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Changes in Erythrocyte Metabolism Following Acute Blood Loss in Chickens1
Changes in Erythrocyte Metabolism Following Acute Blood Loss in Chickens1 SIDNEY R. JONES 2 , JOSEPH E. SMITH, and PHILIP B. BOARD Department of Pathology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506 (Received February 2, 1978)
INTRODUCTION In h u m a n s and animals millions of h e m o globin (Hb) molecules within each e r y t h r o cyte t r a n s p o r t oxygen from t h e lungs t o t h e tissues and carbon dioxide from t h e tissues t o t h e lungs. I n t r a e r y t h r o c y t i c organic phosphates—particularly 2,3-diphosphoglycerate (2,3-DPG), adenosine t r i p h o s p h a t e (ATP), and inositol p e n t a p h o s p h a t e (IP5)—bind t o h e m o g l o b i n molecules and greatly reduce oxygen delivery t o t h e tissues (Benesch and Benesch, 1 9 6 7 ; C h a n u t i n a n d Curnish, 1 9 6 7 ; Vandecasserie et al, 1 9 7 1 ; T y u m a et al, 1 9 7 1 ; Janig et al, 1 9 7 1 ; Oski a n d G o t t l i e b , 1 9 7 1 ; Ochiai et al, 1972). E r y t h r o c y t e s of m a t u r e birds lack 2,3-DPG b u t c o n t a i n high c o n c e n t r a t i o n s of 1, 3, 4 , 5, 6 m y o i n o s i t o l p e n t a p h o s p h a t e (IP5) ( J o h n s o n and T a t e , 1 9 6 9 ) . Considerable evidence n o w
'Contribution No. 78-164-J, Kansas Agricultural Experiment Station. Supported in part by USPHS grants 70119 and 12792. This study was conducted under the auspices of the United States Air Force Institute of Technology. The views expressed herein are those of the authors and do not necessarily reflect the views of the US Air Force or the Department of Defense. 2 Military designation and current assignment are Lt. Col., USAF, VC, Department of Veterinary Pathology, Armed Forces Institute of Pathology, Washington, DC 20306. 1978 Poultry Sci 57:1667-1674
s u p p o r t s IP5 as t h e major regulator of o x y g e n t r a n s p o r t in a d u l t birds just as 2,3-DPG apparently does in m a n y m a m m a l s (Oshima et al., 1 9 6 4 ; Bartels et al, 1 9 6 6 ; Mission and Freem a n , 1 9 7 2 ; Ochiai et al, 1 9 7 2 ; Isaacks et al, 1 9 7 6 a , b , c , d , e ; Borgese and Nagel, 1 9 7 7 ) . T h e discovery of 2,3-DPG in t h e e r y t h r o c y t e s of chicken e m b r y o s (Isaacks a n d Harkness, 1 9 7 5 ) a n d d u c k e m b r y o s (Borgese a n d L a m p e r t , 1 9 7 5 ) renewed interest in t h e organic p h o s phates in t h e red cells of a n u m b e r of n o n - m a m malian vertebrates. In kind and c o n c e n t r a t i o n , i n t r a e r y t h r o c y t i c organic p h o s p h a t e s in birds change during emb r y o n i c d e v e l o p m e n t and after h a t c h i n g . A T P in chickens a n d d u c k s is high during e m b r y o n i c life. It rapidly falls t o l o w levels t h e week before t h e y h a t c h , increases for a few days afterward, and t h e n levels off at a high c o n c e n t r a tion ( a b o u t 12 //moles/ml of e r y t h r o c y t e s ) in adult d u c k s or decreases t o a lower c o n c e n t r a tion ( a b o u t 3 m o l e s / m l e r y t h r o c y t e s ) in a d u l t chickens. 2,3-DPG increase t o 10 t o 12 /imoles/ ml of e r y t h r o c y t e s and t h e n falls precipitously during t h e week before hatching. In b o t h chickens and ducks, IP5 appears a week before hatching and t h e n increases steadily for a b o u t t w o m o n t h s , reaching m o r e t h a n 15 /Umoles/ml of e r y t h r o c y t e s in adult birds (Bartlett and Borgese, 1 9 7 6 ) . In t u r k e y s , 2,3-DPG is t h e major organic p h o s p h a t e only in t h e 2 3 - and 25-day e m b r y o . ATP p r e d o m i n a t e s during t h e last part of t h e last week of e m b r y o n i c life and t h e first 2 9 days after hatching; IP5 b e c o m e s a
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ABSTRACT The effect of acute blood loss on erythrocyte metabolism has been studied in a number of mammals. This study was designated to provide comparative data in chickens. Twenty to 30 ml of blood were removed from 7 chickens for 3 successive days. Packed red cell volumes were restored at the rate of 1.46% per day, and a maximum reticulocyte count (26.7%) occurred on the third day after the initial phlebotomy. Fourteen of the 16 erythrocyte enzymes measured became elevated significantly. Enzymatic activity was increased when the greatest numbers of young red cells were in the circulation and declined as the erythrocytes aged. Initial enzymatic activity was not related to the subsequent magnitude of increased activity. Inositol pentaphosphate, adenosine 5'-triphosphate, and reduced glutathione, but not 2,3-diphosphoglycerate, increased significantly in the postphlebotomy period. The markedly rapid and significant rise of inositol pentaphosphate suggested that it was controlled. That could be the way hemoglobin function is controlled during changing physiologic requirements.
