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In Vivo effect of dihydroxyacetone on oxygen-hemoglobin affinity in rhesus monkeys
JOURNAL
OF SURGICAL
ln Vim
RESEARCH
Effect
20, 309-311 (1976)
of Dihydroxyacetone Affinity
on Oxygen-Hemoglobin
in Rhesus
Monkeys
THOMAS W. POLLOCK, M.D.,’ ERNEST F. ROSATO, M.D.,2 MARIA DELIVORIA-PAPADOPOULOS, M.D., AND LEONARD D. MILLER, M.D.4 University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania Submitted for publication November 20, 1975
INTRODUCTION The potential benefit of a right shift in the oxygen-hemoglobin dissociation curve was first hypothesized in 1967 with the realization that increased red cell 2,3-Diphosphoglycerate (DPG) concentrations resulted in decreased affinity of hemoglobin for oxygen as reflected by an increased P,, [ 1, 61. The clinical importance of a DPG-related right shift was demonstrated by Oski et al. 191, who showed increased exercise tolerance and a better hemodynamic response to exercise in a patient with red cell pyruvate kinase deficiency who had increased levels of 2,3-DPG. Valeri et al. [13] have shown a better hemodynamic response in anemic baboons transfused with blood rich in 2,3-DPG. We have previously demonstrated both in viva and in vitro that inosine, pyruvate and phosphate will increase 2,3-DPG and P,, to supranormal levels [lo, 121.The accumulation of uric acid from inosine metabolism, however, limits the clinical potential of this combination in humans. Dihydroxyacetone (DHA) is a three carbon precursor of 2,3-DPG which prolongs maintenance of normal levels of DPG in banked blood in vitro [5]. Beutler identified ‘Instructor in Surgery, University of Pennsylvania, School of Medicine. 2Professor of Surgery, University of Pennsylvania, School of Medicine. “Associate Professor of Physiology and Pediatrics, University of Pennsylvania, School of Medicine. ‘Professor of Surgery, Director, Harrison Department of Surgical Research, University of Pennsylvania, School of Medicine.
the triokinase enzyme in red cells responsible for phosphorylation of DHA to DHA-P and showed that Cl4 labeled DHA is metabolized to 2,3-DPG, fructose and lactate [3]. He also showed, in vitro, that addition of pyruvate and inorganic phosphate to DHA increased red cell 2,3-DPG to supranormal levels in rabbit blood [4]. No toxic side effects of DHA have been described in animal models. The purpose of these experiments is to assess the in vivo effects of DHA on oxygen-hemoglobin affinity in subhuman primates. MATERIALS AND METHODS Adult male rhesus monkeys (Macaca mulatta), weighing 6.0 to 8.5 kg, were adapted to chronic restraint in primate chairs. Each monkey was equipped with an indwelling inferior vena caval catheter for blood sampling and the administration of drugs. Control DPG level in duplicate, P,, and serum lactate were determined. Before each blood sample was taken, 3 ml of blood was withdrawn into a flush syringe to clear catheter dead space. After sampling, this blood was reinfused and the catheter was flushed with heparinized saline between samples to maintain patency. Drugs were infused over a 45 min period in equal volumes (20 ml/Kg body weight). There were five experimental groups: physiologic saline (6 animals), 1 molar DHA5 (6 animals), 1 M DHA + 0.05 M pyruvate + 0.05 M bisodium phosphate (6 animals) (1 M DHA + PP), 2 M DHA + 0.05 M pyruvate + 0.05 M pyruvate + 0.05 M bisodium phosphate (9 animals) (2 M DHA + PP), 4 M
309 Copyright o 1976b Academic Press, Inc. All rights of repro d.uctlon m any form reserved.
“Sigma Chemical, St. Louis, Missouri.
