The Oxyhemoglobin Dissociation Curve in Acute Disease

Symposium on a Physiologic Approach to Critical Care

The Oxyhemoglobin Dissociation Curve in Acute Disease Rita McConn, PhD. *

While clinicians have always regarded the red cell as an important link in the oxygen transport chain, they tend to evaluate it in terms of the concentration of hemoglobin. From the therapeutic viewpoint, comparatively little attention has been paid to the physiologic significance of the position of the dissociation curve other than those changes induced by the patient's condition, such as the presence of fever or an acidotic or alkalotic pH. In 1967, it was found that the organic phosphate, 2,3 diphosphoglyceric acid, could influence the affinity of oxygen for hemoglobin. 20 ,34 Three years later, Perutz described the structural changes which occur in the hemoglobin molecule upon oxygenation.95 These findings initiated a new look at the role of the dissociation curve in oxygen transport and unloading by the red cell in health and disease. An important aspect of this "new interest" in the dissociation curve was the re-evaluation of current methods of blood storage in terms of the respiratory function of the blood. This article aims to review these recent findings in terms of their relevance to oxygen transport in the acutely ill patient. The oxyhemoglobin dissociation curve is an expression of the reaction of oxygen with hemoglobin in terms of the per cent saturation of hemoglobin versus the partial pressure of oxygen. Classically one is taught that the oxyhemoglobin dissociation curve has a sigmoid shape as first described by Bohr in 1892.25 It is only with the advent of sophisticated techniques in comparatively recent times that the structure of hemoglobin has been unravelled and the'S-shaped dissociation curve explained in terms of the change in the stereochemistry of the respiratory protein on oxygenation. Proteins have a primary structure, i.e., amino acid composition and order of sequence, which is determined genetically, while the secondary and tertiary structure are influenced by both the intrinsic components and the environment. Human hemoglobin consists of two unlike pairs of polypeptide chains, each chain containing one binding site for oxygen, the heme group. Over 90 per cent of the hemoglobin in adult erythrocytes is hemoglobin A, which is composed of two alpha chains and two "'Associate Professor of Surgery, Albert Einstein College of Medicine, Bronx, New York

Surgical Clinics of North America- Vol. 55, No.3, June 1975

627

628

RITA MCCONN

Figure 1. Tertiary structure of the '" chain of human hemoglobin A. A, B, C, D, E, F, G, and H indicate the alphabetical parts of the molecule. The porphyrin ring is shown as a disk. The oxygen molecule is bound between the disk and the E-helix. (Figs. 1 and 2 reproduced from Rorth, M.: Hemoglobin Interactions and Red Cell Metabolism, Series Haematologica V, 1972. Used with permission.)

beta chains. In adult man, hemoglobin A 2, two alpha and two delta chains, accounts for only about 2.5 per cent while there is less than 1 per cent of hemoglobin F, two alpha and two gamma chains. The alpha chain has 141 amino acids and the beta chain 146 amino acids, and in 1961 the primary structure of these chains was determined. 27,67 The secondary structure of these chains has a relatively large proportion of alpha helices-there are seven distinct segments of 7 to 21 amino acids in the alpha helix conformation in the alpha chains (named A, B, C, E, F, G, and H), and eight segments of alpha helix in the beta chains (named A, B, C, D, E, F, G, and H). The helices are linked by stretches of amino acids in a random coil arrangement. The helical segments have clearly defined position in space-the tertiary structure-thus, e.g., the A helix is close to the G and H helices. The E and F helices form a hydrophobic cleft into which the heme moiety is inserted (Fig. 1). The spatial relationship of the four subunit chains is termed the quaternary structure of hemoglobin. The fact that oxyhemoglobin crystals were different in shape from crystals of reduced hemoglobin, the former being needle-shaped and the latter hexagonal,57 gave the first clue that the reaction of hemoglobin with oxygen is accompanied by a change in the structure of the hemoglobin on oxygenation.85 Perutz proposed that combination of an oxygen molecule with a heme group alters the position of the ferrous ion in the heme ring. This in turn triggers a series of physicochemical events which alters the position of the peptide chain such that the salt bridge with a neighboring chain is broken, and this results in a change of the oxygen affinity of the heme group in this neighboring subunit chain. Each combination of an oxygen molecule with a heme group thus facilitates the binding of the next one. Studies suggest that on oxygenation of the third heme, the quaternary structure of hemoglobin changes. 54 ,115 These molecular events are the cause of the changing oxygen affinity of hemoglobin on oxygenation and cause the dissociation curve to have a sigmoid shape. In addition to the shape, the position of the dissociation curve is also an indicator of the affinity of oxygen for hemoglobin. Figure 2 shows that a monomeric hemoglobin has a much higher affinity for oxygen than a tetrameric hemoglobin. Thus the quaternary structure of the molecule superimposes constraints on the monomers and, according to

629

OXYHEMOGLOBIN DISSOCIATION CURVE IN ACUTE DISEASE

Perutz these constraints are six oxygen-linked salt bridges (whose actual position has been identified), which must be broken before oxygenation of the tetramer is complete. Hence the dissociation curve of the monomer does not have a sigmoid shape and has a higher affinity for oxygen due to the absence of the salt bridges. In physiologic studies of the dissociation curve of human blood, it is generally assumed that the shape of the dissociation curve is invariant. Although studies by Roughton in 1972 show a slight deviation in shape at the top of the curve with temperature,t°6 for all practical purposes it can be regarded as insignificant in comparison to the variance which can occur in the position of the curve. Until recently, with the exception of the genetic variants, the position of the dissociation curve had been regarded as a constant physiologic parameter, only varying with temperature and pH. It is now well established that this is a gross oversimplification. The factors influencing the position of the hemoglobin dissociation curve are complex and this complexity is increased even further when the protein is enclosed within the red cell in vivo and travelling through the circulation. The advent of chronic and acute disease is an additional complicating factor, the extent of which is not fully understood. Temperature Paul Bert in 1872 found that blood was 90 per cent saturated at an oxygen tension of 15 mm Hg at room temperature whereas it was only 50 per cent saturated at the same tension at body temperature. 23 Barcroft and King (1909) confirmed these observations in hemoglobin solutions and whole blood. 14 The decrease in oxygen affinity (a right shift of the curve) with a rise in temperature is in agreement with the fact that the reaction of oxygen with hemoglobin is an exothermic reaction. A number of investigators have determined the magnitude of the shift of the dissociation curve of whole blood with temperature in terms of an equation relating the change in P0 2 with temperature at constant

HbA 25°C

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,,002 Figure 2. Oxyhemoglobin dissociation curve of monometric hemoglobin (m) and tetramerle hemoglobin (t).

*

630

RITA MCCONN

saturation. 8everinghaus l08 in the preparation of his slide-rule blood gas calculator revised this work and utilized the following equation: A log P0 2

=

0.024 AT

Ligands of Hemoglobin Other Than Oxygen A number of other entities, besides oxygen, can form bonds (ligands) with hemoglobin, the site of binding being to different groups in the globin molecule. Of these ligands, some show a preferential binding for one form of hemoglobin over another, Le., bind preferentially to Hb02 or Hb. This differential binding of ligands results in a change of hemoglobin affinity for oxygen and a shift in the position of the oxyhemoglobin dissociation curve. Those ligands which bind more readily to reduce hemoglobin lower the oxygen affinity of hemoglobin and cause a right shift in the curve. The relationship between the preferential binding of ligands and the oxygen affinity of hemoglobin can be solved on the basis of fundamental equilibrium thermodynamics in terms of linkage-equations. lo3 For physiologic purposes it is sufficient however, to be aware that hydrogen ions, CO2, inorganic anions, such as C1 and 804 , and organic phosphates bind more readily to reduce hemoglobin and therefore lower the oxygen affinity of hemoglobin and shift the dissociation curve to the right.

