the lactate anion was also investigated. Two concentrations of sodium lactate (DL-lactic acid, sodium salt; Sigma Chemical) were used, 10 and 20 mmol.L -1, to represent levels of whole blood lactate found in the horses during medium and high intensity exercise (Butler et al., 1993). Curves were also constructed where lactic acid [L( + )lactate; Sigma Chemical, UK] was used to alter pH in order to mimic the in vivo conditions. As no change in 2,3-BPG concentration was detected in these horses during exercise, the effects of 2,3-BPG on the OEC or fixed acid Bohr coefficient were not determined. The effect of haemoglobin concentration [Hb] was examined by allowing a blood sample to settle, on ice, removing sufficient plasma to increase the [Hb] by 50-100~o,
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remixing the sample and then constructing the OEC. Hill's 'n'-value was calculated as the slope of the line given by plotting log [y/(100 - y)] against log P (where y = ~o saturation and P = Po2), from 20-80~o saturation. Trend analysis (analysis of variance) was used to determine if there was a significant saturation dependence of the temperature and Bohr coefficients. Mean values are give + SE of mean and the Student 't'-test was used to determine any significant difference between two mean values. P < 0.05 was used as the level of significance.
3. Results
The standard Ps0 for the Thoroughbred horse (2.83 + 0.04 kPa) is significantly (P<0.001) lower than that reported for the Hanoverian horse (3.17 + 0.03 kPa) and lower than that for humans (Fig. 1). The amount of oxygen combined to Hb at 100~o saturation ( 0 2 combining capacity) was 1.397 _+0.008 ml.g -1. When calculated at Ps0,
100 Thoroughbred
(this study) \ ~
80 l--
Hanoverian
co
°~ ..II--
cl
horse
~Humon 60
-I ,,41--
CS =I,--
c w
~0
O..
T = 37°C
20
pH= 7.t, PE0 = 5.33 kPa
0 t_~-.~ I
0
,
I
2
,
I
l,
,
I
6
,
I
8
,
I
10
,
I
J
12
Pcxrfial pressure of oxygen (kPa) Fig. 1. Oxygen equilibrium curves under standard conditions (37 °C, pH 7.4, Pco_, 5.33 kPa) for the Thoroughbred racehorse (present study, []), Hanoverian horses (Clerbaux et al., 1986, /X) and humans (Kelman, 1966, O).
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297
Table 1 M e a n ( _+ S E M ) v a l u e s o f t h e t e m p e r a t u r e coefficient ( T C ) t h e fixed a c i d B o h r coefficient ( B C ) a n d t h e C O 2 saturation
effect for t h e b l o o d o f T h o r o u g h b r e d r a c e h o r s e , f r o m 1 0 - 9 0 % °J % Saturation
TC
BC
C O o effect
10 20 30 40 50 60 70 80
0.012__+0.003 0.013 -+ 0.002 0 . 0 2 0 -+ 0 . 0 0 2 0.022 + 0.001 0.019_+0.001 0.023 + 0.003 0.020 _+ 0.003 0 . 0 2 0 +__0.003
-0.10+0.07 - 0.25 + 0.06 - 0.36 -+ 0.06 - 0.41 -+ 0.06 -0.41 +0.07 - 0.44 _+ 0.07 - 0.46 + 0.07 - 0.40 -+ 0 . 0 9
- 0.46 - 0.48 - 0.53 - 0.53 -0.51 - 0.50 - 0.50 - 0.48
90
0.021 -+ 0.003
- 0.24 _+ 0.08
- 0.35 _+ 0.05
-+ 0.02 + 0.02 __+0.01 + 0.01 _+0.02 + 0.02 _+ 0.02 -+ 0.04
the values for TC, BC and the CO2 effect were 0.019+0.001; -0.41 +0.07, and -0.51 + 0.02, respectively. There was no difference between the BCs calculated from curves where pH had been altered by lactic acid as opposed to HC1. Thus, no effect of the lactate anion on the OEC was noted. However, the coefficients proved to be saturation-dependent. When calculated as 61og Po2/~5{factor} for the full range of saturation values, a significant trend of saturation-dependence appeared, see Table 1. TC was lower at low levels of saturation, particularly below 30~o saturation. Both the BC and the CO2 effect were significantly saturation dependent: (a) BC was dependent upon saturation at the extreme ends of the range, where it was less negative than in the middle of the saturation range; and (b) the CO 2 effect was more negative at low levels of saturation. Haemoglobin concentration had no effect on the position or the shape of the curve and there was no significant difference in the value of Hill's n with the different treatments, i.e., temperature, pH or Pco2. The overall mean value for Hill's n was 2.54 + 0.04.
