The Role of Organic Phosphates in Erythrocytes on the Oxygen Dissociation of Hemoglobin

Transcription

1 A n n a l s o f C l i n i c a l L a b o r a t o r y S c i e n c e, V o l. 1, N o. 2 C o p y r ig h t , I n s titu te f o r C lin ic a l S c ie n c e The Role of Organic Phosphates in Erythrocytes on the Oxygen Dissociation of Hemoglobin FRANK A. OSKI, M.D. C hildrens H ospital of Philadelphia, Philadelphia, PA Human cellular metabolism is critically dependent on an adequate supply of oxygen. The oxygen transport system in man is the erythrocyte, and its primary function is to bring oxygen to the tissues at a sufficient partial pressure to permit its rapid diffusion from the blood in adequate quantities. Normal oxygen transport depends on a number of factors which include the fraction of oxygen in the inspired air, the partial pressure of oxygen in the inspired air, alveolar ventilation, the relation of ventilation to perfussion in the lungs, arterial ph and temperature (Bohr effect), cardiac output, blood volume, hemoglobin concentration, and the affinity of the hemoglobin for oxygen. In the past, the major focus of clinical interest in oxygen transport has centered on factors governing oxygen uptake. Little attention has been paid to the factors governing oxygen release, the unloading of oxygen in the tissue capillaries. It had been assumed that the affinity of hemoglobin for oxygen, as reflected by the position of the oxygen-hemoglobin equilibrium curve, was a fixed param eter that was mainly influenced by changes in ph and tem perature. W ithin the past several years, newly accumulated information indicates that the affinity of hemoglobin for oxygen is altered in a variety of situations and that the red cell delicately and efficiently regulates these changes in oxygen affinity. The Oxygen-Hemoglobin Equilibrium Curve The oxygen-hemoglobin equilibrium curve (figure 1) reflects the affinity of hemoglobin for oxygen. As blood circulates in the normal lung, the arterial oxygen tension rises from 40 mm H g and reaches approximately 110 mm Hg, sufficient to ensure at least 95 percent saturation of the arterial blood. The shape of the oxygen-hemoglobin equilibrium curve is such that a further increase in the oxygen tension in the lung results in only a very small increase in the degree of saturation of the blood. The oxygen tension falls as blood travels from the lungs and oxygen is released from the hemoglobin. In the normal adult when the oxygen tension has fallen to approximately 27 mm Hg at a ph of 7.4 and a tem perature of 37 C, 50 percent of the oxygen bound to hemoglobin has been released. The P50, the whole blood oxygen tension at 50 percent oxygen saturation, would be 27 mm Hg. W hen the affinity of hemoglobin for oxygen is reduced, more oxygen is released to the tissues at a given oxygen tension. In such situations the oxygenhemoglobin equilibrium curve is shifted to the right of normal. Alternatively, if the affinity of hemoglobin for oxygen is increased, the oxygen tension must drop lower than normal before the hemoglobin releases an equivalent amount of oxygen, and Presented at the A pplied Seminar on Chem ical Hem atology, Novem ber,

2 THE ROLE OF ORGANIC PHOSPHATES IN ERYTHROCYTES 163 Oxygen Dissociation 80 T r t O2 Affinity * O2 Release *!< 0 60 cs* "p 50" Values POg mmhg F i g u r e 1. The oxygen dissociation curve of normal adult blood. The PB0, the oxygen tension at 50 percent oxygen saturation, is approximately 27 mm Hg. As the curve shifts to the right, the oxygen affinity of hemoglobin is decreased and more oxygen is released at a given oxygen tension. W ith a shift to the left, the opposite effects are observed. A decrease in ph or an increase in temperature decreases the affinity of hemoglobin for oxygen. thus the equilibrium curve appears shifted to the left. Hemoglobin, the oxygen carrying pigment of man, consists of a protein ( globin ) containing four prosthetic heme groups. The sigmoidal shape of the oxygen-hemoglobin equilibrium curve can be explained only if two assumptions are made: that the hemes react with oxygen in a definite order and that the oxygenation and deoxygenation of one profoundly effects the oxygenation and deoxygenation of the others. This phenomenon has been termed heme-heme interaction and is reflected in the shape of the curve which indicates that as each heme group accepts oxygen, it becomes progressively easier for the next heme group to pick up oxygen. X-ray crystalographic studies have confirmed the fact that oxygenated and deoxygenated hemoglobin differ in their confirmation.18 The Role of Organic Phosphates It is recognized that the oxygen affinity of adult hemoglobin in solution is considerably greater than that of the intact fresh erythrocyte. This difference suggests