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EXPERIMENTAL
Seven young adult, female White Leghorn chickens, all of the same genetic stock were taken from the Kansas State University flock. All were fed a standard poultry ration without any additional iron. The birds were divided into 2 groups: group 1 consisted of 3 birds, and group two consisted of 4 birds. Experimental Design. Birds were sampled 4 or 5 times in a 9-day period to establish control levels for the various parameters. At day 0, blood was withdrawn from each chicken at the rate of 20 to 30 ml for 3 successive days. Because blood taken for the various assays might have imposed a significant additional loss of blood, we did not do every assay on every day. Samples for hematologic examination were taken on days 0 through 9, 12, and 15; IP5 on days 0, 1, 2, 5, 8, 12, and 15; and erythrocyte enzymes and remaining metabolites on days 0, 1,2, 5, 6, 7, 8, 9, 12, and 15. Blood was collected by wing-vein or jugular venapuncture with ethylenediaminetetraacetic acid as an anticoagulant. Hematological examination required blood .5 ml; IP5, 6 ml; and erythrocyte enzymes and remaining metabolites, 1.0 ml. Hematologic Examination. Hemoglobin (Hb) was measured as the cyanmethemoglobin derivative (Henry et al., 1974), and packed cell volume (PCV) was determined in a microhematocrit centrifuge. Reticulocytes were counted in blood films stained supravitally with new methylene blue (Schalm et al, 1975). Reticulocytes were classified as described by Lucas and Jamroz(1961). Inositol Pentaphosphate (IP5) Determinations. IP5 was extracted from washed erythro-
cytes with trichloracetic acid (TCA), fractioned on anion exchange columns, identified by elution position, and quantitated by phosphate assay (Jones et al., 1978). Other Metabolites (Glycolytic Intermediates and Glutathione). Adenosine-5'-triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG) were measured according to Beutler's methods (1971). Reduced glutathione (GSH) concentration was measured as the 5,5'-dithiobis (2-nitrobenzoic acid) derivative (Beutler et al., 1963). Enzyme Assays. Leukocytes and platelets, removed by passing whole, anticoagulated blood through cotton balls, were washed with .154 M KC1 (Beutler, 1975). Red cells diluted in .154 M KC1 were then carefully layered on top of a .25 M sucrose solution and centrifuged at 3,000 g for 15 min in a refrigerated centrifuge (Blincoe, 1974). The supernatant layer of KCl and sucrose was decanted and discarded. Washed red cells (.2 ml) were added to 2.0 ml of ice-cold lysing solution, mixed at 4 C for 15 min, and then centrifuged at 17,000 g for 15 min at 4 C. The lysing solution consisted of 1 part water, 1 part .05 M imidazole, .7 part .154 M KCl, and .25 part saturated digitonin. Hemolyzate solutions for enzyme assays were made in various dilutions with ice-cold deionized water. Enzyme concentrations in hemolyzates were assayed by methods described previously (Smith and Kiefer, 1973; Smith et al, 1970), except that phthalate esters were not used to remove leukocytes. Also, the .5 M imidazole buffer (pH 7.2) was substituted with TrisEDTA buffer (1 M Tris-HCl, 5 mM EDTA, pH 8.0) in some of the systems (Beutler, 1975). Statistical Evaluation. For statistical evaluation, the experiment was divided into 5 time periods (Fig. 1 and 2): A, control, or prephlebotomy period; B, phlebotomy period with low PCV and increased reticulocytes; C, period of increasing PCV and decreasing reticulocytes; D, period of normal or near normal PCV and reticulocytes; and E, period of high PCV and normal reticulocytes. The variation between groups and between days within periods was evaluated, and the effects of phlebotomy were compared with the control period by analysis of variance. A separate analysis of variance for 2,3-DPG was carried out on group 2 because our control period for group 1 was misssing. RESULTS
The initial day of phlebotomy was desig-
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major phosphate 2 or 3 weeks after hatching (Isaacks et al, 1976e). Birds have a more dynamic hemopoietic system than do mammals. Regeneration from phlebotomy or anemia is vigorous and rapid, requiring only about one week in chickens but 3 weeks in most mammals (Wirth, 1950). Reticulocytes in phlebotomized chickens rise spectacularly from 0 to 33%. The objectives of this experiment were to measure a number of erythrocyte factors in chickens, to study age-dependent changes in glycolytic and other enzymes of chicken erythrocytes, and to detect any reappearance of 2,3-DPG in young erythrocytes of adult chickens following blood loss.
ERYTHROCYTE METABOLISM AND ACUTE BLOOD LOSS
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FIG. 1. Packed cell volume (PCV), reticulocytes, and hemoglobin (Hb) before, during and after phlebotomy in chickens from group 1. For statistical evaluation, the experiment was divided into five time periods, A,B,C,D,E.
nated as day 0. Phlebotomies on the 3 successive days decreased the PCVs from 30.1 ± .69 to 20 or below (Fig. 1 and 2). In most birds, hemoglobin levels were lowest (4.86 g/ 100 ml ± .23) on the second or third day after the initial phlebotomy (day 0). In all birds, reticulocytes reached their maximum (mean 26.7%, range 19 to 39%) on the third day after initial phlebotomy (Fig. 1 and 2). PCVs increased linearly (1.46% ± .094) per day for 9 or 10 days from their nadir during the phlebotomy period (period B) to levels significantly (P<.001) higher in period E than in the phlebotomy control period (Fig. 1 and 2). All enzymes measured, except phosphofructokinase and glucose-6-phosphate dehydrogenase, had significantly elevated concentrations during one or more periods after the initial phlebotomy (Table 1; Figs. 3 and 4). Enzymes with elevations significant at . 1 % confi-
- 1 0 1 2 3 4 5 6 7 8 9 DAYS (0 - I I I DAY OF PHLEBOTOMY)
FIG. 2. Packed cell volume (PCV), reticulocytes and hemoglobin (Hb) before, during and after phlebotomy in chickens from group 2.
dence limit were glucose phosphate isomerase, aldolase, phosphoglycerate kinase, monophosphoglycerate mutase, lactate dehydrogenase, glutathione peroxidase, and adenylate kinase. Those with maximum elevations significant at 1.0% confidence limit included hexokinase, triose phosphate isomerase, glyceraldehyde-3phosphate dehydrogenase, enolase, and glutathione reductase. Maximum elevations of pyruvate kinase and 6-phosphogluconate dehydrogenase were significant at 5.0% confidence limit. The concentration of phosphofructokinase, though insignificantly elevated in period C, was slightly lower in periods B, D, and E than in the control period. The concentration of glucose-6-phosphate dehydrogenase was significantly (P<.05) lower in periods B and E and only slightly elevated in periods C and D. Of
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2
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JONES ET AL. TABLE 1. Control erythrocyte enzyme activity in chickens Mean a
SEt>
3.4 20.9 10.3 2.18 291. 76.5 29.3 19.7 18.8 11.6 60.0
.29 .74 .5 .11 23.3 1.89 .845 .92 .49 3.1 1.8
10.7 1.31 1.88 90.4 368.