310
JOURNAL OF SURGICAL
RESEARCH: VOL. 20, NO. 4, APRIL 1976
TABLE 2 DHA + 0.05 M pyruvate + 0.05 M bisodium Mean Change in Pse 5 hr after phosphate (2 animals) (4 M DHA + PP). 2,3Intravenous Administrationa DPG level was determined hourly to 6 hr and Mean change at 24 and 48 hr postinfusion. Venous lactate in Pso Agent Mean % change and P,, were determined at 5 hr post infuin Psu (20 ml/kg) (mm Hg) sion. Total blood aspirated per experiment Saline (6) 0.0 0 equalled 22 ml. To avoid anemia, each +1.6 +5.2 1MDHA (6) monkey received 1 ml of intravenous Imferon IMDHA + PP (6) +2.0* +7.0 (Lakeside Laboratories) for each 100 ml of ZMDHA + PP (9) +2.7* t9.3 to.4 blood withdrawn. Red cell 2,3-DPG was de- 4MDHA + PP (2) to.1 termined by the method of Krimsky [8] as aNumbers in parentheses refer to number of exmodified by Schroter and VonHeyden [I 11. periments in each group. *Significant Difference from control P < O.OlP,, was determined by a tonometric technique previously described [7]. Plasma Student’s r-test. lactates were measured by a modification of those given 2 M DHA + PP increased mean_ the fluourmetric technique of Holzer and 5 hr DPG by 1490 puM/ml RBC (32%)Sohring [2]. P < 0.01 Student’s f-test (Table 1). The 2,3DPG response over time for the 2 M DHA + RESULTS PP group is plotted in Figure 1. It was Mean control values for 2,3-DPG, PsO,and maximal at 5 hr but still significantly elelactate were 4850 & 113 puM/ml RBC, 29.3 vated at 48 hr post infusion. P,, at 5 hr + 0.4 mm Hg, and 1.45 f 0.14 uM/L respec- increased by 2.0 mm Hg (7%) in the 1 M tively (all normal for our lab). We have pre- DHA + PP group and by 2.7 mm Hg (9.3%) viously shown that pyruvate and phosphate in the 2 M DHA = PP group-P < O.Olalone cause no significant change in 5 hr Student’s t-test (Table 2). Plasma lactate DPG or P,, [12]. All animals receiving DHA was unchanged except in the 4 M DHA + PP developed transient retching approximately group where mean 5 hr lactate increased by 30 min after infusion. Changes in DPG were 12.7 uM/ml (900%) and one of the two animaximal at 5 hr in all study groups. Animals mals died at 6 hr with severe metabolic acireceiving saline and 1 M DHA had insignifi- dosis (Table 3). No significant changes in cant changes in 5 hr DPG, P,, and lactate venous pH or carbon dioxide tension were (Tables 1, 2 and 3). Those animals which seen at 5 hr post infusion except in the 4 M received 1 M DHA + PP increased mean 5 DHA + PP group. Hematocrit remained unhr DPG by 1040 puM/ml RBC (25%) while changed at 5 hr in all experimental groups.
TABLE 1 Mean Increase in 2,3-Diphosphoglycerate Intravenous Administration4 Agent (20 ml/kg) Saline (6) 1 M DHA (6) lMDHA+PP(6) 2MDHA+PP(9) 4MDHA+PP(2)
5 hr after
Mean increase in 2,3-DPG (rnp M/ml RBC)
Mean % increase in 2,3 DPG
+45 1 +834 +1040* +1490* +1240*
i-9 +15 +25 +32 +24
QNumbers in parentheses refer to number of experiments in each group. *Significant Difference from control P < 0.01 Student’s t-test.
TABLE 3 Mean Change in Lactate Concentration Intravenous Administration Agent (20 ml/kg) Saline (6) 1 M DHA (6) lMDHAtPP(6) 2MDHA+PP(9) 4MDHA+PP(2)
Mean change in lactate
5 Hours after
(PWml)
Mean % change in lactate
- 1.1 - 1.0 - 0.4 + 0.4 +12.7*
-50 -48 -20 +22 +900
aNumbers in parentheses refer to number of experiments in each group. *Significant Difference from control P < O.OlStudent’s f-test.