3 164 OSKI that the intact red cell contains a substance or substances that are capable of interacting w ith hemoglobin and reducing its affinity for oxygen. In 1967 Benesch and Benesch8 and C hanutin and Curnish10 dem onstrated that the affinity of a hemoglobin solution for oxygen could be decreased by its interaction w ith a number of organic phosphates. Of the organic phosphates tested, 2, 3-diphosphoglycerate (2, 3-DPG) and adenosine triphosphate (ATP) were most effective in lowering oxygen affinity - while adenosine diphosphate, adenosine monophosphate, pyrophosphate and inorganic phosphate showed a progressively decreasing degree of effectiveness. The highly charged anion, 2, 3-diphosphoglycerate (2, 3-DPG), was demonstrated by Benesch and co-workers5 to bind to deoxyhemoglobin but not to the oxygenated form of the molecule. It was found th at one mole of 2, 3-DPG bound reversibly to one mole of deoxyhemoglobin tetram er under physiologic conditions of solute concentration and ph. Of the organic phosphates normally found in the human erythrocyte, 2, 3-DPG is the one found in largest concentration and thus is quantitatively the most important with respect to modulation of hemoglobin oxygen affinity. The content of 2, 3-DPG in the red cell averages 4.5 / moles per ml RBC s (range 3.4 to 5.2 / moles per ml RBC s) and ATP 1.0 / moles per ml of RBC s (range 0.8 to 1.4 / moles per ml RBC s ) while the remainder of the organic phosphates generally total less than 0.4 / moles per ml. The Binding Site W hen either 2, 3-DPG or ATP are added to solutions of fetal hemoglobin, the decrease in oxygen affinity produced by these compounds is significantly less than that observed in the adult. Table I, taken from the work of Bauer and colleagues,2 illustrates this point. The difference in interaction between adult and fetal hemoglobin with 2, 3-DPG has helped facilitate provisional determina Table I. Effect of Added DPG and ATP on Oxygen Affinity of Adult and Fetal Hemoglobin7 Maternal (n 8 ) Fetal (n = 8) x SD x SD P50 W hole Blood 31.0 ± ± 2.4 P50 Dialysed H b 19.7 ± ± 0.9 P50 Dialysed H b + DPG 29.4 ± ± 1.4 P50 Dialysed H b + ATP 21.1 ± ± 1.8 Half Saturation Pressures (P 50) in mm Hg at ph 7.20 (in Blood ph c = 7.20) and 37 C, in Maternal and Fetal Blood and Hemoglobin Solutions without and with DPG (30 / moles/per gm H b) or ATP (8 / moles/per gm H b). n = Number of Samples, x = Mean Value, SD = Standard Deviation

