MATERIALS AND METHODS

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1 Oxygen and Carbon Monoxide Equilibria of Human Adult Hemoglobin at Atmospheric and Elevated Pressure By F. LEE RODKEY, JOHN D. O NEAL, AND HAROLD A. COLLISON T HE FUNDAMENTAL LAW describing the competitive combination of oxygen and carbon monoxide with hemoglobin was defined by Douglas, Haldane, and Haldane in At equilibrium the concentration of these two gases 1)ound to hemoglobin and in the gaseous phase were related by the equation : ( COHb) X ( 02)/( O2Hb) X ( CO ) K where K is the relative affinity constant of hemoglobin for CO and oxygen. The value of K was the same for hemoglobin in solution or in the red cells and was not altered by the presence of CO2, minor changes in ph, salt concentration, or dilution of the hemoglobin solution. Small variations in K were observed within a given species with larger changes between species and at different temperatures. Sendroy and his co-workers2 3 have reported the value of K = 210 for human hemoglobin at 38#{176}and atmospheric pressure. Conflicting reports4 5 have described changes in K as a function of plasma ph in human blood both in vivo and in vitro. Lilienthal, et al.,#{176}demonstrated that decreased barometric pressure did not alter the equilibrium distribution of CO and 02 in man. When blood was diluted 1:20 with water and equilibrated with per cent CO in air at total pressures from 1 to 4 atmospheres, tile same percentage of COHb was observed at all pressures of equilibration.7 No other data are available on the effects of elevated pressure on the relative affinity constant. Our interest in the effect of pressure and various inert gases on the value of K arose from the peculiar problems of maintaining men under the sea for prolonged periods. In this situation tile men are provided with a respiratory gas containing less oxygen and nitrogen than is present in air in order to avoid the development of oxygen toxicity and nitrogen narcosis. Helium is often used as the major inert gas at higher pressures. The hazards of contamination of the inspired air with CO from exogenous sources or from metabolism of the divers can only be predicted if the value of K is known in such gas mixtures or is not changed by the nature of the inert gas used. Blood and Ilenioglobin Solutions MATERIALS AND METHODS Measurements on whole blood were performed with heparinizeci blood from a(lults. hemoglobin solutions were prepared from heparinized blood and from out-dated ACD From Laboratory of Analytical Biochemistry, U. S. Naval Medical Research Institute, National Naval Medical Center, Bethesda, Md. Supported by the Bureau of Medicine and Surgery, Navy Department Research Task MROO5.04-O103, M The opinions or assertion.r contained herein are those of the authors and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large. First submitted June 28, 1968; accepted for publication September 9, BLOOD, VOL. 33, No. 1 (JANUARY),