4. Discussion
The Ps0 for the Thoroughbred horse in this study (2.83 kPa) is lower than that obtained from previous studies on the horse, at standard conditions of 37 °C, pH = 7.4 and Pco2 = 5.33 kPa (3.33 kPa, Kitchen and Bunn, 1975; Bunn and Kitchen, 1973), (3.07 kPa, Schmidt-Nielsen and Larimer, 1958), 3.47 kPa, Kitchen and Bunn, 1975), (3.17 kPa, Clerbaux et al., 1986), (3.24 kPa, Lykkeboe et al., 1977). The first seven studies did not specify the breed of horse whereas the study by Clerbaux et al. (1986) used the Hanoverian horse and the study by Lykkeboe et al. (1977) used trotting horses, which are assumed to be Standardbreds. These data indicate that the haemoglobin of the Thoroughbred horse has a greater affinity for oxygen than that of other breeds. A greater haemoblobin-oxygen affinity means that at those values of Po2 where the Thoroughbred may be considered hypoxaemic, the actual percent saturation is greater than it would be for humans or for the Hanoverian horse. Whether this can be seen
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as any kind of adaptation to intense exercise is blurred by the significant increase in haemoglobin concentration during exercise which means that, whatever the Po2, particularly on the arterial side, the Thoroughbred has a much greater oxygen content than either humans or the Hanoverian horse. Thus, at the low Po: values seen during exercise (Butler et al., 1993), the increase in haemoglobin concentration may be seen as a positive adaptation enabling sufficient oxygen supply to the exercising muscles. Resting 2,3-BPG concentration was lower than that reported for the Hanoverian horse by Clerbaux et al. (1986) of 16.9 + 1.1 #mol.g Hb -1, so it is unlikely that the difference in haemoglobin-oxygen affinity between these two breeds is caused by 2,3BPG. Although it is well known that 2,3-BPG concentration effects both the fixed acid and the CO2 Bohr coefficients (Arturson et al., 1974), any small variation around the normal level should not have introduced any appreciable error (Hlastala and Woodson, 1975; Kwant et al., 1988). The fact that increasing the haemoglobin concentration had no effect on the OEC is important to note, because of the large increase in haemoglobin concentration that occurs during exercise in the thoroughbred. The TC is comparable to those of the Hanoverian horse and the pig although it is lower than that generally found in humans. BC is similar to that reported for humans. Although both coefficients showed a marked saturation dependence, there are marked differences in the amount of saturation dependency for the different coefficients reported in the literature for various species. A greater TC at low saturation levels has been reported in humans (Hlastala et al., 1977) and the pig (Willford and Hill, 1986), whereas Reeves (1980) and Boning et al. (1978) found that TC in humans did not vary with saturation. However, Boning et al. (1978) did find that TC was significantly higher in men than in women. It is unclear why the TC should be lower at low saturations in the horse as found this study and that of Clerbaux et al. (1986), but higher at low saturations in humans. In humans, varying degrees of saturation dependency of the BC have been reported by several authors (Hlastala and Woodson, 1975; Garby et al., 1972; Arturson et al., 1974; Meier et al., 1974; Kwant et al., 1988). For example, Boning et al. (1978) found a saturation dependency of the fixed acid Bohr coefficient that could be approximated by a second order polynomial, with the maximum values between 40 and 700/0 saturation, Meier et al. (1974) reported that the fixed acid Bohr coefficient was maximum at the middle range of saturation, Hlastala and Woodson (1975) and Hlastala et al. (1977) reported a rise in the BC of 15~o between 50 and 10~o saturation, Garby et al. (1972) and Arturson et al. (1974) reported rises of approximately 40~o between 50 and 10~o saturation, Meier et al. (1974) reported that BC increased still further at saturations higher than 50~o, whereas Hlastala and Woodson (1975) did not find any significant effect of saturation on the BC. Willford and Hill (1986) found no specific saturation dependence on the BC in the pig. The variance of the BC according to saturation lends weight to the suggestion that the four 0 2 binding sites in the haemoglobin molecule are not equivalent in terms of behaviour towards the N + ion, i.e., there is a variable number of protons released per molecule of oxygen bound, dependant upon the subunit. Imai and Yonetani (1975) suggested the individual subunit coefficients for the BC and TC in humans varied as follows: (BC) -0.46, -0.65, -1.09, -0.08; (TC) 0.023, 0.015, 0.018, 0.040 at pH 7.2
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in haemoglobin solutions and without 2,3-BPG. Therefore it seems likely that the differences in Bohr coefficients between species could be explained by variations in the binding of individual subunits. It has also been suggested that the saturation dependence of the BC could be due to interaction of Hb with 2,3-BPG and CO 2 under physiological conditions and the pH dependence of their binding constants. The fact that the fixed acid coefficient is identical to that in humans lends weight to the idea that a major adaptation to exercise in the Thoroughbred is the increase in haemoglobin concentration.
