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Ferrihemoglobin formation and reduction in erythrocytes of several wild birds
('.rap. Biea'llem. Physiol.. Vol. 63C. pp. 173 to 175
0306-4492,,'79/037d)-OI73502.00/0
Pergamon Press Ltd 1979 Printed in Great Britain
FERRIHEMOGLOBIN FORMATION AND REDUCTION IN ERYTHROCYTES OF SEVERAL WILD BIRDS B. J. BLAAUBOER,C. W. M. VAN HOLSTEIJN and J. G. WtT Institute of Veterinary Pharmacology and Toxicology, State University, Biltstraat 172. Utrecht. The Netherlands
(Receired 24 August 1978) Abstract--l. Ferrihemoglobin formation by nitrosobenzene and o- and p-aminophenol was measured in erythrocyte suspensions of 7 common Dutch bird species, 2. Ferrihemoglobin reduction is determined in nitrite-treated red cells of 8 avian species. 3. The effect of plasma constituents on HbFe 3* formation and reduction as well as glucose and /Lhydroxybutyrate plasma levels were determined.
INTRODUCTION
were taken from a wing vein and heparinized. Only in the case of starlings was it sometimes necessary to pool blood samples of 2 birds: in that case the birds belonged to the same sex. Preparation of crythrocyte suspensions, incubation conditions, determination of Hb and HbFe ~* and the source of chemicals were described before (Blaauboer et al., 19751. For HbFe j ÷ formation measurements, red cells were suspended in a Krcbs-Ringer phosphate buffer (Blaauboer et al., 1976). HbFe 3+ reduction rates were determined in nitrite-treated erythrocytes, resuspended in Krebs-Ringer bicarbonate buffer (Blaauboer et al, 1979a). Plasma glucose was determined colorimetrically by the o-toluidine method (Dubowski, 1962). fl-Hydroxybutyrate was determined according to Williamson & Mellanby (1974).
Ferrihemoglobin (HbFe 3 *) is formed by many chemicals (Smith & Olson, 1973; Kiese, 1974). Among these compounds are nitrite, chlorate, aromatic amines. aromatic nitro compounds, aminophenols and :V-hydroxyarylamines. However, only a limited number of studies has been published on H b F e 3+ formation in birds (Sell et al, 1963; Stoewsand, 1970; Blaauboer et al., 1976). These studies all deal with HbFe 3* formation in granivorous birds, which are mostly of economic value in animal husbandry fchicken, turkey), as experimental animals (Japanese quail) or as prize winners (pigeons). No data exist on chemical-induced H b F e 3 + formation in wild-living avian species. During the last few decades, increasing attention has been paid to toxic effects of chemicals in wild-liv• ing species. On the one hand there is a growing concern about protecting wild animals from unwanted effects of chemicals emitted into the environment (Koeman, 1972). On the other hand avicides are being developed to kill plague birds (DeCino, 1966). In this study we investigated some parameters of HbF'e 3 + formation and reduction in erythrocytes of eight common Dutch avian species. The H b F e 3+forming properties of 3 monohydroxy aniline derivatives were determined as well as the effects of glucose and lactate on this formation (Biaauboer et al., 1979b). H b F e 3* reduction was measured in nitritetreated red cells, according to Blaauboer et al. (1979a).