Hydrogen Ion and Carbon Dioxide The Bohr effect relates to the influence of the hydrogen ion concentration on the oxygen affinity of hemoglobin. The name dates back to the observations of Bohr, Hasselbalch, and Krogh,24 who noted that CO 2 influenced the position of the curve and hence gave the first indication that the "acidity" of the environment could change the position of the dissociation curve. A molecular explanation of the Bohr effect is now possible due to the refined studies of recent years. As explained earlier, oxygenation of hemoglobin is associated with breakage of salt bridges in the hemoglobin molecule. Rupture of two of these bridges involves the release of an acidic grouping which results in a fall in pK and protons are liberated more easily. Thus, oxyhemoglobin is a stronger acid than reduced hemoglobin and, as a result, protons are preferentially bound by the reduced form; this preferential binding, as stated above, is associated with a lowering of oxygen affinity and a right shift in the curve. The effect of CO2 on the curve can be divided into the classical Bohr effect as above and a CO 2 effect per se since CO 2 can combine with uncharged amino groups to form carbamino compounds. The magnitude of the Bohr effect can be expressed for a constant oxygen saturation by the change in log P0 2 caused by a unit change in pH. For many years it was thought that the numerical value relating these factors was a constant; however, it appears to be variable. 86 Currently the following values are known: (1) Alog P0 2 = -0.48 when the pH change is induced by a change in CO 2 concentration. (2) A log P0 2 = -0.40 when the pH change is induced by an acid or base other than CO 2 , and when the concentration of CO 2 and the organic phosphate 2,3 DPG remains constant. 2,3 DPG concentration is now known to influence the numerical value of the Bohr effect. 121 Obviously, under physiologic conditions pH,

OXYHEMOGLOBIN DISSOCIATION CURVE IN ACUTE DISEASE

631

PC0 2 and 2,3 DPG are all variable parameters and the magnitude of the Bohr factor is currently unknown when these factors are present in various concentration in relation to each other.

2,3 Diphosphoglyceric Acid (2,3 DPG) and Adenosine Triphosphate (ATP) In most mammals, 2,3 DPG is the most abundant organic phosphate within the red cell. In man it is present in approximately four times the concentration of ATP. Although it was known to be produced by a side reaction of glycolysis, for many years no one was able to determine any biochemical or physiologic role for this compound in the red cell. In 1967, Benesch and Benesch in New York 20 and Chanutin and Curnish in Virgina34 showed that 2,3 DPG could influence the position of the oxyhemoglobin dissociation curve. This compound was found to be preferentially bound by reduced hemoglobin2. 21. 52 and hence it lowers the oxygen affinity of hemoglobin. The binding sites were subsequently identified and it appears that the binding of this organic phosphate is influenced by pH,103 PC0 2,17 temperature,22 other anions,22 and the concentration of hemoglobin.53 It is therefore not surprising that precise quantitation of the 2,3 DPG effect on the oxygen dissociation curve is not yet available. Adenosine triphosphate also interacts with hemoglobin presumably in the same way as 2,3 DPG73 but the effect is somewhat less. 102 Salts The effect of inorganic cations and anions on the oxyhemoglobin dissociation curve will be influenced by both the size of the ion and its charge. While there has been a fair amount of work on the influence of salts in hemoglobin solutions, comparatively little work has been done in blood and what is available is inconclusive. It is therefore not possible to make any firm statements with respect to the influence of inorganic salts in the red cell on the oxyhemoglobin dissociation curve.

THE DISSOCIATION CURVE OF WHOLE BLOOD Enclosure of hemoglobin within the erythrocyte places it in a milieu where the ligands, other than O2, are present at varying concentrations under different conditions. Thus, it is a very complex situation which is not yet fully understood. Hemoglobin is present in the red cell in very high concentrations and there is some work to suggest that this in itself influences the oxygen affinity of hemoglobin, a high mean cellular hemoglobin concentration being associated with a decrease in affinity.1s The concept of the erythrocyte being a passive cell, merely acting as a transport agent for O2, is a gross oversimplification. Although the red cell has no mitochondria, it can metabolize glucose by glycolysis and the hexose monophosphate shunt and is an actively metabolizing unit (Fig. 3). The finding that metabolites, such as 2,3 DPG and ATP, can influence the oxygen affinity of hemoglobin, related for the first time the metabolism of the erythrocyte to its physiologic function.

632

RITA MCCONN

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OXY-HEMOGLOBIN DISSOCIATION CURVE ON BLOOD FROM A NORMAL INDIVIDUAL

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633

OXYHEMOGLOBIN DISSOCIATION CURVE IN ACUTE DISEASE

The oxyhemoglobin dissociation curve in whole blood is shown in Figure 4. For comparative purposes the position of the curve is defined as the Po~ at 50 per cent saturation under standard conditions, namely 37.5°C and plasma pH of 7.4 and for normal man the P 50 is 26.52 mmHg.66 The pH which influences the oxygen affinity of hemoglobin is, of course, not the serum pH, but the intraerythrocytic pH. There is a pH gradient across the red cell membranes which in normal man is of the order of 0.2 units; thus, at serum pH 7.4 intraerythrocytic pH is 7.2. This gradient results from a differential distribution of ions between the plasma and the red cell and is due to the Gibbs-Donnan equilibrium, i.e., when there is a large nondiffusible ion on one side of a semipermeable membrane, the remaining ions distribute themselves so that there is electroneutrality. At physiologic pH, hemoglobin is a large nondiffusible anion which is largely responsible for the gradient; however, the organic phosphate 2,3 DPG also contributes since it is a nondiffusible anion. 46 Increasing the concentration of any nondiffusible anion lowers Table 1. Factors Influencing the Position of the Dissociation Curve of Whole Blood EXAMPLE OF CHANGE IN

P 5•

INDUCED IN NORMAL BLOOD

P 50 26.52 AT 37°C AND pH 7.4 UNDER VARIOUS

WITH FACTOR

Temperature

RELATIONSHIP

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t

T

t

MAGNITUDE

A log Po,

Bohr effect

2,3 DPG

P 50

t

2.3 DPG i

ATP

P,O

t

ATP

McHc

P 50 i

t

McHc

0.024A T

Increase in temp. from 37.5°C to 40°C. P 50 changes from 26.52 to 30.3 mmHg. Decrease in temp. from 37.5°C to 35°C. P 50 changed from 26.52 to 23.09 mmHg

A log Po, ApH

-0.40

A log Po, ApH

-0.48

Change in pH from 7.4 to 7.15; P 50 changes from 26.52 to 33.38 mmHg Change in pH from 7.4 to 7.65; P 50 changes from 26.52 to 21.06 mmHg Change in pH from 7.4 to 7.15; P 50 changes from 26.52 to 34.95 mmHg Changes in pH from 7.4 to 7.65; P,o changes from 26.52 to 20.11 mmHg

=

A 0.440IL moles per ml blood P 50 increases 1 mmHg" (normal subjects and chronic disease) Magnitude unknown

i

CONDITIONS

Magnitude unknown

''Conditions of pH. Pco" temperature, etc., not defined.

634

RITA MCCONN

the intraerythrocytic pH and through the Bohr effect lowers the oxygen affinity of hemoglobin. Thus, an increase in 2,3 DPG concentration within the red cell lowers the oxygen affinity of hemoglobin by two means: (1) a direct effect through binding to hemoglobin, (2) indirectly through lowering red cell pH-the Bohr effect.16, 18, 45 Table 1 summarizes the relationship (and the magnitude wherever known) between these various factors known to affect the oxygen affinity of hemoglobin and P 50 values for whole blood. In order to provide the reader with a realistic idea of the amount of change in the position of the curve that these factors can produce in normal blood, a number of examples are given, e.g., the change in P 50 in normal blood (P 50 26.52) produced by an increase in temperature from 37.5°C to 40°C would be an increase of 3.92, i.e., P 50 30.44 mm Hg.