POLLOCK ET AL.: OXYGEN-HEMOGLOBIN 6500r
Effect
of 2M DHA l PPon
23
DPG
DISCUSSION DHA alone does not significantly increase 2,3-DPG concentration or blood P,, in vivo. Both 1M DHA + PP and 2 M DHA + PP significantly decrease the affinity of hemoglobin for oxygen in a species phylogenetically close to man without significant changes in pH, PCO, or hematocrit. The occurrence of lactic acidosis impaired the reponse to 4M DHA + PP and was a dose limiting toxic side effect. Increases in 2,3-DPG and P,, attained with 2 M DHA + PP are comparable to those seen with inosine, pyruvate and phosphate, although with DHA the potential danger of hyperuricemia does not exist. With adequate pulmonary function and arterial oxygenation an acute right-shift in the oxygen-hemoglobin dissociation curve may result in an increased arterio-venous oxygen content difference if venous oxygen tension is unchanged. This results in increased tissue oxygen delivery without an increase in blood flow or cardiac work. The other possible pattern of response to an acute right-shift would be an unchanged oxygen extraction and increased venous oxygen tension allowing for normal tissue oxygen delivery with a decrease in blood flow and cardiac work. These experiments demonstrate that 1 or 2 M DHA = PP may be a useful therapeutic regimen for pharmacologically decreasing the affinity of hemoglobin for oxygen in clinical states where oxygen demand exceeds supply, for example, hypermetabolic or low blood flow states.
AFFINITY
311
REFERENCES 1. Benesch, R., and Benesch, R. E. The Effect of Organic Phosphates from the human erythrocyte on the allosteric properties of hemoglobin. Biochtem. Biophy. Res. Comm. 26:162, 1967. 2. Bergmeyer, H. V. (Ed.) Methods of Enzymafic Analysis, Academic Press, New York, 1965, pp. 275-277. 3. Beutler, E. and &into, E. Dihydroxyacetone metabolism by human erythrocytes: demonstration of triokinase activity and its characterization. Blood, 41:559,1973. 4. Beutler, E. and Guinto, E. The metabolism of dihydroxyacetone by intact erythrocytes. J. Lab. Clin. Med. 82:534, 1973. 5. Brake, J. M. and Deindoerfer. Preservation of red blood cell 2,3-diphosphoglycerate in stored blood containing dihydroxyacetone. Transfusion 13:84, 1973. 6. Chanutin, A. and Curnish, R. R. Effect of organic and inorganic phosphates on the oxygen equilibrium of human erythrocytes. Arch. Biochem. Biophys. 96:121, 1967. 7. Delivoria-Papadopoulos, M., Oski, F. A. and Gottlieb, A. J. Oxygen-hemoglobin dissociation curves: effect of inherited enzyme defects of the red cells. Science, 165:601, 1969. 8. Krimsky, I. D-2,3-diphosphoglycerate. In: H. V. Bergmeyer: “Methods of Enzymatic Analysis,” Academic Press, New York, 1963,p. 238. 9. Oski, F. A., Marshall, B. E., Cohen, P. J., Sugerman, H. J. and Miller, L. D. Exercise with anemia. The role of the left-shifted or right-shifted oxygen-hemoglobin equilibrium curve. An. Intern. Med. 14:44,1971.
10. Oski, F. A., Travis, S. F., Miller, L. D., DelivotiaPapadopoulis, M. and Cannon, E. The in vitro restoration of red cell 2,3-diphosphoglycerate levels in banked blood. Blood37:52, 1971. 11. Schroter, W. and VonHeyden, H. Kinetik des 2,3diphosphoglyceratumsatzes in menschlichen erythrocyten. Biochem. Z. 341:387, 1965. 12. Sugerman, H. J., Pollock, T. W., Rosato, E. F., Delivoria-Papadopoulos, M., Miller, L. D. and Oski, F. A. Experimentally induced alterations in affinity of hemoglobin for oxygen. II In vivo effect of inosine, pyruvate and phosphate on oxygen-hemoglobin affinity in Rhesus monkey. Blood 39:525, 1972. 13. Valeri, C. R., Rorth, M., Zaroulis, C. G., Jakubowski, M. S. and Vescera, S. V. Physiologic effects of transfusing red blood cells with high or low affinity for oxygen to passively hyperventilated, anemic baboons: systemic and cerebral oxygen extraction. Ann. Surg. 181:106, 1975.