.36 .037 .128 8.86 14.4
Glycolytic pathway enzymes
Hexose monophosphate pathway and other enzymes Glucose-6-phosphate dehydrogenase 6-phosphogluconate dehydrogenase Glutathione reductase Glutathione peroxidase Adenylate kinase a
Mean of 4 samples from each of 7 chickens expressed as /umole/g hemoglobin/min.
be
Standard error.
the significantly elevated enzymes, all except triosephosphate isomerase increased most significantly in periods C, D, and E or all three. Triosephosphate isomerase increased significantly during periods B and C. Adolase, the enzyme with the third lowest initial levels (2.18 + .11 yumoles/g Hb/min), and glutathione peroxidase, the enzyme with the third highest initial levels (90.4 ± 8.86 /Umoles/g Hb/min), made the greatest postphlebotomy increases: 205% and 240%, respectively. For all enzymes, the highest postphlebotomy levels were all in period C or D and the lowest, in period B or E—after significant postphlebotomy elevations; triosephosphate isomerase, monophosphoglycerate kinase, and 6-phosphogluconate dehydrogenase were significantly lower than were control values at the end of the study. As described above, glucose-6-phosphate dehydrogenase also was decreased significantly in period E. Glutathione peroxidase was the only enzme still markedly elevated at the end of the study period, 15 days after the initial phlebotomy. The phosphorylated metabolic intermediates IP5 and ATP increased significantly in periods B and C, respectively; 2,3-DPG increased slightly in period B then declined (Table 2, Fig. 5). After the significant peak period, ATP declined
to slightly below prephlebotomy levels; IPS gradually declined but generally remained slightly above prephlebotomy control concentrations. Reduced glutathione was significantly elevated in periods C and D (Table 2; Fig. 5). DISCUSSION Erythropoiesis following phlebotomy was as rapid and vigorous in our chickens as in chickens previously studied (Wirth, 1950; Lucas and Jamroz, 1961). The rapid postphlebotomy increase in the PCV of our chickens (1.46% per day) was comparable to that of dogs (1.57% per day, Smith and Agar, 1975). Such rapid restoration of PCV, and thus the oxygen-carrying capacity of the blood of phlebotomized chickens and dogs, is considerably greater than that of horses (.67% per day; Smith and Agar, 1976) and sheep (.87% per day; Todd and Ross, 1968). Two postphlebotomy phenomena in our chickens indicated that erythrocyte enzymatic activity was age dependent and declined as age of avian red cells increased. First, enzymatic activity increased significantly in all but two enzymes concomitant with the decrease in mean erythrocyte age caused by the influx of young erythrocytes from the bone marrow. Second,
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Hexokinase Glucose phosphate isomerase Phosphofructose kinase Aldolase Triose phosphate isomerase Glyceraldehyde-3-phosphate dehydrogenase Phosphogiycerate kinase Monophosphoglyceromutase Enolase Pyruvate kinase Lactic dehydrogenase
ERYTHROCYTE METABOLISM AND ACUTE BLOOD LOSS * « 0.1%
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FIG. 3. Erythrocytic enzyme activity before, during and after phlebotomy. Each point represents the mean of all seven chickens for all samples during the period, expressed as a percentage of the control. HK is hexokinase; GPI, glucose-6-phosphate isomerase; PFK, phosphofructokinase; TPI, triose phosphate isomerase; GAPD, glyceraldehyde-3-phosphate dehydrogenase; PGK, phosphoglycerate kinase; MPGM, monophosphoglycerate mutase. *, a = significant elevation at indicated probability.
the concentration of almost all enzymes decreased to near or below prephlebotomy levels by the end of the experiment (period E). Similar results have been observed in most mammalian erythrocytes following loss of blood (Smith and Agar, 1975).
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FIG. 4. Erythrocytic enzyme activity before, during, and after phlebotomy. PK is pyruvate kinase; LDH, lactate dehydrogenase; G6PD, glucoses-phosphate dehydrogenase; 6PGD, 6-phosphogluconate dehydrogenase; GR, glutathione reductase; GSH-Px, glutathione peroxidase; AK, adenylate kinase. *, o, + = significant elevation at indicated probability.
Those enzymes most consistently higher in concentration in young erythrocytes of humans (hexokinase, pyruvate kinase, and glucosesphosphate dehydrogenase) were not elevated exceptionally in chickens. In that regard, avian red cells apparently respond in a manner dissimilar to that in human cells. In dogs (Smith and Agar, 1975) and humans
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JONES ET AL.
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FIG. 5. Erythrocytic metabolite concentrations before, during and after phlebotomy. IP5 is inositol pentaphosphate; ATP, adenosine-5'-triphosphate; 2,3-DPG, 2,3-diphosphoglycerate; GSH, reduced glutathione. * = significant elevation at indicated probability.
(Chapman and Schaumburg, 1967), glycolytic enzymes with the lowest initial levels tend to TABLE 2. Control levels of IPS, ATP, DPG, and GSH undergo the greatest postphlebotomy percentage increases. No such trend could be identified SEb Mean a Metabolite in our chickens. Prephlebotomy GSH levels in our chickens IP5 .636 15.9 (10.4 /xmoles/g Hb) were not as high as in 3.50 .08 ATP chickens of a previous study (18.5 £!moles/g DPG .0181 .0024 Hb; Smith, 1974). Dramatic GSH increases in .42 GSH 10.4 anemic sheep are known to be associated with Mean of 4 samples from each of 7 chickens ex- increased substrate (ie, glutamate) rather than pressed as |umole/g hemoglobin except DPG in only 4 with changes in the glutathione-synthesizing enbirds (8 to 11). zymes (Smith, 1977). Elevated GSH levels in chickens are associated with young cells (reticuStandard error.