RESULTS
(a) Ferrihemoglobin formation The amount of H b F e 3. in erythrocyte suspensions after 30 min of incubation with nitrosobenzene (NOB, 5 #M) is given in Table 1. There are differences in susceptibility between species. The cormorant, the carrion-crow and the oyster-catcher are relatively insensitive to NOB, compared with the more sensitive heron, the wild duck and the starling. Glucose enhances NOB-induced H b F e s * formation (Kiese & Waller, 1959; Blaauboer et al., 1979b) but here too, great species differences exist. In the heron and the starling this glucose effect is remarkable. Lactate
Table 1. Ferrihemoglobin formation in erythrocyte suspensions by nitrosobcnzen¢ (NOB, 5 #M) and the effect of glucose (11 raM) and lactate (22 raM)
MATERIALS AND METHODS
Birds were selected from several families and with various diets. Buzzards (Buteo buteo) were kept in cages in zoos (Artis Zoo, Amsterdam and Burgers Zoo, Arnhem, the Netherlands). Cormorants (Phalacrocorax carbo) and grey herons (Ardea cinerea) were obtained from Burgers Zoo. Wild ducks (Anas platyrhinchos), herring gulls (Larus (argentatus) and oyster-catchers (Haematopus ostralegus) belonged to Artis Zoo. Carrion crows (Corvus corone) and starlings (Sturnus rulgaris) were caught in several places in the Netherlands. To obtain blood, starlings were decapitated and blood was collected in a heparinized tube. By means of the apparatus of Dorrestein et al. 0978) all other blood samples
Buzzard Carrion-crow Cormorant Heron Oyster-catcher Starling Wild duck d' Wild duck ~
Blank
Glucose
Lactate
3.8 + 0.8 2.9 -t- 0.4 1.5 + 0.1 10.7 + 3.6 1.4 + 0.3 6.8 -l- 1.1 8.2 + 0.8 7.2 -t- 0.7
4.9 + 0.7 3.8 + 0.3 1.7 + 0.2 17.8 + 2.1 2.7 + 0.3 16.2 -f- 3.2 10.3 + 0.6 9.2 ± 0.7
1.7 + 0.3 0.7 + 0.4 0.5 _+ 0.2 7.8 + 2.1 0.3 -t- 0.2 2.5 + 1.0 3.1 + 0.3 3.3 ± 0.4
Mean + S.E, n = 5. Percentage of HbFe s÷ formed. 173
174
B.J. BLAAUnOER.C. W. M. VAN HOt,STHJN and J. G. Wlr
Table 2. Ferrihemoglobin formation in erythrocyte suspensions by o-aminophenol 150/~M) and the effect of glucose Ill raM) and lactate (22mM)
Buzzard Carrion-crow Cormorant Heron Oyster-catcher Starling Wild duck ,~ Wild duck ~"
Blank
Glucose
Lactate
18.7 + 2.5 29.4 +__4.9 23.1 + 2.0 28.0 + 5.5 18.9 ± 0.9 26.0 + 4.1 14.1 +_ 1.0 16.2 + 0.9
11.3 + 1.5 24.8:1:3.8 17.2 ± 3.8 20.2 + 4.9 10.5 ± I.I 18.1 ± 4.8 11.5 + 1.4 9.3 ± 0.7
II.0 -t- 1.3 18.4 -t- 3.4 10.7 ± 1.6 14.4 ± 4.1 3.1 -t- 1.5 4.6 + 2.4 7.1 ± 0.4 6.6 + 0.7
Mean + S.E., n = 5. Percentage of HbFe a* formed. diminishes H b F e ~* levels in all species (see a l s o . Table 4). In Tables 2 and 3 the effect of o - a m i n o p h e n o i (50#M) and p - a m i n o p h e n o l (50/aM) o n erythrocyte suspensions is shown. Here, species differences are not very extreme. Glucose as well as lactate decreases H b F e 3+ levels with marked species differences. (b) Ferrihemoglohin reduction There are vast species differences in H b F e 3 ÷ reduction rates (Table 4). Addition of glucose (I mM). lactate (22 raM) or fl-hydroxybutyrate (22 raM) gives different effects as well. Glucose enhances H b F e a÷ reduction in all species except in the carrion-crow and in the starling. Lactate gives higher reduction rates in all species. N o stimulating effect on H b F e a+ was found in the case of malate. With fl-hydroxybutyrate Table 3. Ferrihemoglobin formation in erythrocyte suspensions by p-aminophenol (50I~M) and the effect of glucose Ill raM) and lactate (22 mM) Blank Buzzard Carrion-crow Cormorant Heron Oyster-catcher Starling Wild duck ,3 Wild duck ~
3.0 4.2 6.7 4.0 5.2 6.0 2.6 3.4
+ + + + ± + ± +
0.6 0.6 0.6 0.6 0.4 1.7 0.3 0.4
Glucose
Lactate
2.7 3.1 6.8 3.4 4.5 5.2 2.8 3.2
1.7 1.7 1.3 1.4 1.7 1.2 1.1 1.3
+ 0.6 + 0.7 _+ 0.4 + 0.6 ± 0.7 + 1.0 +_ 0.7 + 0.8
+ + ± + + + ± +
0.3 0.4 0.4 0.7 0.3 0.4 0.3 0.3
Mean + S.E.. n = 5. Percentage of HbFe s+ formed.
there is a higher reduction rate in c o r m o r a n t red cells, but n o e n h a n c e m e n t was found in other red cell suspensions. W h e n nitrite-treated cells are resuspended in plasma of the same animal, reduction rates are very high (Table 4). Therefore it is of interest to measure plasma concentrations of constituents that influence H b F e 3÷ reduction. In some cases we were able to determine glucose and fl-hydroxybutyrate plasma values in the same blood samples that were used for H b F e 3+ formation and reduction measurements. Table 5 summarizes these plasma values. According to Bell (1971), plasma glucose levels are high in birds, as c o m p a r e d with mammals. The c o r m o r a n t and the heron have lower glucose levels than other birds. fl-Hydroxybutyrate was present in all samples.