OF SURGICAL
ln Vim
RESEARCH
Effect
20, 309-311 (1976)
of Dihydroxyacetone Affinity
on Oxygen-Hemoglobin
in Rhesus
Monkeys
THOMAS W. POLLOCK, M.D.,’ ERNEST F. ROSATO, M.D.,2 MARIA DELIVORIA-PAPADOPOULOS, M.D., AND LEONARD D. MILLER, M.D.4 University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania Submitted for publication November 20, 1975
INTRODUCTION The potential benefit of a right shift in the oxygen-hemoglobin dissociation curve was first hypothesized in 1967 with the realization that increased red cell 2,3-Diphosphoglycerate (DPG) concentrations resulted in decreased affinity of hemoglobin for oxygen as reflected by an increased P,, [ 1, 61. The clinical importance of a DPG-related right shift was demonstrated by Oski et al. 191, who showed increased exercise tolerance and a better hemodynamic response to exercise in a patient with red cell pyruvate kinase deficiency who had increased levels of 2,3-DPG. Valeri et al. [13] have shown a better hemodynamic response in anemic baboons transfused with blood rich in 2,3-DPG. We have previously demonstrated both in viva and in vitro that inosine, pyruvate and phosphate will increase 2,3-DPG and P,, to supranormal levels [lo, 121.The accumulation of uric acid from inosine metabolism, however, limits the clinical potential of this combination in humans. Dihydroxyacetone (DHA) is a three carbon precursor of 2,3-DPG which prolongs maintenance of normal levels of DPG in banked blood in vitro [5]. Beutler identified ‘Instructor in Surgery, University of Pennsylvania, School of Medicine. 2Professor of Surgery, University of Pennsylvania, School of Medicine. “Associate Professor of Physiology and Pediatrics, University of Pennsylvania, School of Medicine. ‘Professor of Surgery, Director, Harrison Department of Surgical Research, University of Pennsylvania, School of Medicine.
the triokinase enzyme in red cells responsible for phosphorylation of DHA to DHA-P and showed that Cl4 labeled DHA is metabolized to 2,3-DPG, fructose and lactate [3]. He also showed, in vitro, that addition of pyruvate and inorganic phosphate to DHA increased red cell 2,3-DPG to supranormal levels in rabbit blood [4]. No toxic side effects of DHA have been described in animal models. The purpose of these experiments is to assess the in vivo effects of DHA on oxygen-hemoglobin affinity in subhuman primates. MATERIALS AND METHODS Adult male rhesus monkeys (Macaca mulatta), weighing 6.0 to 8.5 kg, were adapted to chronic restraint in primate chairs. Each monkey was equipped with an indwelling inferior vena caval catheter for blood sampling and the administration of drugs. Control DPG level in duplicate, P,, and serum lactate were determined. Before each blood sample was taken, 3 ml of blood was withdrawn into a flush syringe to clear catheter dead space. After sampling, this blood was reinfused and the catheter was flushed with heparinized saline between samples to maintain patency. Drugs were infused over a 45 min period in equal volumes (20 ml/Kg body weight). There were five experimental groups: physiologic saline (6 animals), 1 molar DHA5 (6 animals), 1 M DHA + 0.05 M pyruvate + 0.05 M bisodium phosphate (6 animals) (1 M DHA + PP), 2 M DHA + 0.05 M pyruvate + 0.05 M pyruvate + 0.05 M bisodium phosphate (9 animals) (2 M DHA + PP), 4 M
309 Copyright o 1976b Academic Press, Inc. All rights of repro d.uctlon m any form reserved.
“Sigma Chemical, St. Louis, Missouri.