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o c O O
ERYTHROCYTE METABOLISM AND ACUTE BLOOD LOSS
REFERENCES Bartels, M., G. Hiller, and W. Reinhardt, 1966. Oxygen affinity of chicken blood before and after hatching. Resp. Physiol. 1:345-356. Bartlett, G. R., and T. A. Borgese, 1976. Phosphate compounds in red cells of the chicken and duck embryo and hatching. Comp. Biochem. Physiol. 55A:2O7-210. Benesch, R., and R. E. Benesch, 1967. The effect of organic phosphates from human erythrocytes on the allosteric properties of hemoglobin. Biochem. Biophys. Res. Commun. 26:162-167. Beutler, E., 1971. Red cell metabolism. A manual of biochemical methods. Grune and Stratton, New York. Beutler, E., 1975. Red cell metabolism. A manual of biochemical methods. 2nd ed. Grune and Stratton, New York. Beutler, Ei, O. Duron, and B. M. Kelly, 1963. Improved method for the determination of blood glutathione. J. Lab. Clin. Med. 6 1 : 8 8 2 - 8 8 8 . Blincoe, C , 1974 . The simultaneous separation and washing of erythrocytes. Clin. Chem. Acta 57: 297-300. Borgese, T. A., and L. M. Lampert, 1975. Duck red cell 2,3-diphosphoglycerate: Its presence in the embryo and its disappearance in the adult. Biochem. Biophys. Res. Commun. 65:822-827. Borgese, T. A., and R. L. Nagel, 1977. Differential effects of 2,3-DPG, ATP, and inositol pentaphosphate (IPS) on the oxygen equilibria of duck embryonic, fetal, and adult hemoglobins. Comp. Biochem. Physiol. 56A:539-543. Bunn, H. F., U. S. Seal, and A. F. Scott, 1974. The role of 2,3-diphosphoglycerate in mediating hemoglobin functions of mammalian red cells. Annu. N.Y. Acad. Sci. 241:498-512. Chanutin, A., and R. R. Curnish, 1967. Effect of organic phosphates on the oxygen equilibrium of human erythrocytes. Arch. Biochem. Biophys. 121:96-102. Chapman, R., and L. Schaumburg, 1967. Glycolysis and glycolytic enzyme activity of aging red cells in
man. Brit. J. Haematol. 13:665-678. Henry, R. J., D. C. Cannon, and J. W. Winkelman, 1974. Clinical chemistry principles and technics. 2nd ed. Harper and Row, New York. Isaacks, R. E., and D. R. Harkness, 1975. 2,3-Diphosphoglycerate in erythrocytes of chick embryos. Science 189:393-394. Isaacks, R. E., D. R. Harkness, J. L. Adler, and P. H. Goldman, 1976c. Studies on avian erythrocyte metabolism. III. Effect of organic phosphates on oxygen affinity of embryonic and adult-type hemoglobins of the chick embryo. Arch. Biochem. Biophys. 173:114-120. Isaacks, R. E., D. R. Harkness, J. L. Adler, C.-Y. Kim, P. H. Goldman, and S. Roth, 1976e. Studies on avian erythrocyte metabolism. V. Relationship between the major phosphorylated metabolic intermediates and whole blood oxygen affinity in embryos and poults of the domestic turkey (Meleagris gallopavo). Poultry Sci. 55:1788-1794. Isaacks, R. E., D. R. Harkness, G. A. Froeman, P. H. Goldman, J. L. Adler, S. A. Sussman, and S. Roth, 1976b. Studies on avian erythrocyte metabolism. II. Relationship between the major phosphorylated metabolic intermediates and oxygen affinity of whole blood in chick embryos and chicks. Comp. Biochem. Physiol. 53A:151-156. Isaacks, R. E., D. R. Harkness, G. A. Froeman, and S. A. Sussman, 1976a. Studies on avian erythrocyte metabolism. I. Procedure for separation and quantitation of the major phosphorylated metabolic intermediates by anion exchange chromatography. Comp. Biochem. Physiol. 53A:95-99. Isaacks, R. E., D. R. Harkness, R. N. Sampsell, J. L. Adler, C.-Y. Kim, and P. H. Goldman, 1976d. Studies on avian erythrocyte metabolism. IV. Relationship between the major phosphorylated metabolic intermediates and oxygen affinity of whole blood in adults and embryos in several Galliformes. Comp. Biochem. Physiol. 55A:29-33. Janig, G. R., K. Ruckpaul, and F. Jung, 1971. Interaction of hemoglobin with ions binding of inositol hexaphosphate to human hemoglobin. Fed. of Europe. Biol. Sci. Letters 17:173-176. Johnson, L. F., and M. E. Tate, 1969. Structure of myo-inositol pentaphosphates. Annu. N.Y. Acad. Sci. 165:526-523. Jones, S. R., J. E. Smith and P. B. Board, 1978. An assay procedure for inositol pentaphosphate in avian erythrocytes. Poultry Sci. 57:0000. Lucas, A. M., and C. Jamroz, 1961. Atlas of avian hematology. USDA Monograph 25, Washington, DC. Mission, B. H., and B. M. Freeman, 1972. Organic phosphates and oxygen affinity of chick blood before and after hatching. Resp. Physiol. 14:343— 352. Ochiai, T., T. Gotch, and K. Shikama, 1972. Effect of intracellular organic phosphates on the oxygen equilibrium curve of chicken hemoglobin. Arch. Biochem. Biophys. 149:316-322. Oshima, M., T. G. Taylor, and A. Williams, 1964. Variations in the concentration of phytic acid in the blood of the domestic fowl. Biochem. J. 92: 42-46. Oski, F. A., and A. J. Gottlieb, 1971. The interrelaships between red blood cell metabolites, hemo-
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locytes) rather t h a n with old cells taking u p glutamate. After t h e initial p h l e b o t o m y , IP5 increased before n e w cells reached circulation. Increases in IP5 s h o u l d have shifted t h e hemoglobinoxygen dissociation curve t o t h e right and allowed t h e available h e m o g l o b i n t o deliver m o r e oxygen t o t h e tissues. T h e rapid rise of IP5 suggested t h a t it was controlled metabolically. This control m e c h a n i s m is quite sensitive t o t h e changing needs of chickens, just as t h e 2,3-DPG regulation of o x y g e n binding and delivery is sensitive t o changing needs of m a m m a l s (Bunn et ai, 1 9 7 4 ) . T h e exact m e c h a n i s m regulating t h e c o n c e n t r a t i o n of IP5 apparently is d u e entirely t o a metabolic effect of t h e m a t u r e red cells present in circulation although elevation of e n z y m e levels is clearly a new-cell effect.
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JONES ET AL. cells. Enzyme 1 4 : 7 6 - 8 1 . Smith, J., M. McCants, P. Parks and E. Jones, 1970. The influence of age on bovine erythrocyte enzymes. Biol. Neonate 15:169-175. Todd, J., and J. Ross, 1968. Biochemical and hematological changes in the blood of normal sheep following phlebotomy. Brit. Vet. J. 127:353. Tyuma, I., K. Imai and K. Shimizu, 1971. Effect of inositol hexaphosphate and other organic phosphates on the cooperativity in oxygen binding of human hemoglobins. Biochem. Biophys. Res. Commun. 44:682-686. Vandecasserie, C , A. G. Schnek, and J. Leonis, 1971. Oxygen affinity studies of avian hemoglobins. Chick and pigeon. Europ. J. Biochem. 24:284— 287. Wirth, D., 1950. Grundlagen einer klinischen hematologic der haustiere. Cited from Lucas and Jamroz, Avian hematology. USDA Monograph, 1961, Washington, DC.