DISCUSSION H b F e 3+ is formed by N O B and o- and p-aminophenol in erythrocytes of all selected species of birds. N O B shows species differences in susceptibility. While glucose enhances the effect of NOB. it is likely that the m e c h a n i s m of H b F e 3 + formation is the same as in m a m m a l i a n a n d in quail erythrocytes (i.e. a N A D P H - d e p e n d e n t redox cycle) (Blaauboer et al.. 1975). Since we found n o great species differences in the effect of aminophenols, it is unlikely that structural differences in the hemoglobin molecule are responsible for the differences reported here in the effect of NOB. W e therefore suggest that these differences are due to dissimilarities in the cellular metabolism (e.g. glucose utilization and hexose m o n o p h o s p h a t e shunt activity: Blaauboer et al., 1979b). Lactate diminished H b F e ~ ~ levels in N O B - t r e a t e d red cells, while glucose and lactate do the same when a m i n o p h e n o l s are added. In nitrite-treated erythrocytes these substrates enhance H b F e 3+ reduction rates. Only in c o r m o r a n t red cells fl-hb stimulates H b F e ~ + reduction rates. These results are all consistent with the existence of an N A D H - d e p e n d e n t H b F e 3+ reduction system (Blaauboer et al., 1979a). Malate does not stimulate H b F e ~+ reduction in any of the erythrocyte suspensions used. This is in accordance with our findings in other avian species (Blaauboer et al., 1979a), from which we concluded that mitochondrial metabolism is not important for H b F e 3+ reduction.
Table 4. Ferrihemoglobin reduction rates in erythrocyte suspensions and the effect of glucose (11 raM), lactate (22 raM), malate (22 raM). fl-hydroxybutyrate (22 raM) and of plasma Blank Buzzard Carrion-crow Cormorant Gull Heron Oyster-catcher Starling Wild duck 3 Wild duck 9
10.7 2.7 10.8 i.9 3.8 3.8 -0.5 1.4 8.9
+ 2.0 + 0.9 + 3.6 + 2.9 + 2.1 _+ 1.3 + 3.6 + 0.8 + 1.0
Glucose 29.3 1.3 13.9 20.6 17.6 16.5 5.3 16.6 17.3
+ 4.2 ___2.1 ± 4.1 ± 5.6 4- !.7 :t: 2.1 + 2.1 ± 4.7 ± 3.7
Lactate 28.5 54.1 48.9 43.5 27.4 34.2 66.1 24.3 34.5
+ 4.2 ± 3.1 + 8.6 + 5.3 + 3.7 ± 2.6 + 5.5 + 4.5 :t: 7.3
Malate 5.8 + 2.0 + 13.6 ± -5.6 + 1.4 ± 0.7 ± 6.5 ± 3.2 + 9.6 ±
Percentage of HbFe s ÷ reduced per hour. Mean + S.E., n = 5.
1.6 2.6 2.5 4.4 0.7 1.0 3.1 1.9 1.1
B-Hydroxy butyrate 11.1 3.2 29.6 0.6 4.2 5.4 6.2 4.6 9.5
+ 2.5 __. 2.6 ± 3.3 ± 3.0 ± 1.9 :'- 2.3 +__3.0 + 1.7 -t- 1.5
Plasma 33.7 29.4 31.4 38.6 32.4 37.1 56.6 29.8 40.3
+ 4.3 ± 4.6 ± 7.6 + 5.0 + 3.2 ± 4.1 ___6.0 ___3.3 + 3.4
175
Ferrihemoglobin formation and reduction in erythrocytes Table 5. Plasma/~-hydroxybutyrate (/~-hb) and glucose values in some birds fl-hb (#M) Mean S.E. n Buzzard Carrion-crow Cormorant Gull Heron Starling
394 914 639 + 52 923
2
558
i
3
5 2
Glucose (mM) (370, 418) (193-1303) (513-675) (920, 926)
In the carrion-crow and the cormorant, glucose has little or no effect on l-lbFe 3 + formation and reduction. compared with the other species. This is not reflected in plasma glucose levels. These levels are high in all bird species as compared with mammalian species (Bell, 1971), but there are considerable differences between avian species. (Erlenbach, 1939). probably reflecting dissimilarities in diet. The cormorant and the heron are both fish-eating species and are lowest in plasma glucose levels. However, these levels are still twice as high as in mammals. Glucose stimulates the H b F e 3 + formation mammals (Blaauboer et al., 1979b). Therefore, it is more likely that the differences in the effect of glucose on H b F e 3+ formation and reduction are due to dissimilarities in glucose utilization and metabolism.