4 THE BOLE OF ORGANIC PHOSPHATES IN ERYTHROCYTES 165 tion of the site on the hemoglobin molecule at which 2, 3-DPG is bound. Benesch and co-workers originally proposed that 2, 3- DPG was bound to adult hemoglobin somewhere on the beta chains.4 This hypothesis was based on the finding that 2, 3-DPG did bind to deoxyhemoglobin in a 1:1 molar ratio, did bind to beta4 (Hemoglobin H ) both in the oxy or deoxy state, but did not bind to isolated alpha chains. Since hemoglobin is a symmetrical molecule, it was reasoned that the most likely binding site would be a positively charged area somewhere in the central cavity of the molecule along its diad axis of symmetry. The beta- 143 histidine was suggested as a likely binding site by deverdier and Garby.13 This positively charged amino acid residue is located at the entrance to the central cavity and could form electrostatic bonds with anions such as 2, 3-DPG and ATP. In fetal hemoglobin, this position on the gamma chain is occupied by the neutral amino acid serine which does not bind negatively charged compounds such as 2, 3-DPG. Bunn and Briehl have indicated that beta-143 histidine (B-H 21 histidine) cannot be the sole site of binding because fetal hemoglobin does show some interaction with 2, 3-DPG.8 Experimental evidence indicates that 2, 3-DPG is bound at the beta-143 histidine and to the N-terminal amino groups of the non-alpha chains (beta and gamma chains). Bunn and Briehl indicated8 that Greer and Perutz fit a model of 2, 3-DPG into an atomic model of h u man deoxyhemoglobin. Greer and Perutz were able to place one molecule of 2, 3- DPG in the internal cavity in such a way that the phosphates were within hydrogen bonding distance of the two N-terminal amino groups of the beta chains, the imidazoles of the B-H21 histidines and the e- amino groups of b-ef6 lysines. In contrast, the internal cavity of oxyhemoglobin was too narrow to allow binding at this site. Upon oxygenation, the N-terminal groups of the beta chains moved further apart, making it impossible for both to bind to the same molecule of 2, 3-DPG. Such a difference between the binding of 2, 3-DPG to deoxy and oxyhemoglobin would account for the effect of 2, 3-DPG on oxygen affinity. Regulation of Red Cell 2, 3-DPG Concentration Although the human erythrocyte, unlike most other cells of the body, contains large quantities of 2, 3-DPG, its role within the red cell and the factors regulating its m e tabolism have remained a puzzle.15 The red cell synthesizes 2, 3-DPG from 1, 3- DPG in the presence of the enzyme 2, 3- diphosphoglycerate mutase (figure 2). The 2, 3-diphosphoglycerate formed is eventually hydrolyzed to 3-phosphoglycerate and inorganic phosphate by the enzyme 2, 3-diphosphoglycerate phosphatase. The conversion of glyceraldehyde-3-phosphate to 1, 3-diphosphoglycerate is controlled by the NAD/NADH ratio within the cell. The conversion of 1, 3-DPG to either 2, 3-DPG or 3-PGA is governed in part by the concentration of unbound 2, 3-DPG within the cell, the level of 3-PGA and the adenosine diphosphate/adenosine triphosphate ratio. Increased concentrations of adenosine diphosphate facilitate conversion of 1, 3- DPG to 3-PGA. High levels of 3-PGA appear to inhibit the phosphoglycerate kinase reaction and direct 1, 3-DPG to 2, 3-DPG synthesis. Studies of 2, 3-diphosphoglycerate m u tase indicate that it is inhibited by very low concentrations of its product 2, 3-DPG (Ki = 0.85 /im),25 The observation that deoxygenated hemoglobin binds 2, 3-DPG provides one explanation for the ability of the cell to synthesize this compound. The binding of 2, 3-DPG by deoxyhemoglobin relieves the product inhibition of the enzyme responsible for its synthesis and facilitates further production. This binding of 2, 3-DPG by deoxyhemoglobin appears

5 166 OSKI THE PHOSPHOGLYCERATE CYCLE PG Kinase 2,3-DPG Mutase ATP 3-PG 2,3-DPG P i" 2,3-DPG Phosphatase 2,3-DPG + DeoxyHb 2,3-DPG Hb I +0, 2,3-DPG + HbOi F i g u r e 2. The 2, 3-diphosphoglycerate (2, 3-D PG ) cycle in the human red cell. W hen 2, 3-DPG is formed it can combine reversible with deoxyhemoglobin, thus relieving the product inhibition of 2, 3-diphosphoglycerate mutase (2, 3-DPG m utase) and facilitating further synthesis of 2, 3-DPG from 1, 3-DPG. ABBREVIATIONS: G-3-P, glyceraldehyde-3-phosphate; G-3-PD, glyceraldehyde-3-phosphate dehydrogenase; 3-PG, 3-phosphoglycerate; 2-PG, 2-phosphoglycerate; Pi; inorganic phosphate; PG kinase, phosphoglycerate kinase; PG mutase, phosphoglycerate mutase. to be a partial explanation for the increased levels of 2, 3-DPG observed in situations of hypoxemia such as cyanotic heart disease,23 chronic lung disease,21 exposure to high altitude,16 and chronic anemia.28 All are conditions in which the concentrations of deoxyhemoglobin are increased. Intracellular ph profoundly influences 2, 3-DPG metabolism either by directly regulating red cell glycolysis or possibly by direct effects on the activities of 2, 3-DPG mutase and phosphatase. Increases in ph stimulate both red cell glycolysis and 2, 3-DPG production while acidosis has the opposite effect. Relationship of Red Cell 2, 3-DPG Levels to the Position of the Oxygen Equilibrium Curve The relationship of red cell 2, 3-DPG levels to the oxygen affinity of whole blood were examined in a wide variety of clinical conditions (table II) and a close correlation between these two variables was observed. It was demonstrated previously that in a number of clinical disorders associated with hypoxemia, the blood s affinity for oxygen, was decreased.14,19 This shift to the right in the position of the curve is rec-