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globin, and the oxygen-equilibrium curve. Prog. Hematol. 7:33-67. Schalm, 0 . W., N. C. Jain, and E. J. Carroll, 1975. Veterinary hematology. 3rd ed. Lea and Febiger, Philadelphia, PA. Smith, J. E., 1974. Relationship of in vivo erythrocyte glutathione flux to the oxidized glutathione transport system. J. Lab. Clin. Med. 83:444-450. Smith, J. E., 1977. Elevated erythrocyte glutathione associated with elevated substrate in high- and lowglutathione sheep. Biochem. Biophys. Acta 496: 516-520. Smith, J. E., and N. S. Agar, 1975. The effect of phlebotomy on canine erythrocyte metabolism. Res. Vet. Sci. 18:231-236. Smith, J. E., and N. S. Agar, 1976. Studies on erythrocyte metabolism following acute blood loss in the horse. Equine Vet. J. 8:34-37. Smith, J. E., and S. Kiefer, 1973. Comparative erythrocyte metabolism breed differences using canine
INTRODUCTION In h u m a n s and animals millions of h e m o globin (Hb) molecules within each e r y t h r o cyte t r a n s p o r t oxygen from t h e lungs t o t h e tissues and carbon dioxide from t h e tissues t o t h e lungs. I n t r a e r y t h r o c y t i c organic phosphates—particularly 2,3-diphosphoglycerate (2,3-DPG), adenosine t r i p h o s p h a t e (ATP), and inositol p e n t a p h o s p h a t e (IP5)—bind t o h e m o g l o b i n molecules and greatly reduce oxygen delivery t o t h e tissues (Benesch and Benesch, 1 9 6 7 ; C h a n u t i n a n d Curnish, 1 9 6 7 ; Vandecasserie et al, 1 9 7 1 ; T y u m a et al, 1 9 7 1 ; Janig et al, 1 9 7 1 ; Oski a n d G o t t l i e b , 1 9 7 1 ; Ochiai et al, 1972). E r y t h r o c y t e s of m a t u r e birds lack 2,3-DPG b u t c o n t a i n high c o n c e n t r a t i o n s of 1, 3, 4 , 5, 6 m y o i n o s i t o l p e n t a p h o s p h a t e (IP5) ( J o h n s o n and T a t e , 1 9 6 9 ) . Considerable evidence n o w
'Contribution No. 78-164-J, Kansas Agricultural Experiment Station. Supported in part by USPHS grants 70119 and 12792. This study was conducted under the auspices of the United States Air Force Institute of Technology. The views expressed herein are those of the authors and do not necessarily reflect the views of the US Air Force or the Department of Defense. 2 Military designation and current assignment are Lt. Col., USAF, VC, Department of Veterinary Pathology, Armed Forces Institute of Pathology, Washington, DC 20306. 1978 Poultry Sci 57:1667-1674
s u p p o r t s IP5 as t h e major regulator of o x y g e n t r a n s p o r t in a d u l t birds just as 2,3-DPG apparently does in m a n y m a m m a l s (Oshima et al., 1 9 6 4 ; Bartels et al, 1 9 6 6 ; Mission and Freem a n , 1 9 7 2 ; Ochiai et al, 1 9 7 2 ; Isaacks et al, 1 9 7 6 a , b , c , d , e ; Borgese and Nagel, 1 9 7 7 ) . T h e discovery of 2,3-DPG in t h e e r y t h r o c y t e s of chicken e m b r y o s (Isaacks a n d Harkness, 1 9 7 5 ) a n d d u c k e m b r y o s (Borgese a n d L a m p e r t , 1 9 7 5 ) renewed interest in t h e organic p h o s phates in t h e red cells of a n u m b e r of n o n - m a m malian vertebrates. In kind and c o n c e n t r a t i o n , i n t r a e r y t h r o c y t i c organic p h o s p h a t e s in birds change during emb r y o n i c d e v e l o p m e n t and after h a t c h i n g . A T P in chickens a n d d u c k s is high during e m b r y o n i c life. It rapidly falls t o l o w levels t h e week before t h e y h a t c h , increases for a few days afterward, and t h e n levels off at a high c o n c e n t r a tion ( a b o u t 12 //moles/ml of e r y t h r o c y t e s ) in adult d u c k s or decreases t o a lower c o n c e n t r a tion ( a b o u t 3 m o l e s / m l e r y t h r o c y t e s ) in a d u l t chickens. 2,3-DPG increase t o 10 t o 12 /imoles/ ml of e r y t h r o c y t e s and t h e n falls precipitously during t h e week before hatching. In b o t h chickens and ducks, IP5 appears a week before hatching and t h e n increases steadily for a b o u t t w o m o n t h s , reaching m o r e t h a n 15 /Umoles/ml of e r y t h r o c y t e s in adult birds (Bartlett and Borgese, 1 9 7 6 ) . In t u r k e y s , 2,3-DPG is t h e major organic p h o s p h a t e only in t h e 2 3 - and 25-day e m b r y o . ATP p r e d o m i n a t e s during t h e last part of t h e last week of e m b r y o n i c life and t h e first 2 9 days after hatching; IP5 b e c o m e s a
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ABSTRACT The effect of acute blood loss on erythrocyte metabolism has been studied in a number of mammals. This study was designated to provide comparative data in chickens. Twenty to 30 ml of blood were removed from 7 chickens for 3 successive days. Packed red cell volumes were restored at the rate of 1.46% per day, and a maximum reticulocyte count (26.7%) occurred on the third day after the initial phlebotomy. Fourteen of the 16 erythrocyte enzymes measured became elevated significantly. Enzymatic activity was increased when the greatest numbers of young red cells were in the circulation and declined as the erythrocytes aged. Initial enzymatic activity was not related to the subsequent magnitude of increased activity. Inositol pentaphosphate, adenosine 5'-triphosphate, and reduced glutathione, but not 2,3-diphosphoglycerate, increased significantly in the postphlebotomy period. The markedly rapid and significant rise of inositol pentaphosphate suggested that it was controlled. That could be the way hemoglobin function is controlled during changing physiologic requirements.