Acknowledgement K.S. was in receipt of a SERC CASE award and the authors are grateful to the Horserace Betting Levy Board and Sheikh Mohammed al Maktoum for their generous support.
References Adair, G.S. (1925). The haemoglobin system. VI. The oxygen dissociation curve of haemoglobin. J. Biol. Chem. 65: 529-545. Adams, A.P. and C.E.W. Hahn (1979). Principles and Practice of Blood-Gas Analysis, London: Franklin Scientific Projects Ltd. Arturson, G., L. Garby, M. Robert and B. Zaar (1974). The oxygen dissociation curve of normal human blood with reference to the influence of physiological effector ligands. Scand. J. Clin. Lab. Invest. 34: 9-13. Boning, D., W. Druade, F. Trost and U. Meier (1978). Interrelation between Bohr and temperature effects on the oxygen dissociation curve in men and women. Respir. Physiol. 34: 195-207. Bunn, H.F. and H. Kitchen (1973). Haemoglobin function in the horse; the role of 2,3-Diphosphoglycerate in modifying the oxygen affinity of maternal and fetal blood. Blood 42: 471-479. Butler, P.J., A.J. Woakes, L.S. Anderson, K. Smale, C.A. Roberts and D.H. Snow (1991). The effect of cessation of training on cardiorespiratory variables during exercise. Equine Exercise Physiology 3, edited by S.G.B. Persson, A. Lindholm and LB. Jeffcott, Davis, CA: ICEEP Publications, pp. 71-76. Butler, P.J., A.J. Woakes, K. Smale, C.A. Roberts, C.J. Hillidge, D.H. Snow and D.J. Marlin (1993). Respiratory and cardiovascular adjustments during exercise of increasing intensity and during recovery in thoroughbred racehorses. J. Exp. Biol. 179: 159-180. Clerbaux, Th., D. Serteyn, E. Willems and L. Brasseur (1986). D6termination de la courbe de dissociation standard de l'oxyh6moglobine du cheval et influence, sur cette courbe, de la temp6rature, du pH et du diphosphoglyc6rate. Can. J. Vet. Res. 50 188-192. Garby, L., M. Robert and B. Zaar (1972). Proton- and carbamino-linked oxygen affinity of normal human blood. Acta Physiol. Scan& 84: 482-492. Hill, A.W. (1910). The possible effects of the aggregation of the molecules of haemoglobin on its dissociation curve. J. Physiol. (London) 40: iv-vii. Hlastala, M.P. and R.D. Woodson (1975). Saturation dependency of the Bohr effect: interactions among H +, Co, and DPG, J. Appl. Physiol. 38: 1126-1131. Hlastala, M.P., R.D. Woodson and B. Wranne (1977). Influence of temperature on hemoglobin-ligand interaction in whole blood. J. Appl. Physiol. 43: 545-550. Imai, K. and T. Yonetani (1975). pH dependence of the Adair constants of human hemoblobin. J. Biol. Chem. 250:2227-2231. Kelman G.R. (1966). Digital computer subroutine for the conversion of oxygen tension into saturation. J. Appl. Physiol. 21: 1375-1376.