Acknowledgements--We wish to thank Mr D. Dekker and Mr R. den Hartogh of Artis Zoo. Amsterdam and Mr J. Wensing of Burgers Zoo, Amhem, for their assistance and their kind permission to take blood samples from birds. Mr J. van Tussenbroek, Mr F. Alta, Mr H. Alta and Mr P. Hoogendoorn were of great help in catching starlings and carrion-crows. The assistance of Dr A. J. H. Schotman, Laboratory of Clinical Biochemistry, Clinic for Large Animal MediOne. State University of Utrecht, is acknowledged for determining glucose and ~-hydroxybutyrate. We also thank Miss M. Loots for correcting the English text. REFERENCES BI!LI. D. J. (1971) Plasma glucose. In Physioloqy and Biochemistry of the Domestic Fowl (Edited by BELL D. J. & FREEMANB. M.), pp. 913-920. Academic Press, New York. BLAAUBOERB. J., HOLSTEUNC. W. M. VAN & WIT J. G. {1975) Nitrosobenzene-induced ferrihemoglobin formation in Japanese quail erythrocytes. The significance of ferrihemoglobin reduction. Naunyn-Schmiedebero's Arch. Pharmacol. 289, 127-135.
M e a n S.E.
n
18.7 20.4 4- 2,0 I 1.3 4- 0.6 17.9 4- 0.8 I 1.1 19.9 4- 0.8
2 4 6 3
(18.0, 19.3) (17.3--26.0) (9.9-13.9) (17.1-19.5)
I
3
( 18.4--20.9)
BLAAU~:)ER B. J, HOLSTEUNC. W. M. VAN & WIT J. G. (1976) Ferrihemoglobin formation by monohydroxy aniline derivatives in erythrocytes of some avian species in comparison with mammals. Naunyn-Schmiedeberg's Arch. Pharmacol. 292, 255-258. BLAAUBOERB. J., HOLSTEIJNC. W. M. VAN & WIT J. G. (1979a) Reduction of ferribemoglobin in erythrocytcs of birds and mammals. Comp. Biochem. Physiol. 62A, No. 4. BL,AALmOL~ B. J., HOLS~IJN C. W. M. VAN & Wrr J. G. (1979b) Biochemical processes involved in ferrihemoglobin formation by monohydroxy-aniline derivatives in erythrocytes of birds and mammals. Comp. Biochem. Physiol. 62C, 199-203. Dt'CiNo T. J, CUNNINOHAMD. J. & SCHAFERE. W. (1966) Toxicity of DRC-1339 to starlings. J. Wild. M. 30, 249-253. DORRESTEING. M, BLAAU~ER B. J., MILTENBURGN. A. & DELEY P. P. 0978) A modified method of blood sampling from birds. Lab. Anita. 12, 193-194. DU~OWSKI K. M. (1962) An o-toluidine method for body fluid glucose determination. Clin. Chem. 8, 215. ERLENBACHF. (1939) Experimentelle Untersuchungen fiber den Blutzucker bei ViSgeln. Z. vergl. Physiol. 26, 121-161. KIESE M. (1974) Methemoolobinemia- A Comprehensive Treatise. CRC-Press, Cleveland. KOEMAN J. H. (1972) Side-effects of persistent pesticides and other chemicals on birds and mammals in the Netherlands. TNO-Nieuws 27, 527-632. SELL J. L., ROBBERTSW. K. & WADDELL D. G. (1963) Methemoglobin and reduced feed consumption due to feeding nitrite to chicks. Poultry Sci. 42, 1474-1476. SMITH R. P. & OLSOr~ M. V. 0973) Drug-induced methemogiobinemia. Semin. Hemat. 10, 253-268. STOEWSANDG. S. (1970) Influence of sex and dietary ascorbic acid in nitrite-induced methemoglobinemia in Japanese quail. Proc. Soc. exp. Biol. Med. 133, 1166-1168.
WILLIAM.SON D. H. & MELLANBY J. (1974) In Methoden der Enzymatische Analyse---ll, 3rd edn. (Edited by BERGMEYER H. U.), p. 1883. Verlag Chemie, Weinheim/Bergstrasse.