METABOLIC REGULATION OF THE O 2 AFFINITY OF HEMOGLOBIN The unique role of 2,3 DPG indicates that it could well be the metabolic regulator of O 2 transport by the red cell. For many years it was thought that the only metabolic response of the body to a decreased oxygen supply was the pyruvate-lactate reaction which served as a means for regenerating NAD from NADH to enable the continuation of glycolysis. This reaction is self-limiting,. however, because of the effects of lactic acid on the hemodynamic system. Hypoxia has been found to be associated with an increase in 2,3 DPG in vitro. This observation has led to the current belief that 2,3 DPG plays a major role in the metabolic response to hypoxia and studies are underway to determine exactly how this response is regulated. The concentration of 2,3 DPG is dependent upon the concentration of 1:3 DPG and this in turn is dependent upon the glycolytic rate. 105 There are three rate-limiting enzymatic steps in glycolysis :28 Hexokinase 1. Glucose ~ Glucose 6 phosphate ATP~ADP

Phosphofructokinase 2. Fructose 6 phosphate -----c> Fructose 1:6 diphosphate ADP~ATP

Pyruvic kinase 3. Phosphoenol pyruvate -----c> Pyruvate ADP..---.ATP

Current work indicates that hexokinase is stimulated in the presence of a low circulating level of hemoglobin while phosphofructokinase is stimulated by an alkalotic pH.28 This increase in pH may occur either as a result of hyperventilation and/or an increase in the amount of reduced hemoglobin, since the latter, being less acidic than oxidized hemoglobin, would also raise red cell pH. Thus, in clinical terms anemia, hyperventilation, and/or arterial hypoxemia could all stimulate glycolysis and increase 2,3 DPG levels, and thus trigger the metabolic

OXYHEMOGLOBIN DISSOCIATION CURVE IN ACUTE DISEASE

635

response to hypoxia. The significance of other glycolytic enzymes, substrates, and co-factors such as ATP, ADP, and inorganic phosphate, as well as the influence of preferential binding of 2,3 DPG by reduced hemoglobin, continues to be an active site for research and discussion.50. 103 The in vivo effect of pH on 2,3 DPG level has been quantitated and it appears that an increase in red cell pH of 0.01 unit is associated with a 4 to 5 per cent increase in the normal concentration of '2,3 DPG.'2 In vitro and in vivo studies indicate, however, that this change occurs relatively slowly. Thus the acute effect of an increase in pH is a left shift of the curve due to the Bohr effect. This occurs within less than 1 msec 103 If the pH change is sustained for some hours, this concentration of 2,3 DPG will increase, resulting in a right shift in the curve. In normal man these two effects balance each other, so the overall effect is little or no change in the position of the curve. However, in evaluating the effect of pH changes on the dissociation curve, the time factor is important. 19

METHODS OF MEASURING THE OXYHEMOGLOBIN DISSOCIATION CURVE OF BLOOD 1. In Vitro Equilibration Technique. This requires exposing blood to humidified gases of varying O2 concentration, constant CO 2 and balance nitrogen in a tonometry system. After equilibration the oxygen saturation and pH of the samples are measured. A description of this method is given by Astrup.8 Any desired number of points of the curve may be obtained in this way and the data may be plotted graphically to obtain the dissociation curve. Alternatively the Hill equation may be used: 59

log 10;_ y = log K + n log P y = per cent saturation (Hb0 2 ) K = constant n =2.6 P = partial pressure of oxygen and the P50 values obtained by solving the equation for y=50. This equilibration method may be used for very small quantities of blood if the Astrup micro tonometry system and the Radiometer OSM I Saturation meter are used. 2. The Mixing Technique of Edwards and Martin. 48 This is based on the principle that the P0 2 measured in a blood sample obtained by mixing anaerobically equal volumes of fully oxygenated and fully deoxygenated is equal to the P50 of that blood at the measured pH. Alternatively, by varying the ratios of reduced to oxygenated blood and measuring P0 2 and O 2 saturation of the resultant samples, other points on the dissociation curve may be obtained and a curve drawn as described above. 3. Dissociation Curve AnalyzerY Duvelleroy and colleagues developed an instrument ultilizing oxygen electrodes that enables rapid and complete analysis of the hemoglobin dissociation curve. The technique

636

RITA MCCONN

Pso NOMOGRAM

90

80 70 ~

~ 60

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~ 50 IU

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~ 40 30 20 10

20

30

40

50

60

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OXYGEN TENSION (mm Hg) Figure 5. Nomogram for the calculation of P 50 • The point on the nomogram corresponding to the measured P02 and saturation of a sample of venous blood is traced to the line representing 50 per cent saturation. The intersect represents the estimated P"" e.g., venous blood P0 2 = 39mmHg, O 2 saturation = 65 per cent, P 50 = 31mmHg. (Reproduced from Canizaro, P. C. et al.: A technique for estimating the position of the oxyhemoglobin dissociation curve. Ann. Surg., 180:364,1973. Used with permission.)

offers the advantage that a complete curve is derived from the measurement of oxygen uptake by a sample of deoxygenated blood; the analysis is complete in about 30 minutes and requires approximately 10 ml blood. The only preparation required is complete de saturation of the blood using 95 per cent N z per 5 per cent CO2 gas mixture. The deoxygenated blood is then exposed in a closed cuvette to a gas mixture of 95 per cent O 2 per 5 per cent CO 2 , The blood is stirred by a magnetic slug. Oxygen tensions in both the gas and blood phases within the cuvette are measured continuously by two oxygen electrodes until equilibration. The output voltage from the electrode in the blood phase is fed into the abscissa of an X-Y plotter, and that from the electrode in the gas phase into the ordinate. The X-axis shows changes in blood Pa 2 • The Y-axis is calibrated to read O 2 content (ml O 2 ) based on calculations using PV-RT where V volume of chamber is known. As the blood becomes oxygenated, a plot of O 2 content versus Pa 2 is generated on the X-Y plotter and the dissociation curve is then calculated from this plot. 4. Canizaro's Nomogram. 33 Recently Canizaro and co-workers constructed a nomogram which allows estimation of the P 50 value from Pa 2 and per cent O2 saturation of a single venous blood sample (Fig. 5). They found good correlation between 50 estimated P so values from a variety of severely ill patients compared with P so values derived from a curve obtained using the Martin-Edwards mixing technique as described above.

637

OXYHEMOGLOBIN DISSOCIATION CURVE IN ACUTE DISEASE

Of all the techRiques described, this is the most practical one available to the clinician and therefore merits some further discussion. In using the nomogram, accurate values of PO z and Oz saturation are essential, since a small error in these measurements can result in a large error in the derived value for P 50 • Accurate measurement of blood PO z requires both attention to calibration and meticulous technique in the drawing of the blood and in sample handling. The author recently conducted a survey of the accuracy of blood gas systems available in this country. so An invitation was issued to manufacturers of this equipment and it was requested that each system should be run by the manufacturer's representative. Blood samples equilibrated with humidified gases of known O 2 and CO 2 concentrations were used as test samples. The ,.' results obtained for blood PO z measurements are shown in Figure 6-it can be seen that in the range 50 to 700 mm Hg, the per cent deviation in measured blood P0 2 from actual blood P0 2 ranged from 15 per cent to greater than 30 per cent. In all instances the electrodes were calibrated with humidified gases of known O2 concentrations. It is well known, however, that in the case of the oxygen electrode, there is a difference in readings between gas and liquid samples known to have the same concentration, since it is a diffusion controlled system. However, it is not possible to use arbitrary correction factors for this difference. One solution suggested by Heitmann, Buckles, and Laver is to use glycerine/HzO mixture of the same viscosity as blood, as an equilibration solution.58 Oxygen saturation meters vary in their accuracy'l 7 and, furthermore, the error of O2 saturation measurements increases as the per cent saturation falls (Fig. 7). In order to minimize error in P so estimation, Canizaro recommends that the venous O2 saturation should ideally be between 40 and 75 per cent. 5. Severinghaus Slide Rule. 108 Alternatively the P 50 may be obtained from P0 2 and O 2 saturation of venous blood using the Severinghaus formula: Estimated P 50

=

26.6 P P02 OZ sat

where P0 2 sat is the P0 2 on the standard dissociation curve corresponding to the measured saturation. An even simpler method to obtain P 50

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Figure 6. Actual deviation in mmHg of measured P02 obtained from actual P0 2 in a variety of blood samples prepared by tonometry with gases of known O2 concentration. The results are shown for three different commercial blood oxygen electrodes: Radiometer, In· strumentation Laboratories (I.L.) , and Corning (analyzed by the manufacturers' representa· tives) and the hospital clinical laboratory which has an I.L. O2 electrode.