6 THE BOLE OF ORGANIC PHOSPHATES IN ERYTHROCYTES 167 Table II. Clinical Conditions Associated with Changes in the Oxygen Affinity of Blood A. Increased red cell 2, 3-DPG, increased P 50 Adaptation to high altitude Hypoxemia associated w ith chronic pulmonary disease Hypoxemia associated with cyanotic heart disease Anemia Secondary to iron deficiency Secondary to chronic renal disease Caused by sickle cell anemia Decreased red cell mass Chronic liver disease Hyperthyroidism Red cell pyruvate kinase deficiency B. Decreased red cell 2, 3-DPG, decreased P 50 Septic shock Severe acidosis Following massive transfusions of stored blood Neonatal respiratory distress syndrome C. Increased P 50, no consistent alteration in red cell DPG Abnormal hemoglobins (Kansas, Seattle, Hammersmith, Tacoma, E ) Vigorous exercise D. Decreased P 50, no consistent alteration in red cell DPG abnormal hemoglobins (Kempsey, Philly, Chesapeake, J. Capetown, Yakima, Rainier) ognized as a useful compensatory mechanism that allowed these patients to unload more oxygen to the tissues at higher partial pressures of oxygen. Intrinsic defects of the red cell may also produce changes in red cell 2, 3-DPG levels. These are not directly related to the level of hypoxemia in the patient. Patients with red cell pyruvate kinase deficiency accumulate large concentrations of 2, 3-DPG within their erythrocytes and have, as a consequence, marked elevations in their P 50 values Conversely, patients with metabolic defects in red cell metabolism, proximal to the step of 2, 3-DPG synthesis, have low levels of this compound and a decrease in the P50 of their blood. In patients with septic shock, the red cell level of 2, 3-DPG is decreased and the oxygen-equilibrium curve is shifted to the left.27 As a consequence of this left shift, the mean central venous oxygen tension of the patients is reduced in order to extract sufficient oxygen from the blood. In this disorder there is a strong correlation between the red cell 2, 3-DPG level and the mean central venous oxygen tension (figure 3). Thus in a wide variety of clinical conditions, the position of the oxygen-hemoglobin equilibrium curve, as reflected by the P50, bears a striking relationship to the red cell 2, 3-DPG content (figure 4). Throughout the physiologic range (2,000

7 168 OSKI 2,3 'D P G ( m Ai moles/ml. RBC's) F igube 3. Correlation of the central venous oxygen tension (P voa) with the red cell 2, 3-DPG concentration in patients with septic shock. (r = 0.8 1, p < 0.001) to 10,000 m/tmoles DPG/m l RBC s), the relationship between P50 and 2, 3-DPG was found to be linear; a change of 430 m^moles of DPG produced a change of 1 mm.hg in the P50.28 Effects of Blood Storage In 1954 Valtis and Kennedy30 found that blood stored for more than several days under conventional blood bank conditions showed a significant increase in its oxygen