0306-4492,,'79/037d)-OI73502.00/0
Pergamon Press Ltd 1979 Printed in Great Britain
FERRIHEMOGLOBIN FORMATION AND REDUCTION IN ERYTHROCYTES OF SEVERAL WILD BIRDS B. J. BLAAUBOER,C. W. M. VAN HOLSTEIJN and J. G. WtT Institute of Veterinary Pharmacology and Toxicology, State University, Biltstraat 172. Utrecht. The Netherlands
(Receired 24 August 1978) Abstract--l. Ferrihemoglobin formation by nitrosobenzene and o- and p-aminophenol was measured in erythrocyte suspensions of 7 common Dutch bird species, 2. Ferrihemoglobin reduction is determined in nitrite-treated red cells of 8 avian species. 3. The effect of plasma constituents on HbFe 3* formation and reduction as well as glucose and /Lhydroxybutyrate plasma levels were determined.
INTRODUCTION
were taken from a wing vein and heparinized. Only in the case of starlings was it sometimes necessary to pool blood samples of 2 birds: in that case the birds belonged to the same sex. Preparation of crythrocyte suspensions, incubation conditions, determination of Hb and HbFe ~* and the source of chemicals were described before (Blaauboer et al., 19751. For HbFe j ÷ formation measurements, red cells were suspended in a Krcbs-Ringer phosphate buffer (Blaauboer et al., 1976). HbFe 3+ reduction rates were determined in nitrite-treated erythrocytes, resuspended in Krebs-Ringer bicarbonate buffer (Blaauboer et al, 1979a). Plasma glucose was determined colorimetrically by the o-toluidine method (Dubowski, 1962). fl-Hydroxybutyrate was determined according to Williamson & Mellanby (1974).
Ferrihemoglobin (HbFe 3 *) is formed by many chemicals (Smith & Olson, 1973; Kiese, 1974). Among these compounds are nitrite, chlorate, aromatic amines. aromatic nitro compounds, aminophenols and :V-hydroxyarylamines. However, only a limited number of studies has been published on H b F e 3+ formation in birds (Sell et al, 1963; Stoewsand, 1970; Blaauboer et al., 1976). These studies all deal with HbFe 3* formation in granivorous birds, which are mostly of economic value in animal husbandry fchicken, turkey), as experimental animals (Japanese quail) or as prize winners (pigeons). No data exist on chemical-induced H b F e 3 + formation in wild-living avian species. During the last few decades, increasing attention has been paid to toxic effects of chemicals in wild-liv• ing species. On the one hand there is a growing concern about protecting wild animals from unwanted effects of chemicals emitted into the environment (Koeman, 1972). On the other hand avicides are being developed to kill plague birds (DeCino, 1966). In this study we investigated some parameters of HbF'e 3 + formation and reduction in erythrocytes of eight common Dutch avian species. The H b F e 3+forming properties of 3 monohydroxy aniline derivatives were determined as well as the effects of glucose and lactate on this formation (Biaauboer et al., 1979b). H b F e 3* reduction was measured in nitritetreated red cells, according to Blaauboer et al. (1979a).