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is to use the Severinghaus slide rule 108 (London Company, Cleveland, Ohio) and align the P0 2 and O 2 saturation measurements on Scale A and B respectively. The estimated P 50 is that P0 2 on Scale A immediately above the point of 50 per cent saturation on scale B. For comparative purposes it is necessary to correct the P 50 obtained by any of the above methods to the standard conditions of temperature and pH. Since it is common practice to measure blood gases at 37.5°C the P 50 obtained will not require correction to standard temperature. The standard conditions for pH are usually stated as blood pH (serum pH) 7.4; assuming a normal red cell pH gradient of 0.2, this is equal to an intraerythrocytic pH of 7.2. It is advisable, however, when determining P 50 value on a blood sample, to measure both the serum and red cell pH, since the gradient can vary for a number of reasons, e.g., the gradient is reduced if citrate is used as an anticoagulant; thus in ACD blood.:l pH is 0.05. The gradient varies in the acutely-ill, and in patients during surgery depending upon intravenous solutions given in these instances it may be higher or lower than normal. Red cell pH is easily measured using the technique of Purcell,98 in which red cells are packed by centrifugation in a capillary tube, hemolyzed by freezing and thawing and the pH of the hemolysate determined using a capillary blood pH electrode. The blood and red cell pH will of course depend upon the acid-base state of the blood. The following example serves to illustrate how to convert P 50 values obtained experimentally to standard conditions: In a blood sample the P 50 was found to be 31 mm Hg at blood pH of 7.25 and red cell pH 7.10. Since the red cell pH is that which influences the oxygen affinity of he-

639

OXYHEMOGLOBIN DISSOCIATION CURVE IN ACUTE DISEASE

moglobin, the P 50 value must be corrected to a red cell pH 7.2. In this example a shift in the curve due to pH arises from a fall in pH of 7.2 (standard red cell pH) -7.10 (red cell pH of blood sample) = 0.1. The correction for the shift in the curve for pH is obtained by use of the Bohr equation.

IO~~Oz

= -0.40

log 3 \-log x =-0.40 0 log 31 - 0.040 = log x = 1.4514 x =·28.29 mm Hg P 50 pHc 7.2 A note of caution is necessary at this point; as stated earlier the numerical value of the Bohr equation is now thought to vary depending upon the concentration of the ligands in the red cell; therefore the calculated P 50 , obtained using the standard factors of 0.40 or 0.48, should be treated conservatively when the intraerythrocytic pH measured is markedly different from 7.20. Furthermore, in reviewing data it is important to determine whether P so was corrected to red cell pH 7.2 or whole blood pH 7.4, since if the latter is used, error may arise if there is a variation in the red cell pH gradient.

THE SIGNIFICANCE OF A CHANGE IN THE OXYGEN AFFINITY OF BLOOD Theoretical Viewpoint For the clinician the outstanding question is whether changes in the O2 affinity of hemoglobin which occur in health and in disease are of academic interest, or whether they have any real meaning in therapeutic terms. Here the reader must again be patient to continue with a little more theory before the practical situation is presented. The amount of oxygen transported per minute from the lungs to the tissues is a function of the cardiac output and the oxygen content of the arterial blood. The oxygen content of the arterial blood depends upon the hemoglobin concentration and the POz of the blood which is a reflection of the inspired mixture and the relationship between ventilation perfusion in the lung. Thus: C a0 2

=

Where Ca0 2 = [Hb] = 1.34 = 0.0031 = P a02 =

[Hb] x 1.34 x per cent O 2 saturation O 2 bound to hemoglobin + O2

+ (0.0031

x P a02)

oxygen content of arterial blood, vols per cent hemoglobin concentration, gm per cent ml O 2 bound by 1 gm of hemoglobin Bunsen solubility coefficient for O2 at 37°C in plasma arterial oxygen tension

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40

Po 2 mmHg

P0 2 mmHg

60

80

100

80

100

Po 2 mmHg

400 Ca-vo2 307vols1.

1

300

E

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3 100 o I

234

5 6 789

1234567

CARDIAC OUTPUT lImon

CARDIAC OUTPUT lImon

Pso 19.2 mm Hg

Pso 25.5 mm Hg

100F-_--,--_----:=_--,--

Pso 30.6 mm Hg Ca- va2 4 60vols %

60 %Sat

20

40

60

80

100

20

P0 2 mm Hg

40

60

80

100

P0 2 mm Hg

20

40

60

P0 2 mm Hg

Figure 8. The effect of a shift in the oxyhemoglobin dissociation curve on arteriovenous oxygen extraction, oxygen consumption, and cardiac output. (For detailed explanation see text).

Thus if a blood sample has a

of 98 mm Hg, and an oxygen saturation of 12 gm per cent its oxygen content would be: C a o 2 = 12 x 1.34 x 19;0 + 0.0031 x 98 = Pa02

15.90 vols per cent.

The amount of oxygen extracted by the tissues per minute, i e., O2 consumption, is a function of the cardiac output and the arteriovenous oxygen content difference. This may be calculated as follows. V0 2 Vo z C a o2 CV o 2

= =

Q

=

= =

(Cao, - C,.oz) x Q x 10 O 2 consumption ml per min. arterial oxygen content mixed venous oxygen content cardiac output 1 per min

These equations are illustrated graphically in Figure S using an example of a patient in whom arterial and mixed venous blood gas values and hemoglobin concentration are known. Figure SA illustrates the influence of a change in the position of the curve in this patient on arteriovenous oxygen saturation and arteriovenous oxygen content. A normal curve (P 50 25.5 mm Hg) and left- (P50 19.2 mm Hg) and right-shifted (P50 30.6 mm Hg) curves are illustrated. It can be seen that for the same

640

OXYHEMOGLOBIN DISSOCIATION CURVE IN ACUTE DISEASE

641

values of arterial and mixed venous P0 2, a left-shifted curve results in a higher mixed venous oxygen saturation than a normal curve, while a right shift results in a lower value for the mixed venous saturation. Arterial P0 2 value in all three instances falls on the flat top portion of the curve and it can therefore be seen that a shift in the curve has comparatively little effect on arterial oxygen saturation. Thus, the overall result is that a lower P 50 results in a decrease in arteriovenous oxygen extraction compared to normal; in this specific instance, 3.07 vols per cent compared to 4.60 vols per cent respectively. In contrast, a raised P 50 results in an increase in oxygen extraction, in this example 5.97 vols per cent compared to 4.60 vols per cent. Figure BB illustrates the influence of cardiac output on oxygen consumption in normal, left- and right-shifted dissociation curves. If we assume a normal basal O 2consumption in man of 250 ml per min, it can be seen that to maintain this O2 delivery with a P 50 of 25.5 mmHg, a C.O. of 5.43 1 per min is required. In the case of a left-shifted curve where the a-V0 2 extraction is only 3.07 vols per cent, the cardiac output must increase to B.14 1 per min to maintain this O2 consumption, while in the case of the right-shifted curve where the a-V02 extraction is 5.97 vols per ·cent, the same O2 delivery can be achieved with a lower cardiac output, namely 4.1B 1 per min. If the cardiac output remains constant at the normal value, in this case of 5.43 1 per min, then O2 consumption can only be maintained at 250 ml per min in the presence of a left-shifted curve by lowering the mixed venous oxygen tension from 36 to 29 mm Hg, increasing a-V02 extraction to .4.60 vols per cent (Fig. Be). In contrast, in the case of a right-shifted curve, an O2 delivery of 250 ml per min can be achieved with a cardiao output of 5.43 1 per min with a mixed venous oxygen tension of 42 mm Hg. In normal man, when an increase in O2 demands arise, the various links in the O2 transport chain are able to compensate to increase O2 delivery, such as an increase in cardiac output and/or a change in the distribution of blood flow resulting in increased O2 extraction. The concentration of 2,3 DPG in normal man is constant (0.75 to O.B moles per mole hemoglobin) in the absence of changes in acid-base state lasting a significant period of time. Thus under normal circumstances, the major influence on the oxygen affinity of hemoglobin in vivo is the Bohr effect resulting from pH changes which occur in the plasma and thus within the red cell as it travels through the circulation. The degree of variation in pH will of course be dependent upon tissue metabolism and blood flow.