8 THE ROLE OF ORGANIC PHOSPHATES IN ERYTHROCYTES 169 affinity. Now this increase in oxygen affinity can be correlated with the fall in red cell 2, 3-DPG levels.9 In blood stored in acid-citrate-dextrose, the 2, 3-DPG level falls to percent of its initial level within seven to ten days. By twenty-one days is only 10 percent of the original level. W hen blood which is poor in 2, 3-DPG is transfused into healthy recipients, a prom pt increase in the level of this compound is observed.29 7 Approximately 25 percent of the normal level is achieved within three hours. By twenty-four hours 50 to 66 percent restoration occurs. The complete return to normal may require six to ten days. During this period of time, the patient may be compromised by possessing blood with a high affinity for oxygen which m ay im pair tissue oxygenation. This procedure of transfusing 2, 3-DPG poor blood into anemic patients is likened to placing a dyspneic man in an oxygen tent and then placing a hand over his face so that he can t breathe. The level of red cell 2, 3-DPG and the oxygen affinity of blood is partially restored to normal by incubating the blood with inosine prior to transfusion.9 1 Com plete re- <o I 1 I F ig u r e 4. Relationship between P50 of whole blood and red cell DPG in a variety of clinical disorders. These include cyanotic heart disease ( o ), red cell enzyme defects ( < > ), septic shock ( o ), hyperthyroidism ( < > ), and chronic liver disease ( A ). Square block indicates normal range for P50 and red cell DPG.

9 170 OSKt Pq 2 (mm Hg, ph 7.4) F ig u b e 5. Oxygen equilibrium curve of blood from term infants at different postnatal ages. The P50 on day 1 is 19.4 ± 1.8 mm H g and has shifted to 30.3 ± 0.7 at age 11 months. (Normal adult 27.0 ± 1.1 mm H g.) turn to normal is accomplished by incubation of blood with a combination of inosine, inorganic phosphate and pyruvate.24 These additives have not received clinical trials in this country in patients receiving large quantities of blood. The Neonate and Postnatal Changes in Oxygen Transport The major reason that the blood of the newborn infant possesses an oxygen-hemoglobin equilibrium curve that shifts to the left of that of a normal adult is the failure

10 THE BOLE OF ORGANIC PHOSPHATES IN ERYTHROCYTES 171 F i g u r e 6. Functioning DPG Fraction (m/imoles/ml RBC) (2,3-DPG x % Adult Hb) The P50 and the functioning (or interacting) DPG fraction of term infants studied at different postnatal ages. of fetal hemoglobin to bind 2, 3-DPG to the same degree as does adult hemoglobin. During the first three months of life, the P50 of the blood of term infants gradually rises. By four to six months of age the oxygen-hemoglobin equilibrium curve is similar to that of the adult.11 In many infants of eight to eleven months of age the curve shifts to the right of that of the normal adult (figure 5). The position of the oxygen equilibrium curve, as reflected by the P50, does not directly relate to the total red cell 2, 3-DPG. Thus, the change in P 50 in these infants does not correlate with either the change in fetal hemoglobin alone or with red cell 2, 3-DPG content alone. The progressive decline in oxygen affinity during the first six months of life does, however, correlate precisely with the functioning or interacting DPG fraction. 11 This fraction is derived by multiplication of the total red cell DPG content (m/xmoles per ml RBC s) by the percent adult hemoglobin. It serves to illustrate the fact that both the 2, 3-DPG concentration and the adult hemoglobin concentration within the cell, w ith which the 2, 3-DPG primarily interacts, are necessary factors in determining the position of the oxygen equilibrium curve (figure 6). This relationship helps to explain why infants, during the first weeks of life, with similar concentrations of fetal and adult hemoglobin may have marked differences in their PB0 s. Infants w ith more adult hemoglobin but less 2, 3-DPG may have a P50 similar to that of an infant with a high red cell 2, 3-DPG content in association with increased quantities of fetal hemoglobin.