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EXPERIMENTAL
Seven young adult, female White Leghorn chickens, all of the same genetic stock were taken from the Kansas State University flock. All were fed a standard poultry ration without any additional iron. The birds were divided into 2 groups: group 1 consisted of 3 birds, and group two consisted of 4 birds. Experimental Design. Birds were sampled 4 or 5 times in a 9-day period to establish control levels for the various parameters. At day 0, blood was withdrawn from each chicken at the rate of 20 to 30 ml for 3 successive days. Because blood taken for the various assays might have imposed a significant additional loss of blood, we did not do every assay on every day. Samples for hematologic examination were taken on days 0 through 9, 12, and 15; IP5 on days 0, 1, 2, 5, 8, 12, and 15; and erythrocyte enzymes and remaining metabolites on days 0, 1,2, 5, 6, 7, 8, 9, 12, and 15. Blood was collected by wing-vein or jugular venapuncture with ethylenediaminetetraacetic acid as an anticoagulant. Hematological examination required blood .5 ml; IP5, 6 ml; and erythrocyte enzymes and remaining metabolites, 1.0 ml. Hematologic Examination. Hemoglobin (Hb) was measured as the cyanmethemoglobin derivative (Henry et al., 1974), and packed cell volume (PCV) was determined in a microhematocrit centrifuge. Reticulocytes were counted in blood films stained supravitally with new methylene blue (Schalm et al, 1975). Reticulocytes were classified as described by Lucas and Jamroz(1961). Inositol Pentaphosphate (IP5) Determinations. IP5 was extracted from washed erythro-
cytes with trichloracetic acid (TCA), fractioned on anion exchange columns, identified by elution position, and quantitated by phosphate assay (Jones et al., 1978). Other Metabolites (Glycolytic Intermediates and Glutathione). Adenosine-5'-triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG) were measured according to Beutler's methods (1971). Reduced glutathione (GSH) concentration was measured as the 5,5'-dithiobis (2-nitrobenzoic acid) derivative (Beutler et al., 1963). Enzyme Assays. Leukocytes and platelets, removed by passing whole, anticoagulated blood through cotton balls, were washed with .154 M KC1 (Beutler, 1975). Red cells diluted in .154 M KC1 were then carefully layered on top of a .25 M sucrose solution and centrifuged at 3,000 g for 15 min in a refrigerated centrifuge (Blincoe, 1974). The supernatant layer of KCl and sucrose was decanted and discarded. Washed red cells (.2 ml) were added to 2.0 ml of ice-cold lysing solution, mixed at 4 C for 15 min, and then centrifuged at 17,000 g for 15 min at 4 C. The lysing solution consisted of 1 part water, 1 part .05 M imidazole, .7 part .154 M KCl, and .25 part saturated digitonin. Hemolyzate solutions for enzyme assays were made in various dilutions with ice-cold deionized water. Enzyme concentrations in hemolyzates were assayed by methods described previously (Smith and Kiefer, 1973; Smith et al, 1970), except that phthalate esters were not used to remove leukocytes. Also, the .5 M imidazole buffer (pH 7.2) was substituted with TrisEDTA buffer (1 M Tris-HCl, 5 mM EDTA, pH 8.0) in some of the systems (Beutler, 1975). Statistical Evaluation. For statistical evaluation, the experiment was divided into 5 time periods (Fig. 1 and 2): A, control, or prephlebotomy period; B, phlebotomy period with low PCV and increased reticulocytes; C, period of increasing PCV and decreasing reticulocytes; D, period of normal or near normal PCV and reticulocytes; and E, period of high PCV and normal reticulocytes. The variation between groups and between days within periods was evaluated, and the effects of phlebotomy were compared with the control period by analysis of variance. A separate analysis of variance for 2,3-DPG was carried out on group 2 because our control period for group 1 was misssing. RESULTS
The initial day of phlebotomy was desig-
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major phosphate 2 or 3 weeks after hatching (Isaacks et al, 1976e). Birds have a more dynamic hemopoietic system than do mammals. Regeneration from phlebotomy or anemia is vigorous and rapid, requiring only about one week in chickens but 3 weeks in most mammals (Wirth, 1950). Reticulocytes in phlebotomized chickens rise spectacularly from 0 to 33%. The objectives of this experiment were to measure a number of erythrocyte factors in chickens, to study age-dependent changes in glycolytic and other enzymes of chicken erythrocytes, and to detect any reappearance of 2,3-DPG in young erythrocytes of adult chickens following blood loss.
ERYTHROCYTE METABOLISM AND ACUTE BLOOD LOSS
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FIG. 1. Packed cell volume (PCV), reticulocytes, and hemoglobin (Hb) before, during and after phlebotomy in chickens from group 1. For statistical evaluation, the experiment was divided into five time periods, A,B,C,D,E.
nated as day 0. Phlebotomies on the 3 successive days decreased the PCVs from 30.1 ± .69 to 20 or below (Fig. 1 and 2). In most birds, hemoglobin levels were lowest (4.86 g/ 100 ml ± .23) on the second or third day after the initial phlebotomy (day 0). In all birds, reticulocytes reached their maximum (mean 26.7%, range 19 to 39%) on the third day after initial phlebotomy (Fig. 1 and 2). PCVs increased linearly (1.46% ± .094) per day for 9 or 10 days from their nadir during the phlebotomy period (period B) to levels significantly (P<.001) higher in period E than in the phlebotomy control period (Fig. 1 and 2). All enzymes measured, except phosphofructokinase and glucose-6-phosphate dehydrogenase, had significantly elevated concentrations during one or more periods after the initial phlebotomy (Table 1; Figs. 3 and 4). Enzymes with elevations significant at . 1 % confi-
- 1 0 1 2 3 4 5 6 7 8 9 DAYS (0 - I I I DAY OF PHLEBOTOMY)
FIG. 2. Packed cell volume (PCV), reticulocytes and hemoglobin (Hb) before, during and after phlebotomy in chickens from group 2.
dence limit were glucose phosphate isomerase, aldolase, phosphoglycerate kinase, monophosphoglycerate mutase, lactate dehydrogenase, glutathione peroxidase, and adenylate kinase. Those with maximum elevations significant at 1.0% confidence limit included hexokinase, triose phosphate isomerase, glyceraldehyde-3phosphate dehydrogenase, enolase, and glutathione reductase. Maximum elevations of pyruvate kinase and 6-phosphogluconate dehydrogenase were significant at 5.0% confidence limit. The concentration of phosphofructokinase, though insignificantly elevated in period C, was slightly lower in periods B, D, and E than in the control period. The concentration of glucose-6-phosphate dehydrogenase was significantly (P<.05) lower in periods B and E and only slightly elevated in periods C and D. Of
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2
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JONES ET AL. TABLE 1. Control erythrocyte enzyme activity in chickens Mean a
SEt>
3.4 20.9 10.3 2.18 291. 76.5 29.3 19.7 18.8 11.6 60.0
.29 .74 .5 .11 23.3 1.89 .845 .92 .49 3.1 1.8
10.7 1.31 1.88 90.4 368.
.36 .037 .128 8.86 14.4
Glycolytic pathway enzymes
Hexose monophosphate pathway and other enzymes Glucose-6-phosphate dehydrogenase 6-phosphogluconate dehydrogenase Glutathione reductase Glutathione peroxidase Adenylate kinase a
Mean of 4 samples from each of 7 chickens expressed as /umole/g hemoglobin/min.
be
Standard error.