THE EFFECT OF ENVIRONMENT AND CHRONIC DISEASE ON THE OXYHEMOGLOBIN DISSOCIATION CURVE In 1917 Hasselbalch studied the dissociation curve of eight patients and in only three of the patients was the dissociation curve in the normal position. 56 These five patients with abnormal curves included pernicious anemia, uremia, gout, and diabetic coma. The ensuing years saw only occasional reports in the literature of the effect of disease on the oxygen affinity hemoglobin but the discovery of the role of 2,3 DPG in

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RITA MCCONN

red cell led a concentrated investigative effort by both physiologists and clinicians to determine if disease had a specific influence on oxygen transport by the red cell. The patient population of most intensive care units is not composed of 100 per cent previously healthy subjects suddenly struck down with an acute life-threatening situation. Indeed, if it were, the survival rates from such centers would perhaps be significantly higher. In fact, a fair number of patients have already some pre-existing chronic diseases which may have developed into an acute situation, e.g., the chronic obstructive lung disease patient who, with the advent of pulmonary infection, is precipitated into acute respiratory failure; or alternatively, the onset of another pathologic condition in a patient who already has a chronic disease, could produce also an acute situation, or the patient with a chronic low cardiac output due to long standing cardiac disease, who falls into septic shock subsequent to an acute abdomen, peritonitis, and surgery. It is therefore important for the clinic an to be aware of what is known on chronic disease in relation to the oxygen affinity of hemoglobin before proceeding to the acutely ill. First however, let us consider the influence of age and environment. After the first months of life, age per se does not appear to have any effect on the dissociation curve. Laver examined the blood of Dr. A. V. Bock at the age of 79 years and found the oxygen affinity of hemoglobin not to be significantly different from that obtained some 41 years earlierY The age of red cells in the circulation, however, does affect the oxygen affinity of hemoglobin since young red cells have higher 2,3 DPG content than old red cells. 52 The influence of the patient's environment may be considered from two points of view, the effect of exposure to the atmospheric pollutant carbon monoxide, and the influence of living at high altitude. Carbon monoxide is a colorless nonirritating gas generated by incomplete combustion and it occurs in industry, in tobacco smoke, in household heating, and in motor vehicle exhaust gases. It is regarded as a health hazard because of its ability to impair oxygen transport by the blood through two mechanisms. First, its oxygen affinity for hemoglobin is 216 times greater than oxygen and thus a small amount of CO can inactivate a substantial portion of the oxygen-carrying capacity of the blood, and second, it causes a shift of the dissociation curve to the left. 13 • 15 In the United States cigarette smoking is probably the major source of CO; the median CO-Hb concentration for a one-pack-a-day cigarette smoker is 5.9 per cent,55 a concentration sufficient to imply a health threat in persons with underlying vascular efficiency. Astrup and co-workers 9 studied the effect of tobacco smoking in patients with occlusive arterial disease and their results showed a left-shifted curve in many of the smokers and a high number of patients with occlusive arterial disease exhibited this phenomenon to a pronounced degree. The displacement could be explained entirely by the effect of absorption of CO. Chronic hypoxia of altitude has been found to be associated with a lowering of the oxygen affinity of hemoglobin and an increase in 2,3 DPGY' 65. 69 The mechanism appears to be related to arterial hypoxemia,29 the presence of respiratory alkalosis,69 and the degree of activity at high altitude. 104 The comparative significance of these various factors is

OXYHEMOGLOBIN DISSOCIATION CURVE IN ACUTE DISEASE

643

not known and the influence of chronic as opposed to acute exposure is also unresolved. The improvement in O 2 delivery achieved by this rightward shift in the curve requires more investigation, since while this shift is associated with an increase in oxygen unloading if mixed venous P0 2 stays constant or falls, at high altitude, the P0 2 in the inspired air may reach such a level that there is a significant decrease in arterial oxygenation in the lung, which is only accentuated by a right , shift in the curve. A shift to the right in the dissociation curve and in increase in 2,3 DPG levels has been reported in patients with chronic low cardiac output,122 cyanotic congenital heart disease,R8 anemia,113 and cirrhosis. 10. 40, 66 In patients with anemia, the concentration of 2,3 DPG increases in proportion to the degree of red cell mass deficiency116 and is inversely related to the oxygen saturation of venous blood. 10 Patients with hereditary spherocytosis and splenomegaly may have lower, 2,3 DPG levels than patients with other types of anemia. 93 Thus there obviously are factors other than hemoglobin concentration, O 2 saturation, and red cell mass involved in the 2,3 DPG response to anemia. It has usually been stated that there is a decrease in the oxygen affinity of hemoglobin with ventilatory failure. 70 However, very recently a group of English workers showed that despite hypoxia and either chronic or acute hypercapnia the average values of 2,3 DPG and P 50 were normal in 27 patients although the range for these variables was wider than normal. 49 Changes in the oxyhemoglobin have been reported in disease states other than those associated with failure in the oxygen transport system. Patients with hyperthyroidism 83 and normal subjects treated with triiodothyronine show an increase in P 50 ;51 it appears that L-thyroxine and tri-iodothyronine stimulate diphosphoglycerate mutase which catalyses 1,3 DPG ~ 2,3 DPG.I09 This increase in O 2 delivery is associated with the increased metabolic rate of hyperthyroidism. The right shift of the curve in uremia first observed by Hasselbalch in 1917 56 is known to be associated with an increase in 2,3 DPG, ATP and, serum inorganic phosphate levels. 63 Hemoglobinopathies mayor may not be associated with a change in the oxygen affinity of hemoglobin~ A genetic defect resulting in a change in anyone of the 574 amino acids may not affect the dissociation curve since many amino acids are similar in size and shape and can be substituted for each other without consequence. On the other hand, there are critical areas where substitution may have a significant effect on O 2 affinity. Two such areas are the interface between alpha and beta chains and the terminal end of the beta chain. Substitution in either of these areas can result in a shift of the dissociation curve to the left and may cause a loss of sigmoid shape. This stimulates compensatory polycythemia, e.g., hemoglobin Chesapeake and Rainier. Inherited enzyme defects of the red cell metabolism may affect P 50 levels through their effect on 2,3 DPG concentration, e.g., a patient with a hexokinase deficiency had a decreased level of 2,3 DPG and low P 50 (19 mm Hg) curve in contrast to a patient with a pyruvic kinase deficiency who had high levels of 2,3 DPG and a raised P 50 (38 mm Hg)Y

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THE EFFECT OF ACUTE DISEASE ON THE OXYHEMOGLOBIN DISSOCIATION CURVE If the reader has stayed with patience to this point in the hope that the influence of acute disease on the oxygen affinity of blood will finally be related in concise and simple manner-he is only to be disappointed. The lengthy preamble has been written to place him in the position to be able to critically evaluate current work in this field. He will thus himself realize that our knowledge in this area is far from complete, and it is to be hoped that he will seek, through his own clinical experience, to add to our understanding on the respiratory function of blood. Before discussing the data obtained by clinical and animal studies in acute disease, let us first consider some of the limitations in this type of investigation. Acutely ill patients form a very heterogeneous group, varying not only in age, race, environment, and pre-existing chronic disease, but also in the syndrome causing the acute state; thus burns, trauma, shock - be it septic hemorrhagic, or cardiogenic in origin - postsurgical, combined renal and respiratory failure, are but some of the conditions seen in the Intensive Care Unit. It is therefore comparatively easy to obtain a group of patients to study the dissociation curve in chronic disease, e.g., anemia in Caucasian females aged 20 to 30 years, but how does one obtain a closely defined group of acutely ill patients? Even if one studies only one particular condition, e.g., sepsis, in addition to the above variables, therapy, duration of the disease, response to treatment, variation in the clinical and nursing staff, are all factors which it is impossible to rigidly control, as well as the innumerable variations possible in the patient's pulmonary and hemodynamic state. In the field of animal studies, different problems arise, such as the question of the similarity of the experimental procedure to the actual clinical situation; e.g., there has long been controversy over the validity of animal shock model and the species of animal used. The duration of the acute period of the experiment is frequently far shorter than seen in the clinical context, and furthermore, there is no underlying aspect of chronic disease. With these limitations in view, together with those pointed out earlier in the derivation of a standard P so from experimental data, it becomes easier to appreciate the different findings by various investigators in their studies on O2 transport by the red cell in acute disease. Serial studies of P so values in man and animals have reported a variety of responses in acute disease. No change was found in P so in rats for the first 6 hours after limb ischemia and 24 hours after a scald of 20 per cent of the body surface,72 nor in rhesus monkeys up to 4 hours after the development of shock induced by administration of endotoxin. 87 In contrast, another group of monkeys, hemorrhaged to a similar degree of shock, showed an increase in P 50 of 2.9 mm Hg within the same time periodY The rats showed a reduction in 2,3 DPG, while no consistent or statistically significant changes were found in 2,3 DPG in either group of monkeys. There was no apparent relationship between acid-base state and 2,3 DPG levels in these studies; e.g., in the rats with ischemic injury the maximum fall in 2,3 DPG occurred during ishcemia before a change in acid-base state, while in the monkeys subjected to endotoxin