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Kitchen, H. and H.F. Bunn (1975). Ontogeny of equine haemoglobins. J. Reprod. Fert. (Suppl.) 23: 595598. Kwant, G., B. Oeseburg, A. Zwart and W.G. Zijlstra (1988). Human whole-blood 02 affinity: effect of Co_,. J. AppL Physiol. 64: 2400-2409. Littlejohn, A. and D.G. Snow (1988). Circulatory, respiratory and metabolic responses in thoroughbred horses during the first 400 metres of exercise. Eur. J. Appl. Physiol. 58: 307-314. Lykkeboe, G., H. Schougaard and K. Johansen (1977). Training and exercise change respiratory properties of blood in race horses. Respir. Physiol. 29: 315-325. Meier, U., D. Boning and H.J. Rubenstein (1974). Oxygenation dependent variations of the Bohr coefficient related to whole blood and erythrocyte pH. Plfigers Arch. 349: 203-213. Persson, S.G.B. (1967). On blood volume and working capacity in horses. Acta Vet. Scand. (Suppl.) 19: 1-189. Persson, S.G.B., P. Kallings and C. Ingvast-Larsson (1987). Relationships between arterial oxygen tensions and cardiocirculatory and pulmonary function at rest and during submaximal exercise in the horse. In: Equine Exercise Physiology 2, edited by J.R. Gillespie and N.E. Robinson. Davis CA: ICEEP Publications, pp. 161-171. Reeves, R.B. (1980). The effect of temperature on the oxygen equilibrium curve of human blood. Respir. Physiol. 42:3 17-328. Schmidt-Nielsen, K. and J.L. Larimer (1958). Oxygen dissociation curves of mammalian blood in relation to body size. Am. J. Physiol. 195: 424-428. Severinghaus, J.W. (1979). Simple, accurate equations for human blood oxygen dissociation computations. J. Appl. Physiol. 46: 599-602. Tucker, V.A. (1967). Method for oxygen content and dissociation curves on microliter blood samples. J. Appl. Physiol. 23: 410-413. Willford, D.C. and E.P. Hill (1986). Modest effect of temperature on the porcine oxygen dissociation curve. Respir. Physiol. 64:113-123.
Respiration Physiology 97 (1994) 293-300
Temperature and pH effects on the oxygen equilibrium curve of the thoroughbred horse K. Smale a'b a n d P.J. Butler b'* aDepartment of Comparative Phystology, Animal Health Trust, Newmarket, Suffolk, UK USchool of Biological Sciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK Accepted 21 March 1994
Abstract A new oxygen equilibrium curve is defined for the Thoroughbred horse under standard conditions of 37 °C, pH = 7.4 and Pco2 = 5.33 kPa. The "standard" Pso for the Thoroughbred is, at 2.83 + 0.04 (SE of mean) kPa, significantly lower than that found for the Hanoverian horse (3.17 + 0.03 kPa) by Clerbaux et al. (Can. J. Vet. Res. 50: 188-192, 1986), and lower than other values for horses in the literature. Using data from Butler et al. (J. Exp. Biol. 179:159-180, 1993), curves were also constructed, in vitro, under simulated conditions of intense exercise to examine the individual effects ofpH, temperature and Pc% on the standard curve. The fixed acid Bohr coefficient is similar to that in humans (-0.41) whereas the temperature coefficient is, at 0.019, lower than that for humans. The coefficients were shown to be saturation dependent. Keywords: Blood, 02 transport, horse, comparative; Bohr effect, blood, horse; Mammals, horse, Thoroughbred vs Hanoverian
1. Introduction Exercise-induced arterial hypoxaemia (EIH) has now been accepted as a normal response to exercise in the performance horse (Persson et aL, 1987; Littlejohn and Snow, 1988; Butler et al., 1993). It could be considered, therefore, that EIH might restrict the exercise potential of the Thoroughbred racehorse. However, the thoroughbred shows a marked increase in haemoglobin concentration with exercise - the equine spleen acting as a reservoir of erythrocytes which is released during exercise or under conditions of stress, via adrenergically induced contraction of the smooth muscle of the splenic capsule and trabeculae (Persson, 1967). Under intense exercise conditions, the oxygen carrying capacity has been shown to increase by more than 50% (Butler et aL, 1993). This could be a major adaptation to exercise in the Thoroughbred. * Corresponding author. Tel:(0)21 414 5470; Fax: (0)21 414 5925. 0034-5687/94/$7.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 3 4 - 5 6 8 7 ( 9 4 ) 0 0 0 3 7 - Z
294
K. Smale and P.J. Butler / Respiration Physiology 97 (1994) 293-300
The relationship between Po: and oxygen content has been studied widely for human blood, resulting in several algorithms to describe the OEC. (Hill, 1910; Adair, 1925; Kelman, 1966; Severinghaus, 1979). Data regarding haemoglobin-oxygen binding in the horse are very limited and, as the relationship between the human and Thoroughbred OEC has not been established, these "human" nomograms cannot be used for blood from Thoroughbred horses with full confidence. In order to establish this relationship, the influence of temperature, pH and Pco2 on the shape and position of the OEC were determined for the blood of the Thoroughbred. Curves were constructed under "standard" conditions of 37 °C, pH 7.4 and Pco~ = 5.33 kPa, as well as under the conditions determined in horses at maximum exercise during a standard exercise test on a treadmill (Butler et al., 1993).