11 172 OSSI Physiologic Implications of the Changes in Oxygen Affinity of Blood A certain partial pressure gradient is needed for the transfer of oxygen from capillary to tissue. The term critical Po2 indicates that level of oxygen pressure below which diffusion is impaired and organ function is disturbed. A critical P02 cannot be a well-defined value that applies to all tissues under all conditions. The oxygen requirements of tissues vary. In some tissues, such as striated muscle, oxygen requirements are dependent on the level of activity. Opitz and Schneider20 show that the oxygen uptake of brain is decreased when the venous oxygen tension falls below 20 to 25 mm Hg and loss of myocardial function is observed at an oxygen tension of mm Hg.6 The mean critical range of oxygen tension appears to fall between mm Hg. At this point the oxygen saturation of normal blood ranges from 35 to 55 percent saturation. If the oxygen-hemoglobin equilibrium curve shifts to the right, more oxygen can be unloaded at any given partial pressure of oxygen. This shift to the right can be accomplished by acidosis, increase in temperature, or by decreasing the affinity of hemoglobin for oxygen by increases in the level of red cell organic phosphates such as 2, 3-DPG or ATP. If adequate tissue oxygen delivery is to occur at tensions above those considered critical, several compensatory mechanisms are physiologically available (table III). The two principal mechanisms immediately available to the human are an increase in flow or an increase in oxygen unloading capacity. The ability to increase flow is limited by the ability of vessels to dilate and by the ability of the myocardium to increase its work. The physiologic effects of curves shifted to the left or right are illustrated by the changes observed with exercise in two patients with similar degrees of anemia.22 Both patients h ad a hemoglobin concentration of approximately 10 gm percent. One of the patients had a P50 of 19 mm Hg. This individual whose curve shifted to the left had a low red cell 2, 3-DPG as a consequence of hexokinase deficiency. The second patient had red cell pyruvate kinase deficiency, an elevated 2, 3-DPG level and Table III. Mechanisms for Increasing Tissue Orientation A. Increased arterial flow 1. Increase in cardiac output 2. Arteriolar dilatation B. Increased volume of oxygen 1. Increase in inspired oxygen concentration or pressure 2. Increase in hemoglobin concentration 3. Increase in release of oxygen from hemoglobin Hypertherm ia Acidosis Increase in red cell 2, 3-DPG content

12 THE ROLE OF ORGANIC PHOSPHATES IN ERYTHROCYTES 173 Work Load (Watts Resistance) F i g u r e 7. Changes in (A ) oxygen saturation and (B ) central venous oxygen tension (Pv02) with exercise in patient (S.N.) with red cell hexokinase deficiency and a left-shifted oxygen-hemoglobin equilibrium curve as contrasted with patient (D.S.) with red cell pyruvate kinase deficiency and a right-shifted curve. a P50 of 38 mm Hg. As illustrated in figure 7, exercise on a bicycle ergometer resulted in a prompt fall in the mean central venous oxygen tension in the individual with the left shift curve and an apparent inability to extract further oxygen. Exercise with its requirem ent for increased oxygen consumption was achieved by a doubling of cardiac output (table IV ). In contrast in the patient whose curve shifted to the right, the oxygen saturation was gradually lowered while exercising as was the mean central venous oxygen tension. This ability to extract oxygen required far less of an increase in cardiac output. These laboratory findings reflect the life patterns of the two individuals. The patient whose blood was left shifted tired easily by merely walking to school. The patient whose blood had a right shifted curve, participated in a full range of athletic endeavors despite a similar degree of anemia.

13 174 OSKI Table IV. Changes in Oxygen Delivery with Exercise in Two Patients with Red Cell Enzyme Defects DIAGNOSIS S.N. Hexokinase Deficiency D.S. Pyruvate Kinase Deficiency AGE WEIGHT (Kg) SURFACE AREA (M2) HEMOGLOBIN (g percent) PB0 (mm Hg) PERCENT SATURATION At rest At work load* _ _ _ Pv0 2 (mm H g) At rest At work load* »» ** yy 7* ft *' ** _ CARDIAC INDEX (L per min per M2) At rest At work load* _ OXYGEN CONSUMPTION (ml per min per M2) At rest At work load* " _ »>» yy * _ LACTATE/PYRUVATE At rest At work load* _ yy» yy yy yy yy ph At rest At work load* yy yy yy yy yy yy V» >»»» ( work load in watts resistance )