RESULTS
(a) Ferrihemoglobin formation The amount of H b F e 3. in erythrocyte suspensions after 30 min of incubation with nitrosobenzene (NOB, 5 #M) is given in Table 1. There are differences in susceptibility between species. The cormorant, the carrion-crow and the oyster-catcher are relatively insensitive to NOB, compared with the more sensitive heron, the wild duck and the starling. Glucose enhances NOB-induced H b F e s * formation (Kiese & Waller, 1959; Blaauboer et al., 1979b) but here too, great species differences exist. In the heron and the starling this glucose effect is remarkable. Lactate
Table 1. Ferrihemoglobin formation in erythrocyte suspensions by nitrosobcnzen¢ (NOB, 5 #M) and the effect of glucose (11 raM) and lactate (22 raM)
MATERIALS AND METHODS
Birds were selected from several families and with various diets. Buzzards (Buteo buteo) were kept in cages in zoos (Artis Zoo, Amsterdam and Burgers Zoo, Arnhem, the Netherlands). Cormorants (Phalacrocorax carbo) and grey herons (Ardea cinerea) were obtained from Burgers Zoo. Wild ducks (Anas platyrhinchos), herring gulls (Larus (argentatus) and oyster-catchers (Haematopus ostralegus) belonged to Artis Zoo. Carrion crows (Corvus corone) and starlings (Sturnus rulgaris) were caught in several places in the Netherlands. To obtain blood, starlings were decapitated and blood was collected in a heparinized tube. By means of the apparatus of Dorrestein et al. 0978) all other blood samples
Buzzard Carrion-crow Cormorant Heron Oyster-catcher Starling Wild duck d' Wild duck ~
Blank
Glucose
Lactate
3.8 + 0.8 2.9 -t- 0.4 1.5 + 0.1 10.7 + 3.6 1.4 + 0.3 6.8 -l- 1.1 8.2 + 0.8 7.2 -t- 0.7
4.9 + 0.7 3.8 + 0.3 1.7 + 0.2 17.8 + 2.1 2.7 + 0.3 16.2 -f- 3.2 10.3 + 0.6 9.2 ± 0.7
1.7 + 0.3 0.7 + 0.4 0.5 _+ 0.2 7.8 + 2.1 0.3 -t- 0.2 2.5 + 1.0 3.1 + 0.3 3.3 ± 0.4
Mean + S.E, n = 5. Percentage of HbFe s÷ formed. 173
174
B.J. BLAAUnOER.C. W. M. VAN HOt,STHJN and J. G. Wlr
Table 2. Ferrihemoglobin formation in erythrocyte suspensions by o-aminophenol 150/~M) and the effect of glucose Ill raM) and lactate (22mM)
Buzzard Carrion-crow Cormorant Heron Oyster-catcher Starling Wild duck ,~ Wild duck ~"
Blank
Glucose
Lactate
18.7 + 2.5 29.4 +__4.9 23.1 + 2.0 28.0 + 5.5 18.9 ± 0.9 26.0 + 4.1 14.1 +_ 1.0 16.2 + 0.9
11.3 + 1.5 24.8:1:3.8 17.2 ± 3.8 20.2 + 4.9 10.5 ± I.I 18.1 ± 4.8 11.5 + 1.4 9.3 ± 0.7
II.0 -t- 1.3 18.4 -t- 3.4 10.7 ± 1.6 14.4 ± 4.1 3.1 -t- 1.5 4.6 + 2.4 7.1 ± 0.4 6.6 + 0.7
Mean + S.E., n = 5. Percentage of HbFe a* formed. diminishes H b F e ~* levels in all species (see a l s o . Table 4). In Tables 2 and 3 the effect of o - a m i n o p h e n o i (50#M) and p - a m i n o p h e n o l (50/aM) o n erythrocyte suspensions is shown. Here, species differences are not very extreme. Glucose as well as lactate decreases H b F e 3+ levels with marked species differences. (b) Ferrihemoglohin reduction There are vast species differences in H b F e 3 ÷ reduction rates (Table 4). Addition of glucose (I mM). lactate (22 raM) or fl-hydroxybutyrate (22 raM) gives different effects as well. Glucose enhances H b F e a÷ reduction in all species except in the carrion-crow and in the starling. Lactate gives higher reduction rates in all species. N o stimulating effect on H b F e a+ was found in the case of malate. With fl-hydroxybutyrate Table 3. Ferrihemoglobin formation in erythrocyte suspensions by p-aminophenol (50I~M) and the effect of glucose Ill raM) and lactate (22 mM) Blank Buzzard Carrion-crow Cormorant Heron Oyster-catcher Starling Wild duck ,3 Wild duck ~
3.0 4.2 6.7 4.0 5.2 6.0 2.6 3.4
+ + + + ± + ± +
0.6 0.6 0.6 0.6 0.4 1.7 0.3 0.4
Glucose
Lactate
2.7 3.1 6.8 3.4 4.5 5.2 2.8 3.2
1.7 1.7 1.3 1.4 1.7 1.2 1.1 1.3
+ 0.6 + 0.7 _+ 0.4 + 0.6 ± 0.7 + 1.0 +_ 0.7 + 0.8
+ + ± + + + ± +
0.3 0.4 0.4 0.7 0.3 0.4 0.3 0.3
Mean + S.E.. n = 5. Percentage of HbFe s+ formed.
there is a higher reduction rate in c o r m o r a n t red cells, but n o e n h a n c e m e n t was found in other red cell suspensions. W h e n nitrite-treated cells are resuspended in plasma of the same animal, reduction rates are very high (Table 4). Therefore it is of interest to measure plasma concentrations of constituents that influence H b F e 3÷ reduction. In some cases we were able to determine glucose and fl-hydroxybutyrate plasma values in the same blood samples that were used for H b F e 3+ formation and reduction measurements. Table 5 summarizes these plasma values. According to Bell (1971), plasma glucose levels are high in birds, as c o m p a r e d with mammals. The c o r m o r a n t and the heron have lower glucose levels than other birds. fl-Hydroxybutyrate was present in all samples.
