Oxygen transport properties of blood in two different bovine breeds

Camp. Biochem. Physiol. Vol. 89A, No. 4, pp. 553-558, 1988 Printed

in Great

0300-9629/88 $3.00 + 0.00 Q 1988 Pergamon Press plc

Britain

OXYGEN

TRANSPORT PROPERTIES OF BLOOD IN TWO DIFFERENT BOVINE BREEDS

P. GUSTIN,* TH. CLERBAUX,~E. WtLLEMs,t P. LEKEUX*,F. LOMBA*and A. FRANst *UniversitC de Liege, Faculte de Medecine Veterinaire, Centre de Physiopathologie Respiratoire des bovins, 45, rue des Vtterinaires, B-1070 Bruxelles Belgique. Telephone: (2)521-6858 and tCardiopulmonary Laboratory, Universite Catholique de Louvain, 1200 Brussels, Belgium (Received 30 June 1987)

Abstract-l. The whole oxygen dissociation curve of oxyhemoglobin has been determined in doublemuscled cattle of the Belgian White Blue breed and in Friesian cattle of different body weight. 2. In calves, P,, values are low and DPG level is high (420pmol/g Hb). 3. P,, values of 25 + 1.4mm Hg (R k SD) and a level of DPG less than I .5 pmol/g Hb have been found in animals weighing more than 80 kg. 4. Effects of temperature and pH on the oxygen dissociation curve have been measured at all levels of saturation. The temperature coefficient (dlog P,,/dT) and the Bohr effect expressed as dlog P,/dpH were 0.017 and - 0.40, respectively. 5. Hematocrit, hemoglobin concentrations and oxygen capacity of hemoglobin have been measured. 6. No difference between both breeds has been observed. 7. These data can be used to correct measured values of oxygen tension for temperature and pH and to measure oxygen content of blood in cattle.

INTRODUCTION Oxygen tension (PO,) in blood, saturation of oxyhemoglobin (So,)and oxygen content of blood (0,C) are often measured in cardio-pulmonary studies. In humans, these parameters are calculated taking into account pH, temperature (T) and 2-3 diphosphoglycerate levels (DPG). Little is known about the oxygen dissociation curve (ODC) in cattle and results are not always consistent (Ross and Romijns, 1938; Bartels and Harms, 1959; Gahlenbeck et al., 1968; Breepol et al., 1981). It is generally agreed that bovine blood oxygen affinity is only slightly lower than that of man and that bovine hemoglobin is much less influenced by DPG than human hemoglobin (Bunn, 1974). Fetal bovine blood oxygen affinity is higher than that of adult cows and erythrocytes of young bovine fetuses have been reported to contain DPG in appreciable amounts (Breepol et al., 1981). The purpose of this study is to determine the entire ODC of bovine oxyhemoglobin and to study the effects of pH, DPG, T and somatic growth on this curve in double-muscled cattle of the Belgian White and Blue breed (BWB) and in Friesian cattle. We have studied these two breeds because arterial PO2is lower in the former than in the latter (Gustin et al., 1986). MATERIALSAND

METHODS

We have investigated 10 BWB and 10 Friesian cattle. As the ODC were initially identical in both groups, we have examined 19 additional BWB cattle in order to determine the effect of age and weight on oxygen affinity. Animals weighed between 40 and 680 kg. All animals were healthy on clinical examination, and had no history of diseases. No cow was more than I month pregnant. A 2&60 ml sample of blood was drawn from the jugular vein of each animal into a glass syringe containing heparin.

The blood samples were immediately placed in ice-water and studies began 1hr later. Oxygen dissociation curve of oxyhemoglobin The oxygen dissociation curve of oxyhemoglobin has been measured with a dynamic method at constant r, pH and PcoJ (Clerbaux et al., 1973; Sottiaux et al., 1976). A sample of 1Oml of blood was deoxygenated in a rotative tonometer with a gas mixture composed of 5.6% CO, and nitrogen. Blood was placed in an analyser, equilibrated with the first gas mixture. For 15 min, the oxygen tension was slowly increased from 0 to 350mm Hg by introducing a second gas mixture composed of 5.6% CO, and oxygen, PO, was measured by a polarographic method (Po, electrode, Eschweiler, Kiel, RFA) and oxygen saturatton (S,) by a photometric method (LED, 660 nm). Values of pH, T and Pcol were kept constant @H = 7.400; T = 37°C and P,, = 40 mm Hg). Data were recorded on magnetic tape and curves were calculated by a computerized method (Modcomp H-25, USA) and graphed by an electrostatic plotter (Versatec, USA). Between a So, of 10 and 80%, reproducibility was 0.1 mm Hg. The 39 samples of blood were used to draw the ODC in standard conditions (PH = 7.4; Pco, = 40 mm Hg; T = 37°C. Student’s t-test for unpaired data was used to compare corresponding data points on the curves obtained in 10 BWB and in 10 Friesian cattle (BMDP: Statistical Software). Effects of pH oxyhemoglobin