the significantly elevated enzymes, all except triosephosphate isomerase increased most significantly in periods C, D, and E or all three. Triosephosphate isomerase increased significantly during periods B and C. Adolase, the enzyme with the third lowest initial levels (2.18 + .11 yumoles/g Hb/min), and glutathione peroxidase, the enzyme with the third highest initial levels (90.4 ± 8.86 /Umoles/g Hb/min), made the greatest postphlebotomy increases: 205% and 240%, respectively. For all enzymes, the highest postphlebotomy levels were all in period C or D and the lowest, in period B or E—after significant postphlebotomy elevations; triosephosphate isomerase, monophosphoglycerate kinase, and 6-phosphogluconate dehydrogenase were significantly lower than were control values at the end of the study. As described above, glucose-6-phosphate dehydrogenase also was decreased significantly in period E. Glutathione peroxidase was the only enzme still markedly elevated at the end of the study period, 15 days after the initial phlebotomy. The phosphorylated metabolic intermediates IP5 and ATP increased significantly in periods B and C, respectively; 2,3-DPG increased slightly in period B then declined (Table 2, Fig. 5). After the significant peak period, ATP declined
to slightly below prephlebotomy levels; IPS gradually declined but generally remained slightly above prephlebotomy control concentrations. Reduced glutathione was significantly elevated in periods C and D (Table 2; Fig. 5). DISCUSSION Erythropoiesis following phlebotomy was as rapid and vigorous in our chickens as in chickens previously studied (Wirth, 1950; Lucas and Jamroz, 1961). The rapid postphlebotomy increase in the PCV of our chickens (1.46% per day) was comparable to that of dogs (1.57% per day, Smith and Agar, 1975). Such rapid restoration of PCV, and thus the oxygen-carrying capacity of the blood of phlebotomized chickens and dogs, is considerably greater than that of horses (.67% per day; Smith and Agar, 1976) and sheep (.87% per day; Todd and Ross, 1968). Two postphlebotomy phenomena in our chickens indicated that erythrocyte enzymatic activity was age dependent and declined as age of avian red cells increased. First, enzymatic activity increased significantly in all but two enzymes concomitant with the decrease in mean erythrocyte age caused by the influx of young erythrocytes from the bone marrow. Second,
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Hexokinase Glucose phosphate isomerase Phosphofructose kinase Aldolase Triose phosphate isomerase Glyceraldehyde-3-phosphate dehydrogenase Phosphogiycerate kinase Monophosphoglyceromutase Enolase Pyruvate kinase Lactic dehydrogenase
ERYTHROCYTE METABOLISM AND ACUTE BLOOD LOSS * « 0.1%
-I
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D - 1.0%
1671 + - 5.0%
1
FIG. 3. Erythrocytic enzyme activity before, during and after phlebotomy. Each point represents the mean of all seven chickens for all samples during the period, expressed as a percentage of the control. HK is hexokinase; GPI, glucose-6-phosphate isomerase; PFK, phosphofructokinase; TPI, triose phosphate isomerase; GAPD, glyceraldehyde-3-phosphate dehydrogenase; PGK, phosphoglycerate kinase; MPGM, monophosphoglycerate mutase. *, a = significant elevation at indicated probability.
the concentration of almost all enzymes decreased to near or below prephlebotomy levels by the end of the experiment (period E). Similar results have been observed in most mammalian erythrocytes following loss of blood (Smith and Agar, 1975).
r
FIG. 4. Erythrocytic enzyme activity before, during, and after phlebotomy. PK is pyruvate kinase; LDH, lactate dehydrogenase; G6PD, glucoses-phosphate dehydrogenase; 6PGD, 6-phosphogluconate dehydrogenase; GR, glutathione reductase; GSH-Px, glutathione peroxidase; AK, adenylate kinase. *, o, + = significant elevation at indicated probability.
Those enzymes most consistently higher in concentration in young erythrocytes of humans (hexokinase, pyruvate kinase, and glucosesphosphate dehydrogenase) were not elevated exceptionally in chickens. In that regard, avian red cells apparently respond in a manner dissimilar to that in human cells. In dogs (Smith and Agar, 1975) and humans
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i
JONES ET AL.
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FIG. 5. Erythrocytic metabolite concentrations before, during and after phlebotomy. IP5 is inositol pentaphosphate; ATP, adenosine-5'-triphosphate; 2,3-DPG, 2,3-diphosphoglycerate; GSH, reduced glutathione. * = significant elevation at indicated probability.
(Chapman and Schaumburg, 1967), glycolytic enzymes with the lowest initial levels tend to TABLE 2. Control levels of IPS, ATP, DPG, and GSH undergo the greatest postphlebotomy percentage increases. No such trend could be identified SEb Mean a Metabolite in our chickens. Prephlebotomy GSH levels in our chickens IP5 .636 15.9 (10.4 /xmoles/g Hb) were not as high as in 3.50 .08 ATP chickens of a previous study (18.5 £!moles/g DPG .0181 .0024 Hb; Smith, 1974). Dramatic GSH increases in .42 GSH 10.4 anemic sheep are known to be associated with Mean of 4 samples from each of 7 chickens ex- increased substrate (ie, glutamate) rather than pressed as |umole/g hemoglobin except DPG in only 4 with changes in the glutathione-synthesizing enbirds (8 to 11). zymes (Smith, 1977). Elevated GSH levels in chickens are associated with young cells (reticuStandard error.