DISCUSSION H b F e 3+ is formed by N O B and o- and p-aminophenol in erythrocytes of all selected species of birds. N O B shows species differences in susceptibility. While glucose enhances the effect of NOB. it is likely that the m e c h a n i s m of H b F e 3 + formation is the same as in m a m m a l i a n a n d in quail erythrocytes (i.e. a N A D P H - d e p e n d e n t redox cycle) (Blaauboer et al.. 1975). Since we found n o great species differences in the effect of aminophenols, it is unlikely that structural differences in the hemoglobin molecule are responsible for the differences reported here in the effect of NOB. W e therefore suggest that these differences are due to dissimilarities in the cellular metabolism (e.g. glucose utilization and hexose m o n o p h o s p h a t e shunt activity: Blaauboer et al., 1979b). Lactate diminished H b F e ~ ~ levels in N O B - t r e a t e d red cells, while glucose and lactate do the same when a m i n o p h e n o l s are added. In nitrite-treated erythrocytes these substrates enhance H b F e 3+ reduction rates. Only in c o r m o r a n t red cells fl-hb stimulates H b F e ~ + reduction rates. These results are all consistent with the existence of an N A D H - d e p e n d e n t H b F e 3+ reduction system (Blaauboer et al., 1979a). Malate does not stimulate H b F e ~+ reduction in any of the erythrocyte suspensions used. This is in accordance with our findings in other avian species (Blaauboer et al., 1979a), from which we concluded that mitochondrial metabolism is not important for H b F e 3+ reduction.
Table 4. Ferrihemoglobin reduction rates in erythrocyte suspensions and the effect of glucose (11 raM), lactate (22 raM), malate (22 raM). fl-hydroxybutyrate (22 raM) and of plasma Blank Buzzard Carrion-crow Cormorant Gull Heron Oyster-catcher Starling Wild duck 3 Wild duck 9
10.7 2.7 10.8 i.9 3.8 3.8 -0.5 1.4 8.9
+ 2.0 + 0.9 + 3.6 + 2.9 + 2.1 _+ 1.3 + 3.6 + 0.8 + 1.0
Glucose 29.3 1.3 13.9 20.6 17.6 16.5 5.3 16.6 17.3
+ 4.2 ___2.1 ± 4.1 ± 5.6 4- !.7 :t: 2.1 + 2.1 ± 4.7 ± 3.7
Lactate 28.5 54.1 48.9 43.5 27.4 34.2 66.1 24.3 34.5
+ 4.2 ± 3.1 + 8.6 + 5.3 + 3.7 ± 2.6 + 5.5 + 4.5 :t: 7.3
Malate 5.8 + 2.0 + 13.6 ± -5.6 + 1.4 ± 0.7 ± 6.5 ± 3.2 + 9.6 ±
Percentage of HbFe s ÷ reduced per hour. Mean + S.E., n = 5.
1.6 2.6 2.5 4.4 0.7 1.0 3.1 1.9 1.1
B-Hydroxy butyrate 11.1 3.2 29.6 0.6 4.2 5.4 6.2 4.6 9.5
+ 2.5 __. 2.6 ± 3.3 ± 3.0 ± 1.9 :'- 2.3 +__3.0 + 1.7 -t- 1.5
Plasma 33.7 29.4 31.4 38.6 32.4 37.1 56.6 29.8 40.3
+ 4.3 ± 4.6 ± 7.6 + 5.0 + 3.2 ± 4.1 ___6.0 ___3.3 + 3.4
175
Ferrihemoglobin formation and reduction in erythrocytes Table 5. Plasma/~-hydroxybutyrate (/~-hb) and glucose values in some birds fl-hb (#M) Mean S.E. n Buzzard Carrion-crow Cormorant Gull Heron Starling
394 914 639 + 52 923
2
558
i
3
5 2
Glucose (mM) (370, 418) (193-1303) (513-675) (920, 926)
In the carrion-crow and the cormorant, glucose has little or no effect on l-lbFe 3 + formation and reduction. compared with the other species. This is not reflected in plasma glucose levels. These levels are high in all bird species as compared with mammalian species (Bell, 1971), but there are considerable differences between avian species. (Erlenbach, 1939). probably reflecting dissimilarities in diet. The cormorant and the heron are both fish-eating species and are lowest in plasma glucose levels. However, these levels are still twice as high as in mammals. Glucose stimulates the H b F e 3 + formation mammals (Blaauboer et al., 1979b). Therefore, it is more likely that the differences in the effect of glucose on H b F e 3+ formation and reduction are due to dissimilarities in glucose utilization and metabolism.