and T on oxygen dissociation curve of

Effects of pH and T were measured in blood samples of two BWB and two Friesian cattle randomly selected and weighing between 110 and 135 kg. ODC (n = 36) were determined at three values of pH (7.2, 7.4.7.6) and at 33,37 and 41°C. The Bohr effect was derived from multiple regressions (BMDP, Statistical Software) of log PO, and PO, on pH at 20 different levels of saturation. Two coefficients were calculated: dlog Po,/dpH and dP,/dpH. 553

P. GUSTIN

554

et al.

The effect of T was determined in the same manner by means of two coefficients: dlog P,,/dT and dP,,/dT. The Hill coefficient (n) and the equilibrium constant K were calculated. A Fisher-Snedecor’s test was used to compare results obtained in Friesian and double-muscled cattle (BMDP: Statistical Software).

are in Tables 2 and 3 and are illustrated in Figs 4 and 5. No significant difference exists between BWB and Friesian data.

Eflecr of DPG on oxygen measurements

The entire ODC of bovine hemoglobin has seldom been determined (Breepoel er al., 1981). It is important to determine the whole ODC rather than P,, only because the first measurement allows a calculation of the true saturation from PO,. pH and DPG and a correction of Po, by appropriate use of temperature. Generally, only values of P,, are given and these values exhibit a very wide range. This great variability of results can be explained by the fact that different techniques have been used for these measurements. The P,, values reported in the present study for 37’C, pH = 7.4 and Pco, = 40 mm Hg are generally lower than those previously given for adult cattle. A P,, ranging between 28 and 37 mm Hg is mentioned by Roos and Romijn (1938). Values reported by Bartels and Harms (1959) and by Gahlenbeck et al. (1968) lie well within this range. Values found in this study are 25 k 1.4 mm Hg (R + SD) and are similar to those mentioned by Sahay and Kohli (1984) (26 mm Hg). There is a relationship across mammalian species between body size and blood oxygen affinity, with oxygen affinity decreasing from the elephant to the mouse (Dhindsa et al., 1974). The high P,, values previously reported in cattle (Roos and Romijns. 1938) contradict this rule, and since there is no reason why cattle should be an exception, we believe that our lower values of P,, are more representative of this species.

dissociation curues and other

Hemoglobin, hematocrit (Hct), oxygen capacity of hemoglobin and DPG were measured by the Drabkin method (Van Kampen and Zijlstra, 1961) by microcentrifugation (Heraeus Christ), with a Lex-Or-con (Lexington, MA, USA). and the enzymatical method (Sigma kit, USA), respectively. Lex-Or-con was calibrated with human blood of non-smokers, saturated with oxygen (Clerbaux, 1981). Oxygen capacity of hemoglobin was assumed to be 1.39 (Zander and Vaupel, 1985). RESULTS Figure I shows the relationship weight and 44 P,, values measured

between

body

in our cattle. Values of DPG are also indicated in this figure but only when they were higher than 1.5 pmol/g Hb. P,, increases and DPG level remains high (range: 4.0-20 pmol/g Hb) until body weight reaches 80 kg. Above this weight, DPG levels are lower than I .5 pmol/g Hb. The effect of somatic growth on the whole ODC measured in BWB cattle is illustrated in Fig. 2. ODC of adult BWB and Friesian cattle, Hill coefficient, equilibrium constant K, values of Hb, Hct and oxygen capacity of hemoglobin are indicated in Table 1. Mean deviations between blood curve calculated in our cattle and in man (Severinghaus, 1966) are shown in Fig. 3; results are expressed as percentages of saturation. Whole ODC obtained in BWB and Friesian adult cattle are also illustrated in this figure. No significant difference exists between BWB and Friesian data. Results of multiple regressions calculated to study the effects of pH and T on oxygen curve dissociation

DISCUSSION Oxygen

Eflect curve

dissociation

of somatic

curves

growth

on the oxygen

dissociation

It is well known that bovine fetal oxygen affinity is higher than that of adult cattle and that erythrocytes of calves contain DPG in appreciable amounts (Smith et al., 1979; Breepoel et al., 1981). This has

o Bolglanwhato

and blue cattle 4 Friestan cattle

WEIGHT

so

100

150

ZOO

250

300

350

um

(

kg

I

“650

Fig. I. Values of P, measured in standard conditions T = 37°C; pH = 7.4; Pco2 = 40 mm Hg) as a function of body weight (kg). Values of DPG concentrations are also indicated (pmol/g Hb). In animals weighing more than 80 kg, DPG level was less than 1.5 pmol/g Hb and was not indicated. (n = 44). The symbol @ indicates that one animal has been investigated twice.