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o c O O
ERYTHROCYTE METABOLISM AND ACUTE BLOOD LOSS
REFERENCES Bartels, M., G. Hiller, and W. Reinhardt, 1966. Oxygen affinity of chicken blood before and after hatching. Resp. Physiol. 1:345-356. Bartlett, G. R., and T. A. Borgese, 1976. Phosphate compounds in red cells of the chicken and duck embryo and hatching. Comp. Biochem. Physiol. 55A:2O7-210. Benesch, R., and R. E. Benesch, 1967. The effect of organic phosphates from human erythrocytes on the allosteric properties of hemoglobin. Biochem. Biophys. Res. Commun. 26:162-167. Beutler, E., 1971. Red cell metabolism. A manual of biochemical methods. Grune and Stratton, New York. Beutler, E., 1975. Red cell metabolism. A manual of biochemical methods. 2nd ed. Grune and Stratton, New York. Beutler, Ei, O. Duron, and B. M. Kelly, 1963. Improved method for the determination of blood glutathione. J. Lab. Clin. Med. 6 1 : 8 8 2 - 8 8 8 . Blincoe, C , 1974 . The simultaneous separation and washing of erythrocytes. Clin. Chem. Acta 57: 297-300. Borgese, T. A., and L. M. Lampert, 1975. Duck red cell 2,3-diphosphoglycerate: Its presence in the embryo and its disappearance in the adult. Biochem. Biophys. Res. Commun. 65:822-827. Borgese, T. A., and R. L. Nagel, 1977. Differential effects of 2,3-DPG, ATP, and inositol pentaphosphate (IPS) on the oxygen equilibria of duck embryonic, fetal, and adult hemoglobins. Comp. Biochem. Physiol. 56A:539-543. Bunn, H. F., U. S. Seal, and A. F. Scott, 1974. The role of 2,3-diphosphoglycerate in mediating hemoglobin functions of mammalian red cells. Annu. N.Y. Acad. Sci. 241:498-512. Chanutin, A., and R. R. Curnish, 1967. Effect of organic phosphates on the oxygen equilibrium of human erythrocytes. Arch. Biochem. Biophys. 121:96-102. Chapman, R., and L. Schaumburg, 1967. Glycolysis and glycolytic enzyme activity of aging red cells in
man. Brit. J. Haematol. 13:665-678. Henry, R. J., D. C. Cannon, and J. W. Winkelman, 1974. Clinical chemistry principles and technics. 2nd ed. Harper and Row, New York. Isaacks, R. E., and D. R. Harkness, 1975. 2,3-Diphosphoglycerate in erythrocytes of chick embryos. Science 189:393-394. Isaacks, R. E., D. R. Harkness, J. L. Adler, and P. H. Goldman, 1976c. Studies on avian erythrocyte metabolism. III. Effect of organic phosphates on oxygen affinity of embryonic and adult-type hemoglobins of the chick embryo. Arch. Biochem. Biophys. 173:114-120. Isaacks, R. E., D. R. Harkness, J. L. Adler, C.-Y. Kim, P. H. Goldman, and S. Roth, 1976e. Studies on avian erythrocyte metabolism. V. Relationship between the major phosphorylated metabolic intermediates and whole blood oxygen affinity in embryos and poults of the domestic turkey (Meleagris gallopavo). Poultry Sci. 55:1788-1794. Isaacks, R. E., D. R. Harkness, G. A. Froeman, P. H. Goldman, J. L. Adler, S. A. Sussman, and S. Roth, 1976b. Studies on avian erythrocyte metabolism. II. Relationship between the major phosphorylated metabolic intermediates and oxygen affinity of whole blood in chick embryos and chicks. Comp. Biochem. Physiol. 53A:151-156. Isaacks, R. E., D. R. Harkness, G. A. Froeman, and S. A. Sussman, 1976a. Studies on avian erythrocyte metabolism. I. Procedure for separation and quantitation of the major phosphorylated metabolic intermediates by anion exchange chromatography. Comp. Biochem. Physiol. 53A:95-99. Isaacks, R. E., D. R. Harkness, R. N. Sampsell, J. L. Adler, C.-Y. Kim, and P. H. Goldman, 1976d. Studies on avian erythrocyte metabolism. IV. Relationship between the major phosphorylated metabolic intermediates and oxygen affinity of whole blood in adults and embryos in several Galliformes. Comp. Biochem. Physiol. 55A:29-33. Janig, G. R., K. Ruckpaul, and F. Jung, 1971. Interaction of hemoglobin with ions binding of inositol hexaphosphate to human hemoglobin. Fed. of Europe. Biol. Sci. Letters 17:173-176. Johnson, L. F., and M. E. Tate, 1969. Structure of myo-inositol pentaphosphates. Annu. N.Y. Acad. Sci. 165:526-523. Jones, S. R., J. E. Smith and P. B. Board, 1978. An assay procedure for inositol pentaphosphate in avian erythrocytes. Poultry Sci. 57:0000. Lucas, A. M., and C. Jamroz, 1961. Atlas of avian hematology. USDA Monograph 25, Washington, DC. Mission, B. H., and B. M. Freeman, 1972. Organic phosphates and oxygen affinity of chick blood before and after hatching. Resp. Physiol. 14:343— 352. Ochiai, T., T. Gotch, and K. Shikama, 1972. Effect of intracellular organic phosphates on the oxygen equilibrium curve of chicken hemoglobin. Arch. Biochem. Biophys. 149:316-322. Oshima, M., T. G. Taylor, and A. Williams, 1964. Variations in the concentration of phytic acid in the blood of the domestic fowl. Biochem. J. 92: 42-46. Oski, F. A., and A. J. Gottlieb, 1971. The interrelaships between red blood cell metabolites, hemo-
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locytes) rather t h a n with old cells taking u p glutamate. After t h e initial p h l e b o t o m y , IP5 increased before n e w cells reached circulation. Increases in IP5 s h o u l d have shifted t h e hemoglobinoxygen dissociation curve t o t h e right and allowed t h e available h e m o g l o b i n t o deliver m o r e oxygen t o t h e tissues. T h e rapid rise of IP5 suggested t h a t it was controlled metabolically. This control m e c h a n i s m is quite sensitive t o t h e changing needs of chickens, just as t h e 2,3-DPG regulation of o x y g e n binding and delivery is sensitive t o changing needs of m a m m a l s (Bunn et ai, 1 9 7 4 ) . T h e exact m e c h a n i s m regulating t h e c o n c e n t r a t i o n of IP5 apparently is d u e entirely t o a metabolic effect of t h e m a t u r e red cells present in circulation although elevation of e n z y m e levels is clearly a new-cell effect.
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JONES ET AL. cells. Enzyme 1 4 : 7 6 - 8 1 . Smith, J., M. McCants, P. Parks and E. Jones, 1970. The influence of age on bovine erythrocyte enzymes. Biol. Neonate 15:169-175. Todd, J., and J. Ross, 1968. Biochemical and hematological changes in the blood of normal sheep following phlebotomy. Brit. Vet. J. 127:353. Tyuma, I., K. Imai and K. Shimizu, 1971. Effect of inositol hexaphosphate and other organic phosphates on the cooperativity in oxygen binding of human hemoglobins. Biochem. Biophys. Res. Commun. 44:682-686. Vandecasserie, C , A. G. Schnek, and J. Leonis, 1971. Oxygen affinity studies of avian hemoglobins. Chick and pigeon. Europ. J. Biochem. 24:284— 287. Wirth, D., 1950. Grundlagen einer klinischen hematologic der haustiere. Cited from Lucas and Jamroz, Avian hematology. USDA Monograph, 1961, Washington, DC.
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globin, and the oxygen-equilibrium curve. Prog. Hematol. 7:33-67. Schalm, 0 . W., N. C. Jain, and E. J. Carroll, 1975. Veterinary hematology. 3rd ed. Lea and Febiger, Philadelphia, PA. Smith, J. E., 1974. Relationship of in vivo erythrocyte glutathione flux to the oxidized glutathione transport system. J. Lab. Clin. Med. 83:444-450. Smith, J. E., 1977. Elevated erythrocyte glutathione associated with elevated substrate in high- and lowglutathione sheep. Biochem. Biophys. Acta 496: 516-520. Smith, J. E., and N. S. Agar, 1975. The effect of phlebotomy on canine erythrocyte metabolism. Res. Vet. Sci. 18:231-236. Smith, J. E., and N. S. Agar, 1976. Studies on erythrocyte metabolism following acute blood loss in the horse. Equine Vet. J. 8:34-37. Smith, J. E., and S. Kiefer, 1973. Comparative erythrocyte metabolism breed differences using canine