14 T H F. BOLE OF OBGANIC PHOSPHATES IN EBYTHROCYTES 175 Conclusion These findings force a re-examination of the teleologic explanation for why man, unlike lower forms of life, carries his hemoglobin packaged in red cells. The often repeated explanation that a hemoglobin solution with the same oxygen capacity as human blood would be highly viscous, and therefore would require much greater cardiac effort for circulation, is false.26 It appears that hemoglobin is contained within erythrocytes for two other primary reasons. In this intracellular environment, hemoglobin is protected from oxidative dénaturation by the red cell s enzyme complement. W ithin the cell, hemoglobin interacts with the metabolic intermediates and co-factors of red cell glycolysis. This interaction results in sensitive changes in its pattern of oxygen release. Certainly judgments about the adequacy of tissue oxygen transport cannot be made from the simple determ ination of the hemoglobin or hem atocrit alone. It is now apparent that the position of the oxygen dissociation curve is influenced not only by changes in ph and tem perature, two rather insensitive regulators of tissue oxygen release, but also by the presence of the organic phosphates within the red cell. The levels of the organic phosphates respond rapidly to metabolic and environmental influences and in turn affect metabolism and the cellular environment. References 1. A k e r b l o m, O., d e V e b d ie r, C. H., G a r b y, L., a n d H ô g m a n, C.: Restoration of defective oxygen-transport function of stored red blood cells by addition of inosine. Scand. J. Clin. Lab. Invest. 21: , B a u e r, C., L u d w ig, I., a n d L u d w ig, M.: Different effects of 2, 3-diphosphoglycerate and adenosine triphosphate on oxygen affinity of adult and fetal human hemoglobin. L ife Sci. 7: , B e n e s c h, R. a n d B e n e s c h, R. E.: The effect of organic phosphates from the human erythrocyte on the allosteric properties of hemoglobin. Biochem. Biophys. Res. Commun. 26: , B e n e s c h, R., B e n e s c h, R. E., a n d E n o k i, Y.: The interaction of hemoglobin and its subunits with 2, 3-diphosphoglycerate. Proc. Nat. Acad. Sci. 61: 1102, B e n e s c h, R., B e n e s c h, R. E., a n d Y u, C. I.: Reciprocal binding of oxygen and diphosphoglycerate by human hemoglobin. Proc. Nat. Acad. Sci. 59: , B e r n e, R. M., B l a c k m a n, J. R., a n d G a r d n e r, T. H.: Hypoxemia and coronary blood flow. J. Clin. Invest. 36: 1101, B e u t l e r, E. a n d W o o d, L.: The in vivo regeneration of red cell 2, 3-diphosphoglyceric acid (DPG) after transfusion of stored blood. J. Lab. Clin. Med. 74: 300, B u n n, H. F. a n d B r i e h l, R. W.: The interaction of 2, 3-diphosphoglycerate with various human hemoglobins. J. Clin. Invest. 49: 1088, B u n n, H. F., May, M. M., K o c h o l a t y, W. F., a n d S h i e l d s, C. R.: Hemoglobin function in stored blood. J. Clin. Invest. 48: , C h a n u t i n, A. a n d C u r n i s h, R. R.: Effect of organic and inorganic phosphates on the oxygen equilibrium of human erythrocytes. Arch. Biochem. Biophys. 121:96-102, D e l i v o r i a - P a p a d o p o u l o s, M. a n d Oski, F. A.: Developmental changes in the oxygen equilibrium curve of infants as related to the functioning DPG fraction and its alteration with disease. Pediat. Res. 4 : 470, D e l i v o r i a - P a p a d o p o u l o s, M., O s k i, F., a n d G o t t l i e b, A. J.: Oxygen-hemoglobin dissociation curves: Effect of inherited enzyme defects of the red cell. Science 165: 601, d e V e r d i e r, C. H. a n d G a r b y, L.: Low binding of 2, 3-diphosphoglycerate to Haemoglobin F. Scand. J. Clin. Lab. Invest. 23: , E d w a r d s, M. J., N o v y, M. J., W a l t e r s, C. L., a n d M e t c a l f e, J.: Improved oxygen release: Adaptation of mature red cells to hypoxia. J. Clin. Invest. 47: , G u e s t, G. M. a n d R a p o p o r t, S.: Role of acid-soluble phosphorus compounds in red blood cells: In experimental rickets, renal insufficiency, pyloric obstruction, gastroenteritis, ammonium chloride acidosis, and diabetic acidosis. Amer. J. Dis. Child. 58: 1072, L e n f a n t, C., T o r r a n c e, J., E n g l i s h, E., F i n c h, C. A., R e y n a f a r j e, C., R a m o s, J., a n d F a u r y, J.: Effect of altitude on oxygen binding by hemoglobin and on organic phosphate levels. J. Clin. Invest. 47: , 1968.