310
JOURNAL OF SURGICAL
RESEARCH: VOL. 20, NO. 4, APRIL 1976
TABLE 2 DHA + 0.05 M pyruvate + 0.05 M bisodium Mean Change in Pse 5 hr after phosphate (2 animals) (4 M DHA + PP). 2,3Intravenous Administrationa DPG level was determined hourly to 6 hr and Mean change at 24 and 48 hr postinfusion. Venous lactate in Pso Agent Mean % change and P,, were determined at 5 hr post infuin Psu (20 ml/kg) (mm Hg) sion. Total blood aspirated per experiment Saline (6) 0.0 0 equalled 22 ml. To avoid anemia, each +1.6 +5.2 1MDHA (6) monkey received 1 ml of intravenous Imferon IMDHA + PP (6) +2.0* +7.0 (Lakeside Laboratories) for each 100 ml of ZMDHA + PP (9) +2.7* t9.3 to.4 blood withdrawn. Red cell 2,3-DPG was de- 4MDHA + PP (2) to.1 termined by the method of Krimsky [8] as aNumbers in parentheses refer to number of exmodified by Schroter and VonHeyden [I 11. periments in each group. *Significant Difference from control P < O.OlP,, was determined by a tonometric technique previously described [7]. Plasma Student’s r-test. lactates were measured by a modification of those given 2 M DHA + PP increased mean_ the fluourmetric technique of Holzer and 5 hr DPG by 1490 puM/ml RBC (32%)Sohring [2]. P < 0.01 Student’s f-test (Table 1). The 2,3DPG response over time for the 2 M DHA + RESULTS PP group is plotted in Figure 1. It was Mean control values for 2,3-DPG, PsO,and maximal at 5 hr but still significantly elelactate were 4850 & 113 puM/ml RBC, 29.3 vated at 48 hr post infusion. P,, at 5 hr + 0.4 mm Hg, and 1.45 f 0.14 uM/L respec- increased by 2.0 mm Hg (7%) in the 1 M tively (all normal for our lab). We have pre- DHA + PP group and by 2.7 mm Hg (9.3%) viously shown that pyruvate and phosphate in the 2 M DHA = PP group-P < O.Olalone cause no significant change in 5 hr Student’s t-test (Table 2). Plasma lactate DPG or P,, [12]. All animals receiving DHA was unchanged except in the 4 M DHA + PP developed transient retching approximately group where mean 5 hr lactate increased by 30 min after infusion. Changes in DPG were 12.7 uM/ml (900%) and one of the two animaximal at 5 hr in all study groups. Animals mals died at 6 hr with severe metabolic acireceiving saline and 1 M DHA had insignifi- dosis (Table 3). No significant changes in cant changes in 5 hr DPG, P,, and lactate venous pH or carbon dioxide tension were (Tables 1, 2 and 3). Those animals which seen at 5 hr post infusion except in the 4 M received 1 M DHA + PP increased mean 5 DHA + PP group. Hematocrit remained unhr DPG by 1040 puM/ml RBC (25%) while changed at 5 hr in all experimental groups.
TABLE 1 Mean Increase in 2,3-Diphosphoglycerate Intravenous Administration4 Agent (20 ml/kg) Saline (6) 1 M DHA (6) lMDHA+PP(6) 2MDHA+PP(9) 4MDHA+PP(2)
5 hr after
Mean increase in 2,3-DPG (rnp M/ml RBC)
Mean % increase in 2,3 DPG
+45 1 +834 +1040* +1490* +1240*
i-9 +15 +25 +32 +24
QNumbers in parentheses refer to number of experiments in each group. *Significant Difference from control P < 0.01 Student’s t-test.
TABLE 3 Mean Change in Lactate Concentration Intravenous Administration Agent (20 ml/kg) Saline (6) 1 M DHA (6) lMDHAtPP(6) 2MDHA+PP(9) 4MDHA+PP(2)
Mean change in lactate
5 Hours after
(PWml)
Mean % change in lactate
- 1.1 - 1.0 - 0.4 + 0.4 +12.7*
-50 -48 -20 +22 +900
aNumbers in parentheses refer to number of experiments in each group. *Significant Difference from control P < O.OlStudent’s f-test.