OXYHEMOGLOBIN DISSOCIATION CURVE IN ACUTE DISEASE

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shock, the metabolic acidosis was only partially compensated and, despite the resultant acid pH, there was no significant change in 2,3 DPG levels. These studies did not report on the values of the red cell hemoglobin concentration, nor were the red cell/plasma pH gradients measured, so it is uncertain that P so values were reduced to standard conditions in terms of red cell pH. It is therefore difficult to draw any definite conclusions from these investigations. Serial studies in acutely ill patients over much longer time periods, from days to weeks, indicate that the position of the dissociation curve varies and the data also reflect, in some instances, similar inconsistencies in 2,3 DPG levels and P so values are not corrected to the standard red cell pH 7.2 but to serum pH 7.4 and there are no studies on the relationship to MCHC. The range in P so values is from 18.2 to 37 mm Hg, though very few are found below 20 mm Hg. The position of the curve in acute disease is undoubtedly influenced by not only the red cell environment, but also the efficiency of other links in the O 2 transport chain, the time course in the development of the pathologic process, and therapeutic interventions. Thus it becomes a remarkably complex situation to determine the significance of each of these factors. Thus, in the early stage of sepsis, the dissociation curve may be normal or to the right of normal in the presence of the hyperdynamic state and is associated with increasing O2 delivery. However, if the septic process continues and the patient becomes more moribund, O 2 consumption falls. There is then an increase in O 2 affinity, i e., the curve shifts to the left74. 84. 120 despite the hyperdynamic state. A different pattern was reported in the severely burned patientS-the oxygen affinity increased during the early phase and decreased during the latter phase of the burn syndrome as well as in the period of refractory insufficiency. Some investigators found a significant relationship between P so and 2,3 DPG values in the septic and burn patients. s• 84 In contrast, other workers have found in these conditions and others that in some acutely-ill patients normal or lowered P so values occurred in the presence of a raised or normal 2,3 DPG respectively.74. 78. 120 The relationship of the oxygen affinity of hemoglobin and the acidbase state of the blood in acute disease is also a matter in which no clear-cut conclusion can be drawn from the present data. It was described earlier how in normal man that when there is a change in pH, e.g., a fall in pH, that in due time the right Bohr shift is equally offset by the left shift produced by the fall in 2,3 DPG so there is no overall change in P so • In contrast, it appears that this is not so in the acutely ill patients but opinions differ with respect to the overall result. There is some work which indicates that in a respiratory or metabolic acidosis the left shift due to the fall in 2,3 DPG is stronger than the right shift induced by the fall in pH - the Bohr effect, and the overall result is a left shift of the curve when it is reduced to standard pH conditions. 78 . 112. 120 In contrast there is another report which indicates the opposite, namely that alkalemic patients fail to increase 2,3 DPG levels and shift P so to compensate for left shift in alkalosis caused by the Bohr effect.38 These discrepancies might well be resolved in the future when the magnitude of the Bohr factor may be more precisely defined under varying condi-

646

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RITA MCCONN

tions and the influence of other factors such as inorganic phosphate and ADP and the duration of the change in acid-base state are also taken into account. The influence of blood oxygenation on 2,3 DPG levels in acute disease appears to be opposite to that found in chronic disease; in other words, it appears that in patients in shock the subjects with the lowest level of venous oxygenation had the lowest levels of 2,3 DPG rather than the highest. 36 • 11o There is definite agreement on two causes of a left shift in the curve in acute disease, namely hypophosphatemia71 . 114. 120 and transfusion of stored blood. 3s • 79 Marked hypophosphatemia may occur in acute disease as a result of hyperalimentation without phosphate supplementation 114 or from oral therapy'with aluminum hydroxide in patients on hemodialysis. 120 Low levels of serum inorganic phosphate are associated with low 2,3 DPG values in the acutely ill. Blood which has been stored at 4°C in an acid medium has red cells which are low in 2,3 DPG; transfusion of these cells results in an increase in oxygen affinity of hemoglobin in the transfusion recipient. In the last 5 years, a great deal of attention has been paid to methods of blood storage and the effect of transfusion "2,3 DPG poor" red cells and it is pertinent to briefly review these data.

Blood Storage and Transfusion The primary purpose of giving whole blood to a patient, rather than plasma or other clear fluids, is to increase the oxygen delivery to the tissues. At the present time, blood drawn routinely for transfusion is stored either in acid citrate dextrose (ACD) or citrate phosphate dextrose (CPD) anticoagulant and stored at 4°C. The pH of freshly drawn blood in ACD is 7.0 and this falls progressively during storage to about 6.6 after 21 days. Blood drawn into CPD has a slightly higher pH of 7.2, due to the lower citric acid content of CPD compared with ACD; however, the pH also falls progressively during storage in CPD to reach about 6.8 after 28 days, which is the shelf-life of this type of blood. It has been known for many years that storage of red cells in an acid medium results in loss of 2,3 DPG99 and the Benesches' findings on the effect of this compound on the dissociation curve initiated a large number of investigations in the field of red cell preservation. It can be seen from Figure 9A that there is a progressive fall in P so and 2,3 DPG in blood during liquid preservation at 4°C. There is, however, a greater fall in P so and 2,3 DPG in ACD compared with CPD due to the lower pH of the ACD anticoagulant. 39 . 42 Thus, transfusing a patient with stored ACD or CPD blood can result in a lowering of the patient's 2,3 DPG concentration and a shift of the dissociation curve to the left, the magnitude of these changes depending upon the age and the amount of the blood transfused. It is not unusual for patients subjected to sudden hemorrhage, who were previously healthy, to have an alkalotic pH in the early phase of shock. Blood transfusion is often accompanied by administration of NaHC0 3 to compensate for the acidosis of the stored blood, in this instance, the left shift in the curve, due to the transfusion of 2,3 DPG poor cells, is further compounded by the left shift of the pre-existing alkalosis and heightened by the NaHC0 3 infusion. 3s

28~N 26-

0

Pso 22 mmH<;I

18~

s

__________

-----------C

,
o -c';7-------!\~.------;2!1

0,

DAYS STORED AT 4'e

A

DAYS OF STORAGE AT 4°(

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31

*

Pso mmHg

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12~

M/MHb 08, 0,4

-"u'p.----O'----:O----O'-... _--O- ___ -
18

ATP !J.M/M Hb

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1.0

62 DAYS STORED AT 4·e

Figure 9. The effect of various modifications in liquid blood storage on P 50 , 2,3 DPG, and ATP levels. A, Acid citrate dextrose (ACD) blood compared with citrate phosphate dextrose (CPD) blood stored at 4° C (n = 3). B, ACD blood stored at 4° C under a flowing stream of oxygen (0), a flowing stream of nitrogen (N), or with prestorage addition of methyl prednisolone sodium succinate 2.5 mg per ml (Solu-Medrol, Upjohn) (5), compared to control ACD blood (C). (n = 7). C, ACD blood stored at 4° C with and without prestorage addition of adenine (ImM) and/or inosine (10 mM). (n = 6). D, CPD-adenine (0.5 mM) blood stored at 4° C with and without prestorage addition of ascorbate (5.9 mM) and dihydroxyacetone (20 mM). (n = 3).