2. M e t h o d s
The pool of experimental animals consisted of 14 Thoroughbred horses (three fillies and 11 geldings, weighing approximately 450 kg), aged between four and 17 years. Blood samples were taken from the jugular vein by needle [21-gauge, Monoject, Sherwood Medical, Crawley, UK) into heparinised (heparin sodium injection BP (mucous), 5000 units in 1 ml, Evans Medical, Sussex, UK)] polypropylene syringes and used immediately for the construction of equilibrium curves. Mixing pumps, (2M301/1-f, WOsthoff, Bochum, Germany) were used to create gas mixtures of oxygen and carbon dioxide with high purity nitrogen (Air products, Ipswich, UK] as the balancing gas. The required gas mixture was passed through a spinning tonometer (IL237, Instrumentation Laboratory, Milan) at a rate of approximately 300 ml.min -~ where it was humidified before equilibrating with the spinning blood. Blood samples were spun for at least fifteen minutes at each gas mixture which ensured equilibration, although a longer time was required for initial deoxygenation and for a larger sample volume. Packed cell volume (PCV) was measured by the micro-haematocrit method. Haemoglobin was converted to cyanmethaemoglobin and measured spectrophotometrically using a haemoglobinometer (Coulter Electronics Ltd, Reading, UK). Haemoglobin concentration in plasma before and after tonometry was below 0.008 raM, confirming that the action of the tonometer did not cause haemolysis. For the analysis of 2,3-bisphosphoglycerate (2,3-BPG), venous blood (1 ml) was added to 3 ml cold trichloroacetic acid (8~o w/v), shaken and kept on ice until the sample was spun at 4000 rpm for 4 min. The pellet was then discarded and the supernatant stored at -20 °C. Analysis was performed using a Sigma kit (procedure number 665, Sigma UK). In 28 samples, 2,3-BPG concentration was measured before and after tonometry and showed no significant change (P<0.05). Mean 2,3-BPG concentration was 14.7 + 0.2 gmol (g Hb) -l. Oxygen content was measured by the method of Tucker (1967), and pH measurements were performed by a blood gas analyser thermostatted at 37 °C (ABL330, Radiometer, Copenhagen, Denmark). The ABL 330 has an inbuilt temperature corAnimals and blood collection
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rection facility which is sufficiently accurate for pH of horse blood (Butler et al., 1991). Po2 and Pco2 were calculated from the composition of the equilibrating gas, barometric pressure, water vapour pressure etc. A single OEC was constructed by completely desaturating the blood and then slowly resaturating it by increasing the oxygen concentration in the gas mixture. Samples were taken at points along the resaturation curve, and curves were originally run in both directions to ensure identical results. For each curve, temperature, pH, P¢o2 and haemoglobin concentration were recorded. Nine levels of oxygen were used for each curve up to a maximum of 25 ~o. The latter gave the same amount of oxygen combined to haemoglobin (CHbo:) as 30 ~o 02, thus 100 ~o saturation of haemoglobin with oxygen was measured using a gas mixture containing 25 ~o 02. Samples were withdrawn from the tonometer, using a syringe which had been flushed with the circulating gas mixture, and introduced into the blood gas analyser and the Tucker apparatus. Total 02 content is the sum of the oxygen dissolved in the plasma (% x Po2, where % is the solubility of O2 in the plasma) and that combined with the haemoglobin: Co = CHbo + ( ~ x Po ). Dissolved oxygen [% x Po2, where ~p = 0.000031 ml O2.m1-1 mmHg-1 (Aclamsand I~Iahn, 