POLLOCK ET AL.: OXYGEN-HEMOGLOBIN 6500r
Effect
of 2M DHA l PPon
23
DPG
DISCUSSION DHA alone does not significantly increase 2,3-DPG concentration or blood P,, in vivo. Both 1M DHA + PP and 2 M DHA + PP significantly decrease the affinity of hemoglobin for oxygen in a species phylogenetically close to man without significant changes in pH, PCO, or hematocrit. The occurrence of lactic acidosis impaired the reponse to 4M DHA + PP and was a dose limiting toxic side effect. Increases in 2,3-DPG and P,, attained with 2 M DHA + PP are comparable to those seen with inosine, pyruvate and phosphate, although with DHA the potential danger of hyperuricemia does not exist. With adequate pulmonary function and arterial oxygenation an acute right-shift in the oxygen-hemoglobin dissociation curve may result in an increased arterio-venous oxygen content difference if venous oxygen tension is unchanged. This results in increased tissue oxygen delivery without an increase in blood flow or cardiac work. The other possible pattern of response to an acute right-shift would be an unchanged oxygen extraction and increased venous oxygen tension allowing for normal tissue oxygen delivery with a decrease in blood flow and cardiac work. These experiments demonstrate that 1 or 2 M DHA = PP may be a useful therapeutic regimen for pharmacologically decreasing the affinity of hemoglobin for oxygen in clinical states where oxygen demand exceeds supply, for example, hypermetabolic or low blood flow states.
AFFINITY
311
REFERENCES 1. Benesch, R., and Benesch, R. E. The Effect of Organic Phosphates from the human erythrocyte on the allosteric properties of hemoglobin. Biochtem. Biophy. Res. Comm. 26:162, 1967. 2. Bergmeyer, H. V. (Ed.) Methods of Enzymafic Analysis, Academic Press, New York, 1965, pp. 275-277. 3. Beutler, E. and &into, E. Dihydroxyacetone metabolism by human erythrocytes: demonstration of triokinase activity and its characterization. Blood, 41:559,1973. 4. Beutler, E. and Guinto, E. The metabolism of dihydroxyacetone by intact erythrocytes. J. Lab. Clin. Med. 82:534, 1973. 5. Brake, J. M. and Deindoerfer. Preservation of red blood cell 2,3-diphosphoglycerate in stored blood containing dihydroxyacetone. Transfusion 13:84, 1973. 6. Chanutin, A. and Curnish, R. R. Effect of organic and inorganic phosphates on the oxygen equilibrium of human erythrocytes. Arch. Biochem. Biophys. 96:121, 1967. 7. Delivoria-Papadopoulos, M., Oski, F. A. and Gottlieb, A. J. Oxygen-hemoglobin dissociation curves: effect of inherited enzyme defects of the red cells. Science, 165:601, 1969. 8. Krimsky, I. D-2,3-diphosphoglycerate. In: H. V. Bergmeyer: “Methods of Enzymatic Analysis,” Academic Press, New York, 1963,p. 238. 9. Oski, F. A., Marshall, B. E., Cohen, P. J., Sugerman, H. J. and Miller, L. D. Exercise with anemia. The role of the left-shifted or right-shifted oxygen-hemoglobin equilibrium curve. An. Intern. Med. 14:44,1971.
10. Oski, F. A., Travis, S. F., Miller, L. D., DelivotiaPapadopoulis, M. and Cannon, E. The in vitro restoration of red cell 2,3-diphosphoglycerate levels in banked blood. Blood37:52, 1971. 11. Schroter, W. and VonHeyden, H. Kinetik des 2,3diphosphoglyceratumsatzes in menschlichen erythrocyten. Biochem. Z. 341:387, 1965. 12. Sugerman, H. J., Pollock, T. W., Rosato, E. F., Delivoria-Papadopoulos, M., Miller, L. D. and Oski, F. A. Experimentally induced alterations in affinity of hemoglobin for oxygen. II In vivo effect of inosine, pyruvate and phosphate on oxygen-hemoglobin affinity in Rhesus monkey. Blood 39:525, 1972. 13. Valeri, C. R., Rorth, M., Zaroulis, C. G., Jakubowski, M. S. and Vescera, S. V. Physiologic effects of transfusing red blood cells with high or low affinity for oxygen to passively hyperventilated, anemic baboons: systemic and cerebral oxygen extraction. Ann. Surg. 181:106, 1975.