In patients the time taken for normal 2,3 DPG and P 50 values to return to normal after transfusion depends upon the "milieu interieur" of the recipient. Studies in volunteers receiving transfusion of stored blood for anemia showed that their 2,3 DPG levels had been restored to 50 per cent or normal within 24 hours, and by the 11 th day after transfusion, the level of 2,3 DPG was approximately that of the recipient cells prior to transfusion. ll7 Serial studies showed that in a group of young trauma patients receiving massive transfusion of stored ACD blood, that the mean P 50 and 2,3 DPG levels were 19 mm Hg and 0.2 M/MHb respectivelyafter transfusion and it took an average of 4 days for the values to return to those of normal control subjects. 79 Currently, many investigators are working on improving methods of red blood cell preservation to prevent changes in the metabolic integrity

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RITA MCCONN

Figure 10. Distribution of frozen blood centers in the U.S.A. (courtesy of W. Miles). (Reproduced from McConn, R.: O2 transport in the elderly and high risk surgical patient. In: Siegel, J. H., and Chodoff, P. (eds.): Surgery in the Elderly and High Risk Patient. New York, Grune and Stratton, in press.)

and oxygen affinity of hemoglobin during storage at 4°C. While the increased use of CPD as an anticoagulant represents some improvement over ACD, it is still far from ideal. Techniques are available for cryopreservation of red cells using either a "low glycerol rapid freeze"lo7 or "high glycerol slow freeze method."60 Erythrocytes can be stored frozen for more than 1 0 years and still retain their viability on thawing and reconstitution. Freezing the red cell maintains the 2,3 DPG level and P 50 values at their prefreeze level; thus from the viewpoint of maintaining the oxygen affinity of hemoglobin, the erythrocytes which are collected in ACD should be frozen as quickly as possible after withdrawal. 79 It is appreciated, however, that the number of frozen blood centers in the United States is limited and that they tend to be concentrated mainly on the east coast with some in the West and South and very few in the Central states H1 (Fig. 10). There is at present considerable controversy whether there should be widespread expansion of frozen storage facilities and processing equipment. However, the majority of arguments in favor of cryopreservation, apart from maintenance of 2,3 DPG levels, have nothing to do with the quality of the red cells but rather depend on incidental properties of the processed red cell suspen'sion such as promotion of component therapy, reduction in hepatitis, etc. 35 Accepting the fact that for many, liquid preserved red cells will be the only method of preservation for sometime to come, investigators continue to explore modifications in order to maintain P 50 and 2,3 DPG levels. Various approaches have been studied such as the prestorage addition of various compounds to ACD or CPD blood such as adenine 1 , 62, 79 and/or inosine,32.79 the buffer THAM (tri-hydroxy amino methane),62 dihydroxy acetone,26. 76 or storage of blood in an atmosphere of O 2 or N2.77 The rationale being based on, either providing an alternate substrate for

OXYHEMOGLOBIN DISSOCIATION CURVE IN ACUTE DISEASE

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2,3 DPG which enters glycolysis at a point which is unaffected by an acid pH or by raising the pH during storage and thereby preventing loss of 2,3 DPG (Fig. 3). While these methods have been successful at maintaining 2,3 DPG and P 50 value"s during storage above, at or near normal levels, this improvement has always occurred at a metabolic cost, loss of ATP, and/or increase in the flux or cellular Na+ and K+ (see Fig. 9).42,76, 77,79 Another approach has been to restore the stored red cells to normal by incubation at 37°C with inosine, pyruvate, and phosphate and then to freeze the rejuvenated cells. 43 , 118 Valeri has recently demonstrated successful transfusion of such rejuvenated frozen cells. While this restoration is of academic interest, its application on a nationwide scale is doubtful. One practical solution to the surgeon contemplating elective surgery is autologous transfusion 7 ,81 or, in emergency surgery, intraoperative autotransfusion. However, these are circumstances where this solution is not possible-there may be no access to fresh or frozen blood and the physician has to transfuse stored ACD or CPD blood into his patient. The degree of stress on the oxygen transport system from the left shift in the curve resulting after transfusion is difficult to evaluate. This point raises the whole question of what is the physiologic significance of the changes in the oxygen affinity of hemoglobin in acute disease and what is the influence of transfusion of stored blood.

THE PHYSIOLOGIC SIGNIFICANCE OF A SHIFT IN THE DISSOCIATION CURVE IN ACUTE DISEASE It is sad but true to state that despite the upsurge in investigation in O 2 transport and its relation to tissue needs, very little progress has been made in terms of practical significance to the clinician. Laver 68 recently likened the search for clues to solve this problem vis-a-vis the relationship between blood flow and the O 2 affinity of hemoglobin and O2 requirements to "Percival's quest for the Holy Grail, like an Arthurian legend each has set forth. .. on a search for the Grail but has forgotten God and spends years on fruitless adventures." The problem of the evaluation of the adequacy of O 2 transport for the whole body in acute disease is not resolved, yet alone the more complex problem of specific O 2 delivery to vital organs in relation to the organs' requirements. Investigations aimed at determining the significance of a shift in the dissociation curve have been based on studies in normal subjects and in those in which there is already some degree of inefficiency in one or more links of the O 2 transport chain. Two types of experimental design have been used; (1) The production of a left-shifted curve by replacement transfusion with blood low in 2,3 DPG. Inhalation of CO has also been used to produce a shift in the dissociation curve to the left although this method also alters the oxygen-carrying capacity as well as P 50• (2) The production of increased 2,3 DPG levels by replacement transfusion of "2,3 DPG-rich" red cells, with a raised P 50 level or attempting to induce an increase in P 50 by increasing 2,3 DPG levels in

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RITA MCCONN

vivo by various means. Significance of the change in P 50 has been determined by a variety of indices, ranging from changes in the hemodynamic system, maximum O 2 uptake, exercise tolerance, tissue Po 2, and the ultimate index, mortality. Replacement transfusion with 2,3 DPG depleted erythrocytes in healthy animals was found to be associated with a fall in mixed venous oxygen tension,lOl no reduction in maximal O 2 uptake,124 and no change in exercise tolerance compared to controls.123 Exposure to the stress of breathing low oxygen mixtures did not cause any fall in cerebral tissue P0 2 as measured by tissue O 2 electrodes. 10o However if the animals were exchange transfused,61 or hemorrhaged and exchange transfused, to hemoglobin levels low enough to be classified as anemia,38 then there was a significant increase in mortality rate in those receiving blood low in 2,3 DPG compared to those given blood with normal or raised 2,3 DPG levels. The measurements of maximum oxygen uptake have been used as an index of the ability of 'the O 2 transport system to provide increased amounts of O 2 to exercising muscles. In man the lowering of the arterial oxygen content by 10 per cent by either inspiration of low O 2 mixture or saturation of 10 per cent Hb by CO caused a similar fall in maximum O 2 uptake compared to the control. Thus, the left shift in the dissociation curve of 6 mm Hg caused by breathing CO appeared to exert no additional detrimental effect in healthy man. 96 • 119 Patients with a similar degree of anemia but with dissociation curves shifted in the opposite direction were studied at rest and during exercise. It was found that those with a left-shifted curve had a lower exercise tolerance, a higher cardiac output, and a lower mixed venous PO z compared to those with a right-shifted curve. In angina pectoris and intermittent claudication, increases in blood flow to the affected muscles during exercise are limited, and this is clearly indicated by the onset of pain. It was found that CO saturation of 3 per cent (a value which is frequently found in cigarette smokers) in these patients subjected to light exercise reduced the time of onset of pain by 20 per cent. 3. 4 However, it is difficult to differentiate to the comparative significance of the reduction in O 2 capacity versus the left shift of the curve caused by carbon monoxide. An overall view of these data would lead to the suggestion that a shift of the dissociation curve to the left which occurs in a patient who has no embarrassment of any other link in the Oz transport chain is of little or no significance. In other words the system is resilient enough to compensate if there is a reduction in efficiency in Oz deli-very by the red cell, not only by an increase in O 2 affinity, but even when there is a reduction in red cell mass. 89 Compensation may be seen as an increase in cardiac output and/or lowering of mixed venous O 2, However, in the acute situation when a left-shifted curve occurs together with another deficiency in the O 2 transport system, such as low hemoglobin from hemorrhage or the presence of a defect in the hemodynamic system, e.g., the hypo- and hyperdynamic state of sepsis, or reduced coronary blood flow as a result of chronic or acute disease, then a left-shifted curve may indeed become very significant. The only drawback is that