Acknowledgements--We wish to thank Mr D. Dekker and Mr R. den Hartogh of Artis Zoo. Amsterdam and Mr J. Wensing of Burgers Zoo, Amhem, for their assistance and their kind permission to take blood samples from birds. Mr J. van Tussenbroek, Mr F. Alta, Mr H. Alta and Mr P. Hoogendoorn were of great help in catching starlings and carrion-crows. The assistance of Dr A. J. H. Schotman, Laboratory of Clinical Biochemistry, Clinic for Large Animal MediOne. State University of Utrecht, is acknowledged for determining glucose and ~-hydroxybutyrate. We also thank Miss M. Loots for correcting the English text. REFERENCES BI!LI. D. J. (1971) Plasma glucose. In Physioloqy and Biochemistry of the Domestic Fowl (Edited by BELL D. J. & FREEMANB. M.), pp. 913-920. Academic Press, New York. BLAAUBOERB. J., HOLSTEUNC. W. M. VAN & WIT J. G. {1975) Nitrosobenzene-induced ferrihemoglobin formation in Japanese quail erythrocytes. The significance of ferrihemoglobin reduction. Naunyn-Schmiedebero's Arch. Pharmacol. 289, 127-135.
M e a n S.E.
n
18.7 20.4 4- 2,0 I 1.3 4- 0.6 17.9 4- 0.8 I 1.1 19.9 4- 0.8
2 4 6 3
(18.0, 19.3) (17.3--26.0) (9.9-13.9) (17.1-19.5)
I
3
( 18.4--20.9)
BLAAU~:)ER B. J, HOLSTEUNC. W. M. VAN & WIT J. G. (1976) Ferrihemoglobin formation by monohydroxy aniline derivatives in erythrocytes of some avian species in comparison with mammals. Naunyn-Schmiedeberg's Arch. Pharmacol. 292, 255-258. BLAAUBOERB. J., HOLSTEIJNC. W. M. VAN & WIT J. G. (1979a) Reduction of ferribemoglobin in erythrocytcs of birds and mammals. Comp. Biochem. Physiol. 62A, No. 4. BL,AALmOL~ B. J., HOLS~IJN C. W. M. VAN & Wrr J. G. (1979b) Biochemical processes involved in ferrihemoglobin formation by monohydroxy-aniline derivatives in erythrocytes of birds and mammals. Comp. Biochem. Physiol. 62C, 199-203. Dt'CiNo T. J, CUNNINOHAMD. J. & SCHAFERE. W. (1966) Toxicity of DRC-1339 to starlings. J. Wild. M. 30, 249-253. DORRESTEING. M, BLAAU~ER B. J., MILTENBURGN. A. & DELEY P. P. 0978) A modified method of blood sampling from birds. Lab. Anita. 12, 193-194. DU~OWSKI K. M. (1962) An o-toluidine method for body fluid glucose determination. Clin. Chem. 8, 215. ERLENBACHF. (1939) Experimentelle Untersuchungen fiber den Blutzucker bei ViSgeln. Z. vergl. Physiol. 26, 121-161. KIESE M. (1974) Methemoolobinemia- A Comprehensive Treatise. CRC-Press, Cleveland. KOEMAN J. H. (1972) Side-effects of persistent pesticides and other chemicals on birds and mammals in the Netherlands. TNO-Nieuws 27, 527-632. SELL J. L., ROBBERTSW. K. & WADDELL D. G. (1963) Methemoglobin and reduced feed consumption due to feeding nitrite to chicks. Poultry Sci. 42, 1474-1476. SMITH R. P. & OLSOr~ M. V. 0973) Drug-induced methemogiobinemia. Semin. Hemat. 10, 253-268. STOEWSANDG. S. (1970) Influence of sex and dietary ascorbic acid in nitrite-induced methemoglobinemia in Japanese quail. Proc. Soc. exp. Biol. Med. 133, 1166-1168.
WILLIAM.SON D. H. & MELLANBY J. (1974) In Methoden der Enzymatische Analyse---ll, 3rd edn. (Edited by BERGMEYER H. U.), p. 1883. Verlag Chemie, Weinheim/Bergstrasse.