15 176 OSSI 17. M o u r d j i n i s, A., W a l t e r s, C., E d w a r d s, M. J., K o l e r, R. D., Va n d e r h e i d e n, B., a n d M e t c a l f e, J.: Improved oxygen delivery in pyruvate kinase deficiency. Clin. Res. 17: 153, M u b h e a d, H., C o x, J. M., M a z z a r e l l a, L., a n d P e r u t z, M. F.: Structure and function of hemoglobin. III. A three-dimensional Fourier synthesis of human deoxy haemoglobin at 5.5 A resolution. J. Molec. Biol. 28: 117, M ü l h a u s e n, R., A s t r u p, P., a n d K j e l d s e n, K.: Oxygen affinity of hemoglobin in patients with cardiovascular diseases, anemia and cirrhosis of the liver. Scand. J. Clin. Lab. Invest. 19: 291, O p itz, E. a n d S c h n e i d e r, M.: Über die Sauerstoffversogung des Gehirns und der mechanisms von Mangeliverkungen. Ergebn. Physiol. 46: 126, O sk i, F. A., G o t t l i e b, A. J., D e l i v o b i a - P a p a d o p o u l o s, M., a n d M i l l e b, W. W.: Red cell 2, 3-diphosphoglycerate levels in subjects with chronic hypoxemia. New Eng. J. Med. 200: , O sk i, F. A., M a r s h a l l, B. E., C o h e n, P. J., S u g e r m a n, H. J., a n d M i l l e r, L. D.: Exercise with anemia. The role of the left or right shifted oxygen-hemoglobin equilibrium curve. Ann. Int. Med. 74: 44-46, O sk i, F. A., M i l l e r, W., D e l i v o r i a - P a p a - d o p o u l o s, M., a n d G o t t l i e b, A. J.: The effects of deoxygenation of adult and fetal hemoglobin on the synthesis of red cell 2, 3- diphosphoglycerate and its in vivo consequences. J. Clin. Invest. 49:400, O s k i, F. A., T r a v i s, S. F., M i l l e r, L. D., a n d D e l i v o r i a - P a p a d o p o u l o s, M.: The in vitro restoration of red cell 2, 3-diphosphoglycerate levels in banked blood. Blood 37: 52-58, R o s e, A. B.: Purification and properties of diphosphoglycerate mutase from human erythrocytes. J. Biol. Chem. 243:4810, S c h m i d t - N i e l s e n, K. a n d T a y l o r, R. R.: Red blood cells: Why or why not? Science 162: 274, S u g e r m a n, H., M i l l e r, L. D., O sk i, F. A., D i a c o, J., a n d D e l i v o r i a - P a p a d o p o u l o s, M.: Decreased 2, 3-diphosphoglycerate (DPG ) and reduced oxygen (Os ) consumption in septic shock. Clin. Research 18: 418, T o r r a n c e, J., J a c o b s, P., L e n f a n t, C., a n d F i n c h, C.: Intraerythrocytic adaptation to anemia. New Eng. J. Med. 283: 165, V a l e r i, C. R. a n d H i r s c h, N. M.: Restoration in vivo of erythrocyte adenosine triphosphate, 2, 3-diphosphoglycerate, potassium ion, and sodium ion concentrations following the transfusion of acid-citrate-dextrose-stored human red blood cells. J. Lab. Clin. Med. 73; , V a l t i s, D. J. a n d K e n n e d y, A. C.: Defective gas-transport function of stored red-blood cells. Lancet 1: , 1954.