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there is no magic index which will indicate to the clinician when his patient is in this type of situation. Furthermore, if he finds a left-shifted curve, what therapeutic regimen should he institute? Practical Approach to Monitoring O 2 Transport The only practical solution is for continual monitoring and evaluation of the efficiency of all links in' the O 2 transport chain. To the physician practicing without the sophisticated resources provided by an academic umbrella, it comes down to a question of having blood gas equipment, an O2 saturation meter, a colourimeter, and an instrument for measuring O 2 content, such as Van Slyke manometric equipment or, much more simple to operate, the Lex-0 2 -Con (Lexington Instruments), which is basically an electric cell. The type of data which can be obtained from this equipment is summarized in Table 2. If there is access to a small digital computer, it becomes a very easy matter to calculate the various hemodynamic indices. A note of caution at this point to the clinician to draw the minimum quantity of blood required for any test and to meticulously record the volume of all blood drawn on patients throughout their hospitalization. In view of the increase in the amount of physiologic and biochemical monitoring of patients, particularly the acutely ill, over-enthusiastic monitoring, especially when cardiac output is measured by the dye dilution method, may precipitate the patient into anemia which the clinician then resolves by transfusion of stored blood! From the viewpoint of therapy with respect to the dissociation curve, it would seem advisable to maintain the patient in a normal acidbase state, and avoid "over-alka1inisation" of a patient should the necessity arise to treat a metabolic acidosis with infusion of NaHCO a• Supplementation of the diet with phosphate should be done if low serum levels of inorganic phosphate are found. While it is important for the physician to be aware of the advantages of having fresh blood for transfusion for the acutely ill patient, it must be appreciated that it is not always possible. It is obvious that the situation produced by transfusion of stored blood may be far more critical in some patients than others; e.g., the heart should be the organ most vulnerable to an acute fall in P 50 because its mixed venous P02 is already close to the minimal Table 2. Measurements Which Aid Evaluation of O 2 Transport in the Acutely III FrO" respiratory rate, minute volume, type of ventilation Temperature, blood pressure, heart rate, CVP Arterial pH, Pco" Po" 0, saturation, 0, content, Hb conc; base excess' Mixed venous pH, Pco" Po" 0, saturation, 0, content Hb conc- base excess" Arterio-mixed venous ,gradients, e.g., Ca-vo, Alveolar-arterial 0, gradient', intrapulmonary shunting Q,/Qt (when FrO, = 1.0),' pHc (red cell pH)" P,o", inorganic phosphate Cardiac index from Fick prinCiple or dye dilution method Hemodynamic parameters: e.g. stroke volume, stroke index, mean ejection rate, stroke work, total peripheral resistance 0, consumption (C.O. x a-vO, x 10) 'Calculated values.

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functionalleveps Thus, if the ability of the coronary vessels to dilate is impaired by disease, theoretically a fall in P 5 () value in these patients could be critical. However, this has yet to be proved experimentally. Until that time when such critical groups of patients can be clearly defined, the choice of blood for transfusion depends upon the clinician's careful evaluation of his patient and the facilities available to him. The importance of a close meaningful relationship with the blood bank staff cannot be overemphasized.

THERAPEUTIC MANIPULATION OF THE OXYHEMOGLOBIN DISSOCIATION CURVE Bioc4emical manipulation of the oxyhemoglobin curve is an attractive, therapeutic modality, i.e., inducing a shift of the dissociation curve to the right to increase O 2 delivery when there is failure in the pulmonary or hem9dynamic link of the transport chain. The finding that the addition of inosine, pyruvate, and phosphate restored 2,3 DPG levels to supranormal values in stored blood 92 suggested that intravenous administration of these compounds could produce in vivo an increase in 2,3 DPG and P 50 .82 This was first verified in primates ll1 and then tried in man using allopurinol, a potent xanthine oxidase inhibitor to prevent hyperuricemia. Using inosine pyruvate and phosphate, IPP (O.lM, O.lM, 0.05M) in the range of 5 to 20 ml per kg, the peak effect in 2,3 DPG in any dosage usually occurred 3 to 6 hours after the infusion and lasted 8 to 72 hours and did not appear to be dose related. The average rise in 2,3 DPG was 1600 mmoles per ml of red cells and the usual P 50 increase 3 to 5 mm Hg. Recently a report was published in which 10 hypoxic patients (P a o 2 > 65 mm Hg despite mechanical ventilation and F]o2 > 0.6) were randomly treated with either intravenous saline or intravenous inosine, pyruvate, and phosphate (O.lM, O.lM, 0.25M solution given 15 ml per kg over 90 minutes) with 300 mg alopurinol and 1 gm calcium gluconate. IPP proved to be an easy nontoxic method of rapidly increasing 2,3 DPG but there was no statistical difference in cardiac output, arterial Po 2, A-V oxygen extraction and oxygen consumption between those receiving IPP and those given saline. 97 In other words, there is no evidence of improved tissue oxygenation in those with increased 2,3 DPG levels. Furthermore, there is some work which shows that glycolysis is profoundly impaired in 2,3 DPG rich red cells and also there is a remarkable inertness in 2,3 DPG concentration once it has increased. 44 ,64 Thus, there is at the moment little support for the usefulness of increased 2,3 DPG levels as a therapeutic modality to increase oxygen delivery in the acutely ill. The steroid methyl prednisolone sodium succinate (Solu-Medrol, Upjohn) which is commonly used in supraphysiologic doses in the treatment of shock (12 to 35 mg per kg) has been found to increase P 50 in the acutely ill patient'1O,74 and after massive transfusionY Increase in P oo ranged from 3 to 8 mm Hg and was not always accompanied by an increase in 2,3 DPG levels. Furthermore, this increase in P 50 was shown to be associated with an improvement in O2 consumption, and a fall in

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mixed venous oxygen saturation, while the cardiac index only showed a transient increase.:lI Thus the steroid administration appeared to increase the efficiency of oxygen transport through improving perfusion and increasing availability of O 2 through its effect on the oxygen affinity of hemoglobin. The steroid propranolol has also been reported to increase P 50 in blood 94 and it has been shown that this compound releases 2,3 DPG bound to the red cell membrane. 91 This may well be the action through which methyl prednisolone sodium succinate increases P 50 in the acutely ill.

SUMMARY Studies in molecular chemistry have provided a rational basis to explain how various physicochemical factors influence the oxygen affinity of hemoglobin. Enclosure of the protein within the red cell introduces a more complex situation since these various factors are interrelated and also can influence each other's relationship with hemoglobin. The oxygen affinity of hemoglobin can change in chronic and acute disease. The concentration of the organic phosphate 2,3 DPG appears to play a significant role in influencing the position of the oxyhemoglobin dissociation curve in chronic disease and in recipients of large volumes of transfused stored blood, 2,3 DPG has been named the metabolic regulator of oxygen transport but the precise relationship between the various factors which appear to trigger the metabolic response to hypoxia, i.e., an increase in 2,3 DPG are not yet known. The position of the dissociation curve varies in an acutely ill patient and cannot be predicted on the basis of red cell 2,3 DPG levels, pH, or acid-base state. Knowledge is incomplete at present on what determines the oxygen affinity of hemoglobin in acute disease and on the physiologic significance of a shift in the curve. It appears at present that a left shift in the curve is only significant when there is failure in another link in the O 2 transport chain. Maintenance of a patient in normal acid-base state and preventing hypophosphatemia should help prevent undesirable shifts in the curve. Therapeutically increasing 2,3 DPG levels in the acutely ill with pulmonary and/or hemodynamic failure does not appear to have any favorable effects on the dissociation curve or oxygen delivery. In contrast, administration of methyl prednisolone sodium succinate in these patients was associated with a right shift in the curve and increased tissue perfusion.

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