Oxygen BELGIAN

WHITE AND

transport

properties

555

of bovine blood

Influence of T on oxygen association curves

BLUE CATTLE

Fig. 2. Entire oxygen dissociation curves measured in three Belgian White and Blue cattle of different ages. Age (days), body weight (kg), DPG concentration (pmol/g Hb) and P,, values (mm Hg) are also indicated.

been observed in other ruminants (Breepoel et al., 1981). In calves, oxygen affinity of hemoglobin is influenced by DPG, which plays an important regulatory role. In adult cattle, values of DPG are less than 1.5 pmol/g Hb and a small interaction between DPG and oxygen hemoglobin affinity has been demonstrated (Breepoel et al., 1981). These effects of somatic growth on oxygen affinity of hemoglobin and DPG concentrations have also been observed in this study in BWB and Friesian cattle. Adult values of P,, and DPG are recorded in animals weighing more than 80 kg. Algorithms proposed in this study would not be used in younger calves.

As in man (Hlastala et al., 1977) and in pig (Willford and Hill, 1986), the temperature coefficients (dlog P,,/dT) depend on oxygen saturation in BWB and Friesian cattle. No difference exists between these breeds. In man, values of dlog P,,/dT are high (0.023 and 0.024) (Severinghaus, 1966; Hlastala et al., 1977) and are greater than values recorded in pig (0.016) (Willford et al., 1986), in horse (0.015) (Clerbaux et al., 1986) and in our cattle (0.017). Values of 0.022 have been reported in dog by Reeves et al. (1986). Effects of Ton the bovine ODC are similar to those found in pig and horse and smaller than those reported in man or dog. Willford et al. (1986) suggested that the low values of dlog P,,/dT recorded in pig were due to the high concentration of DPG in this species. The right-ward shift of the pig oxygen dissociation curve due to such high values of DPG would be advantageous because hematocrit of this animal is naturally low. These hypotheses are certainly not valid in cattle: indeed, bovine hemoglobin sensitivity to temperature is smaller than in man, but both DPG and

Hb concentrations are low and P,, values in cattle (25 mm Hg) are smaller than in pig (35.7 mm Hg). Efect

ofpH

on oxygen dissociation curve

As it has been observed in horse (Clerbaux et al., 1986), the Bohr effect (dlog Po,/dpH) appears to be saturation-dependent in cattle. Our values of dlog P,,/dpH ( - 0.40) are smaller than those found in man < f 0.48) (Severinghaus,

Table I. Oxygen dissociation curves of bovine oxyhemoglobin determined in standard conditions (pH = 7.4; T = 37°C; CO, tension = 40 mm Hg). Oxygen tensions (P,::mm Hg) are given as a function of saturation (S,.:%L Hill coefficient. eauilibrium constant (K). hemoelobin concentration (Hb: g%), hematokrz (Hct: %) are also indkated. Standard dektions are in parentheses Double-muscled (DM) cattle (n = IO)

PO?’

S”, (%)

Friesian cattle (n = IO)

PO2

Student’s f-test’

Friesian and DM cattle (n = 20) P,

5.5 (0.4) 9.2 (0.5) 12.0 (0.6) 14.2 (0.7) 16.1 (0.8) 18.0 (1.0) 19.7 (1.0) 21.4 (1.1) 23.0( I .2) 24.6 (1.3) 26.4 (1.5) 28.3 (1.6) 30.4 (1.7) 32.8 (I .9) 35.6 (2.0) 39.0 (2.4) 43.9 (3.1) 51.1 i4.oj 65.7 (7.5) 71.1 (8.9) 79.4 (10.4) 96.5 (20.0)

5.3 (0.6) 9.0 (0.8) I I .8 (0.8) 14.1 (0.9) 16.1 (1.1) 18.1 (1.3) 19.9 (1.4) 21.7 (1.5) 23.4 (1.5) 25.2 (1.5) 27.0 (1.6) 29.0 (I .6) 31.2 (1.6) 33.6 (I .6) 36.5 (1.8) 40.0 (2.1) 44.8 (2.7) 52.0 (3.6) 67.1 (7.8) 74.3 (12.0) 83.2 (16.7) 99.0 (27.8)