1979)] was subtracted from the total 02 content. Percent saturation was then calculated as: CHbo2/(CHb o . . . . x 100, where CHbo2ma x is the maximum amount of oxygen that can combine chemically with haemoglobin. The effect of temperature, p H and P C o 2 on the standard O E C Standard arterial conditions were taken as 37 °C, pH = 7.4 and P¢o~ = 5.33 kPa. The effects of temperature, pH, and Pco2 were examined within the physiological ranges observed during exercise (Butler et al., 1991; Butler et al., 1993): 37-41°C, pH 7.01-7.4 and P¢o: 6.3-16.9 kPa. Curves were drawn by hand to determine the temperature coefficient (TC), the fixed acid Bohr coefficient (BC) and the effect of CO 2 (CO2 effect) on the OEC. The effect of temperature was examined by altering the temperature of the waterbaths surrounding the tonometer. Three temperatures were used: 37, 39 and 41 °C. The temperature coefficient was calculated as blog Ps0/bT, at constant Pco, (mean pH was taken over the OEC). The fixed acid Bohr coefficient was determined by adding 0.1 mol.L -I HC1 to the blood sample until the required pH was achieved in the tonometer, and calculated as Slog Ps0/cSpH. The CO 2 effect was determined by increasing the P¢o2 in the gas mixture but no attempt was made to keep the pH constant. However, the subtraction of the fixed acid coefficient from the CO2 effect (calculated as Slog Ps0/~SpH) gives the net CO z coefficient. The effect of the lactate anion was also investigated. Two concentrations of sodium lactate (DL-lactic acid, sodium salt; Sigma Chemical) were used, 10 and 20 mmol.L -1, to represent levels of whole blood lactate found in the horses during medium and high intensity exercise (Butler et al., 1993). Curves were also constructed where lactic acid [L( + )lactate; Sigma Chemical, UK] was used to alter pH in order to mimic the in vivo conditions. As no change in 2,3-BPG concentration was detected in these horses during exercise, the effects of 2,3-BPG on the OEC or fixed acid Bohr coefficient were not determined. The effect of haemoglobin concentration [Hb] was examined by allowing a blood sample to settle, on ice, removing sufficient plasma to increase the [Hb] by 50-100~o,
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remixing the sample and then constructing the OEC. Hill's 'n'-value was calculated as the slope of the line given by plotting log [y/(100 - y)] against log P (where y = ~o saturation and P = Po2), from 20-80~o saturation. Trend analysis (analysis of variance) was used to determine if there was a significant saturation dependence of the temperature and Bohr coefficients. Mean values are give + SE of mean and the Student 't'-test was used to determine any significant difference between two mean values. P < 0.05 was used as the level of significance.
3. Results
The standard Ps0 for the Thoroughbred horse (2.83 + 0.04 kPa) is significantly (P<0.001) lower than that reported for the Hanoverian horse (3.17 + 0.03 kPa) and lower than that for humans (Fig. 1). The amount of oxygen combined to Hb at 100~o saturation ( 0 2 combining capacity) was 1.397 _+0.008 ml.g -1. When calculated at Ps0,
100 Thoroughbred
(this study) \ ~
80 l--
Hanoverian
co
°~ ..II--
cl
horse
~Humon 60
-I ,,41--
CS =I,--
c w
~0
O..
T = 37°C
20
pH= 7.t, PE0 = 5.33 kPa
0 t_~-.~ I
0
,
I
2
,
I
l,
,
I
6
,
I
8
,
I
10
,
I
J
12
Pcxrfial pressure of oxygen (kPa) Fig. 1. Oxygen equilibrium curves under standard conditions (37 °C, pH 7.4, Pco_, 5.33 kPa) for the Thoroughbred racehorse (present study, []), Hanoverian horses (Clerbaux et al., 1986, /X) and humans (Kelman, 1966, O).