0.9 0.7 0.7 0.4 0 0.3 0.4 0.6 0.7 0.9 1.0 1.0 I.2 I.0 I .o I.0 0.6 0.5 0.4 0.7 0.6 0.4

5.4 9.1 II.9 14. I 16.1 18.1 19.8 21.6 23.2 25.0 26.7 28.6 30.8 33.2 35.9 39.5 44.4 51.5 66.4 12.7 81.3 98.3

Hb Hct

10.4 (0.9) 34 (3)

10.6 (0.8) 34 (3)

0.6 0.1

10.5 34.0

Hill Coefficient log K

2.8 (0.1) 4.0 (0.2)

2.8 (0.2) 3.Y (0.3)

I.2 0.8

2.8 3.9

5 IO

15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 96 91 98

* All data are statistically

not significant

P. GUSTINet al.

556

1966), horse (kO.45) (Clerbaux et al., 1986) and pig (+ 0.44) (Willford et al., 1986). In conclusion, it seems that the control of oxygen affinity of hemoglobin by pH, T and especially DPG is less important in cattle than in other species like man or dog. Comparison of BWB and Friesian cattle PO2 tmmHg) Oo

20

10

60

80

100

120

140

200

300

Fig. 3. Mean entire oxygen dissociation curves measured in 10 Belgian White and Blue (---) and 10 Friesian cattle (---) in standard conditions (T = 37°C; pH = 7.4; Pco2= 40 mm Hg). Mean deviations between blood curve calculated in cattle and in man are also illustrated; results are expressed as percentages of saturation (A So, %) (Severinghaus, 1966).

Normal cattle have a greater resistance to exercise and to respiratory diseases than double-muscled cattle. Monin and Boccard (1974) have attributed this difference to the fact that oxygen capacity of blood is smaller in the latter. The low oxygen capacity ot double-muscled cattle was explained by their lower value of hemoglobin concentration. Oxygen capacity of hemoglobin was similar in normal and doublemuscled cattle (1.21 and 1.23, respectively). In our study, this value was found to be the same as in man (1.39). Moreover, values of hematocrit and hemoglobin concentration, oxygen dissociation curve,

Table 2. Effects of temperature and pH on the oxygen dissociation curve of bovine hemoglobin (n = 18) at different levels of saturation (So,). These correction factors are derived from multiple regression calculated over a range of pH values (7.2-7.6) and temperatures (3341°C) [d(log PO,) = a, (pH 7.4) + b, (T, 37)]. PO,: oxygen tension (mm Hg); T: temperature (“C). Carbon dioxide tension has been maintained at 40 mm Hg during all experiments. A Fisher test has been used to compare multiple regressions between both breeds. All data are statistically not significant. r : linear correlation coefficient. Coefficients of variation (%) are given in parentheses Double-muscled cattle

(DM)

Friesian cattle

DM and Friesian cattle

So2 (%)

--(I1

6,

-01

b,

5 10 20 30 40 50 60 70 80 90 95 96 97 98

0.43 0.48 0.46 0.44 0.41 0.40 0.38 0.38 0.39 0.38 0.37 0.36 0.35 0.30

0.024 0.020 0.017 0.018 0.017 0.017 0.017 0.017 0.017 0.016 0.015 0.016 0.016 0.014

0.48 0.48 0.42 0.41 0.41 0.40 0.40 0.40 0.41 0.43 0.44 0.43 0.44 0.40

0.022 0.020 0.017 0.016 0.016 0.016 0.016 0.016 0.016 0.015 0.015 0.016 0.014 0.014

r

> 0.95 between 20 and 95% S,.

> 0.95 between and 95% S,

IO

-“I 0.45 0.48 0.44 0.43 0.41 0.40 0.39 0.39 0.40 0.41 0.41 0.40 0.39 0.35

Fisher test P value

b, (14) (8) (6) (5) (4) (4) (4) (4) (4) (5) (6) (8) (9) (15)

0.023 0.020 0.017 0.017 0.017 0.017 0.017 0.016 0.016 0.016 0.015 0.016 0.015 0.014

> 0.95 between 95% s,.