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297
Table 1 M e a n ( _+ S E M ) v a l u e s o f t h e t e m p e r a t u r e coefficient ( T C ) t h e fixed a c i d B o h r coefficient ( B C ) a n d t h e C O 2 saturation
effect for t h e b l o o d o f T h o r o u g h b r e d r a c e h o r s e , f r o m 1 0 - 9 0 % °J % Saturation
TC
BC
C O o effect
10 20 30 40 50 60 70 80
0.012__+0.003 0.013 -+ 0.002 0 . 0 2 0 -+ 0 . 0 0 2 0.022 + 0.001 0.019_+0.001 0.023 + 0.003 0.020 _+ 0.003 0 . 0 2 0 +__0.003
-0.10+0.07 - 0.25 + 0.06 - 0.36 -+ 0.06 - 0.41 -+ 0.06 -0.41 +0.07 - 0.44 _+ 0.07 - 0.46 + 0.07 - 0.40 -+ 0 . 0 9
- 0.46 - 0.48 - 0.53 - 0.53 -0.51 - 0.50 - 0.50 - 0.48
90
0.021 -+ 0.003
- 0.24 _+ 0.08
- 0.35 _+ 0.05
-+ 0.02 + 0.02 __+0.01 + 0.01 _+0.02 + 0.02 _+ 0.02 -+ 0.04
the values for TC, BC and the CO2 effect were 0.019+0.001; -0.41 +0.07, and -0.51 + 0.02, respectively. There was no difference between the BCs calculated from curves where pH had been altered by lactic acid as opposed to HC1. Thus, no effect of the lactate anion on the OEC was noted. However, the coefficients proved to be saturation-dependent. When calculated as 61og Po2/~5{factor} for the full range of saturation values, a significant trend of saturation-dependence appeared, see Table 1. TC was lower at low levels of saturation, particularly below 30~o saturation. Both the BC and the CO2 effect were significantly saturation dependent: (a) BC was dependent upon saturation at the extreme ends of the range, where it was less negative than in the middle of the saturation range; and (b) the CO 2 effect was more negative at low levels of saturation. Haemoglobin concentration had no effect on the position or the shape of the curve and there was no significant difference in the value of Hill's n with the different treatments, i.e., temperature, pH or Pco2. The overall mean value for Hill's n was 2.54 + 0.04.
4. Discussion
The Ps0 for the Thoroughbred horse in this study (2.83 kPa) is lower than that obtained from previous studies on the horse, at standard conditions of 37 °C, pH = 7.4 and Pco2 = 5.33 kPa (3.33 kPa, Kitchen and Bunn, 1975; Bunn and Kitchen, 1973), (3.07 kPa, Schmidt-Nielsen and Larimer, 1958), 3.47 kPa, Kitchen and Bunn, 1975), (3.17 kPa, Clerbaux et al., 1986), (3.24 kPa, Lykkeboe et al., 1977). The first seven studies did not specify the breed of horse whereas the study by Clerbaux et al. (1986) used the Hanoverian horse and the study by Lykkeboe et al. (1977) used trotting horses, which are assumed to be Standardbreds. These data indicate that the haemoglobin of the Thoroughbred horse has a greater affinity for oxygen than that of other breeds. A greater haemoblobin-oxygen affinity means that at those values of Po2 where the Thoroughbred may be considered hypoxaemic, the actual percent saturation is greater than it would be for humans or for the Hanoverian horse. Whether this can be seen
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as any kind of adaptation to intense exercise is blurred by the significant increase in haemoglobin concentration during exercise which means that, whatever the Po2, particularly on the arterial side, the Thoroughbred has a much greater oxygen content than either humans or the Hanoverian horse. Thus, at the low Po: values seen during exercise (Butler et al., 1993), the increase in haemoglobin concentration may be seen as a positive adaptation enabling sufficient oxygen supply to the exercising muscles. Resting 2,3-BPG concentration was lower than that reported for the Hanoverian horse by Clerbaux et al. (1986) of 16.9 + 1.1 #mol.g Hb -1, so it is unlikely that the difference in haemoglobin-oxygen affinity between these two breeds is caused by 2,3BPG. Although it is well known that 2,3-BPG concentration effects both the fixed acid and the CO2 Bohr coefficients (Arturson et al., 1974), any small variation around the normal level should not have introduced any appreciable error (Hlastala and Woodson, 1975; Kwant et al., 1988). The fact that increasing the haemoglobin concentration had no effect on the OEC is important to note, because of the large increase in haemoglobin concentration that occurs during exercise in the thoroughbred. The TC is comparable to those of the Hanoverian horse and the pig although it is lower than that generally found in humans. BC is similar to that reported for humans. Although both coefficients showed a marked saturation dependence, there are marked differences in the amount of saturation dependency for the different coefficients reported in the literature for various species. A greater TC at low saturation levels has been reported in humans (Hlastala et al., 1977) and the pig (Willford and Hill, 1986), whereas Reeves (1980) and Boning et al. (1978) found that TC in humans did not vary with saturation. However, Boning et al. (1978) did find that TC was significantly higher in men than in women. It is unclear why the TC should be lower at low saturations in the horse as found this study and that of Clerbaux et al. (1986), but higher at low saturations in humans. In humans, varying degrees of saturation dependency of the BC have been reported by several authors (Hlastala and Woodson, 1975; Garby et al., 1972; Arturson et al., 1974; Meier et al., 1974; Kwant et al., 1988). For example, Boning et al. (1978) found a saturation dependency of the fixed acid Bohr coefficient that could be approximated by a second order polynomial, with the maximum values between 40 and 700/0 saturation, Meier et al. (1974) reported that the fixed acid Bohr coefficient was maximum at the middle range of saturation, Hlastala and Woodson (1975) and Hlastala et al. (1977) reported a rise in the BC of 15~o between 50 and 10~o saturation, Garby et al. (1972) and Arturson et al. (1974) reported rises of approximately 40~o between 50 and 10~o saturation, Meier et al. (1974) reported that BC increased still further at saturations higher than 50~o, whereas Hlastala and Woodson (1975) did not find any significant effect of saturation on the BC. Willford and Hill (1986) found no specific saturation dependence on the BC in the pig. The variance of the BC according to saturation lends weight to the suggestion that the four 0 2 binding sites in the haemoglobin molecule are not equivalent in terms of behaviour towards the N + ion, i.e., there is a variable number of protons released per molecule of oxygen bound, dependant upon the subunit. Imai and Yonetani (1975) suggested the individual subunit coefficients for the BC and TC in humans varied as follows: (BC) -0.46, -0.65, -1.09, -0.08; (TC) 0.023, 0.015, 0.018, 0.040 at pH 7.2
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in haemoglobin solutions and without 2,3-BPG. Therefore it seems likely that the differences in Bohr coefficients between species could be explained by variations in the binding of individual subunits. It has also been suggested that the saturation dependence of the BC could be due to interaction of Hb with 2,3-BPG and CO 2 under physiological conditions and the pH dependence of their binding constants. The fact that the fixed acid coefficient is identical to that in humans lends weight to the idea that a major adaptation to exercise in the Thoroughbred is the increase in haemoglobin concentration.
Acknowledgement K.S. was in receipt of a SERC CASE award and the authors are grateful to the Horserace Betting Levy Board and Sheikh Mohammed al Maktoum for their generous support.
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Kitchen, H. and H.F. Bunn (1975). Ontogeny of equine haemoglobins. J. Reprod. Fert. (Suppl.) 23: 595598. Kwant, G., B. Oeseburg, A. Zwart and W.G. Zijlstra (1988). Human whole-blood 02 affinity: effect of Co_,. J. AppL Physiol. 64: 2400-2409. Littlejohn, A. and D.G. Snow (1988). Circulatory, respiratory and metabolic responses in thoroughbred horses during the first 400 metres of exercise. Eur. J. Appl. Physiol. 58: 307-314. Lykkeboe, G., H. Schougaard and K. Johansen (1977). Training and exercise change respiratory properties of blood in race horses. Respir. Physiol. 29: 315-325. Meier, U., D. Boning and H.J. Rubenstein (1974). Oxygenation dependent variations of the Bohr coefficient related to whole blood and erythrocyte pH. Plfigers Arch. 349: 203-213. Persson, S.G.B. (1967). On blood volume and working capacity in horses. Acta Vet. Scand. (Suppl.) 19: 1-189. Persson, S.G.B., P. Kallings and C. Ingvast-Larsson (1987). Relationships between arterial oxygen tensions and cardiocirculatory and pulmonary function at rest and during submaximal exercise in the horse. In: Equine Exercise Physiology 2, edited by J.R. Gillespie and N.E. Robinson. Davis CA: ICEEP Publications, pp. 161-171. Reeves, R.B. (1980). The effect of temperature on the oxygen equilibrium curve of human blood. Respir. Physiol. 42:3 17-328. Schmidt-Nielsen, K. and J.L. Larimer (1958). Oxygen dissociation curves of mammalian blood in relation to body size. Am. J. Physiol. 195: 424-428. Severinghaus, J.W. (1979). Simple, accurate equations for human blood oxygen dissociation computations. J. Appl. Physiol. 46: 599-602. Tucker, V.A. (1967). Method for oxygen content and dissociation curves on microliter blood samples. J. Appl. Physiol. 23: 410-413. Willford, D.C. and E.P. Hill (1986). Modest effect of temperature on the porcine oxygen dissociation curve. Respir. Physiol. 64:113-123.