(13) (10) (7) (6) (5) (5) (4) (4) (4) (6) (8) (9) (12) (17)

0.72 0.92 0.91 0.58 0.47 0.33 0.39 0.19 0.63 0.58 0.20 0.08 0.03 0.09

-

IO and

Table 3. Effects of temperature and pH on the oxygen dissociation curve of bovine hemoglobin (n = 18) at different levels of saturation (So,). These correction factors are derived from multiple regression calculated over a range of pH values (7.2-7.6) and temperatures (33-41 “C) [d PO2 = a2 (pH 7.4) + b, (T, 37)]. PO,: oxygen tension (mm Hg); T: temperature (“C). Carbon dioxide tension has been maintamed at 40 mm Hg during all experiments. A Fisher test has been used to compare multiple regressions between both breeds. All data are statistically not sknificant. r: linear correlation coefficient. Coefficients of variation (%) are given in parentheses

1:

20 30 40 50 60 70 80 90 95 96 97 98 r

Double-muscled cattle - 02

(DM) b2

- 02

b,

5.7 10.3 IS.1 18.2 20.3 22.5 24.9 28.5 34.9 43.9 54.9 57.8 63.3 63.4

0.352 0.460 0.602 0.758 0.856 0.992 I.125 1.275 1.537 I .893 2.273 2.617 3.016 3.038

6.0 10.2 14.0 17.5 20.7 23.4 26.6 30.9 37.4 49.9 63.6 66.4 74.9 79.0

0.269 0.408 0.550 0.675 0.819 0.933 I .075 1.202 I.417 1.723 2.206 2.448 2.506 2.810

> 0.95 between 20 and 95% S,

Friesian cattle

z- 0.95 between and 96% S,

IO

DM and Friesian cattle - 01 b> 5.78 (14) 10.2 (8) 14.5 (6) 17.9 (5) 20.5 (5) 23.5 (5) 25.8 (5) 29.8 (4) 36.2 (5) 46.9 (6) 59.2 (7) 61.9 (8) 68.8 (IO) 70.7 (151 I

0.310 0.434 0.576 0.717 0.838 0.965 I.100 1.239 1.477 1.808 2.240 2.532 2.761 2.924

Fisher test P value (13) (10) (8) (7) (6) (6) (5) (5) (6) (7) (IO) (IO) (13) (18)

> 0.95 between 20 and 95% so,

I

0.45 0.75 0.86 0.76 0.69 0.54 0.53 0.35 0.72 0.64 0.35 0.14 0.05 0.1 I

557

Oxygen transport properties of bovine blood

Correction of oxygen tension for T, determination of oxygen saturation and oxygen content of blood

I

/e--J

-0.40

-06OJ

f

xso2 25

50

4

75

100

0,02t+ 0.01

t

I

OO

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25

50

100

75

Our results show that a difference exists between human and bovine hemoglobin. Correction of PO, for T and determination of saturation of oxyhemoglobin and oxygen content of blood performed by current analysers are therefore not valid for cattle. Data found in this study can be used to achieve a more appropriate correction. Blood samples are analysed at a temperature of 37°C and at the pH of the animal blood. PO*can be expressed in standard conditions (P,,st) (pH = 7.4 and T = 37°C) by using the following equation: p,, st = P,,~. 1 oW.41 (PH - 7.4) - 0.~~1 BEI, where P&m is the measured oxygen tension and BE is the base excess. Saturation of oxyhemoglobin can be obtained from Fig, 3. Equations of Tables 2 and 3 allow a calculation of oxygen tension at the temperature of animal blood. Oxygen capacity of hemoglobin of cattle is 1.39. This equation can he used to calculate the oxygen content (0,C) of blood expressed as volume percent: 0,C = 1.39. So>.Hb + 0.003 PO>.

Fig. 4. Evolution of dlog P@/ dpH and dlog PO,/ dT as a function of oxygen saturation (So,%) in cattle.

effects of pH, DPG, T and somatic growth were similar in BWB and Friesian cattle. This difference between our data and results obtained by Monin and Boccard (1974) is probably explained by the small number of animals investigated by these authors. The greater sensitivity of double-muscled cattle to respiratory diseases and to exercise, as compared to normal cattle, can therefore not be explained by hematological factors.

In conclusion, we found the previously used values, derived from human blood, to be inadequate for bovine blood, and we suggest that the presently reported parameters should be applied in algorithms for computing oxygen tension and content in cattle. Adult values of P, and DPG concentration are reached in animals weighing more than 80 kg. The effects of DPG and to some extent of T and pH, are less important in cattle than in man or dog. No differences between double-muscled and normal cattle have been observed. authors wish to thank Dr B. Nemery for correcting the English translation of this work. Thanks are also due to Irene Fumibre, J. F. Deneubourg, J. C. Leroy and R. Malice for their technical assistance. Financial sunoort was orovided bv I.R.S.I.A. (Institut uour 1’Encouragement de - la Rechdrche Scientifique dans 1’Industrie et I’Agriculture), rue de Crayer, 6, 1050, Bruxelles. Acknowledgements-The

1

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0

)

,

,

25

50

75

REFERENCES

v.502 , loo

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IO-

OO

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w

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%502 . 100

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