2 58 RODKEY, O NEAL, COLLISON blood. In each case the red cells were repeatedly washed with 0.9 per cent NaC1 solution to remove plasma constituents. Packed red cells were hemolyzed by addition of 0.1 volume of 10 per cent Sterox in water for each volume of cells. The hemolystate was centrifuged to remove cell stroma and decanted through several thicknesses of gauze. A clear concentrated hemoglobin solution was obtained which was then diluted to the desired concentration by addition of 0.1 M phosphate or borate buffer. Changes in ph of the highly buffered hemoglobin solutions were achieved by addition of NaOH or HC1 to the buffer solution before it was added to the concentrated hemoglobin solution. There was no detectable difference in either the fraction of active hemoglobin present or the equilibrium values observed with such hemoglobin solutions prepared from fresh blood and from outdated ACD blood. Equilibration Two equilibration systems were used. At atmospheric pressure the sample was equilibrated in an open system of the type described by Laue.8 A humidified gas phase of known CO and concentration was continuously passed over the sample until the amounts of CO and 02 bound to hemoglobin remained constant for one hour. Three to 6 hours were often required to reach equilibrium in this system when the original sample was far removed from the equilibrium point. Equilibrations at elevated pressure were performed in a stainless steel cylinder of about 500 ml. capacity. Blood or hemoglobin solution (5 ml. ) was added to the cylinder which had been repeatedly flushed with the desired mixture of CO in air. The cylinder containing the liquid sample was then pressurized with the mixture of CO in air to the desired pressure, submerged in a water bath, and rotated in the horizontal position for 2 hours. Measurements of CO concentration in the gas phase within the steel cylinder showed no detectable change after 1 hour of equilibration with this closed system even when the original sample contained 100 per cent COHb or O9Hb. At the end of the equilibration period the cylinder was removed from the water bath and placed in a vertical position to permit drainage of the liquid phase into an exit port provided at the bottom. Samples of the gas phase were removed from the upper end of the cylinder for CO analysis. The liquid phase was displaced by a small residual gas pressure within the cylinder into a lightly oiled glass syringe attached to the bottom exit port. Approximately 3 to 5 minutes were allowed for liberation of dissolved gases from the liquid before gasometric analysis of bound CO and 02. Analytical Procedures Carbon monoxide and oxygen contents of the equilibrated liquid samples were determined by a modification of the method of Sendroy and Liu.9 Both gases were determined in the same 2 ml. sample. No correction for dissolved CO was required due to the low concentrations (less than 0.2 per cent) employed. Oxygen content was converted to O2Hb content by subtracting 0.28 mm/l. This correction corresponds to the dissolved oxygen of blood 0 in equilibrium with one atmosphere of air at 23#{176}, the temperature attained by the samples before analysis. Total hemoglobin was determined as cyanmethemoglobin at 540 mu by use of the extinction coefficient given by Drabkin and Austin.1 Complete conversion of COHb to cyanmethemoglobin was assured by a three hour reaction time with Drabkin s reagent.12 Total hemoglobin, COHb, and O2Hb are expressed in mm/l, one mm being taken as the amount of hemoglobin (16.7 Cm.) which can combine with one mm of oxygen or CO. Measurements of ph were performed with a glass electrode (Instrumentation Laboratory) at the temperature of sample equilibration. Compressed mixtures of CO in air used for equilibrations were analyzed for oxygen with the Henderson 13 modification of the Haldane apparatus. Carbon monoxide of the original gas mixtures and of the equilibrium gas phase was determined by gas chromatography. The CO was separated from other gases on 5A molecular sieve, catalytically reduced to methane, and detected by hydrogen flame ioinzation as described by Porter and Volman. 4 Although the reproducibility of the chromatographic procedure was about ± 1 per cent,

3 OXYGEN AND CARBON MONOXIDE EQUILIBRIA 59 Table 1.-Relative Affinity Constant of Adult Human Hemoglobin Determined at 37#{176} and Atmospheric Pressure Initial Solution Hb Total COHb pj mm/l. Equilibration time hr. COHb ph + O,Hb mm/l. Equilibrium o,m values Po2 #{149}-. K #{176} Avg Equilibration gas contained 4.21% CO2. the accuracy of the analysis was approximately 2 per cent of the CO concentration in the gas phase. Mea,siirements at Atmospheric Pressure RESULTS AND DISCUSSION A series of equilibrations of COHb-O2Hb mixtures was performed at atmospheric pressure and 37#{176}. The initial ratio of COHb/O2Hb was adjusted to be significantly above or below the equilibration value. A wide range of ph was used and equilibration was continued until the analyzed ratio of COHb/O2Hb remained constant for one hour. The data given in Table 1 indicate that no difference in K was observed from ph 6.8 to 8.8 and the presence of CO2 in the gas phase had no detectable effect. The average value of K=207±5 is in agreement with that reported by Sendroy.23 These data, obtained over physiologically extreme ph values, are in opposition to the large changes reported by Allen and Root4 and the smaller variations observed by Joels and Pugh5 for human blood at different CO2 tensions. Equilibrations performed with the open system required an unacceptable period of 6 hours or more when the ratio of COHb/O2Hb in the original solution deviated widely from the equilibrium value. Comparison of the total hemoglobin measurements on the original solutions with the sum of COHb plus O2Hb at equilibrium precludes appreciable methemoglobin formation in these experiments. To avoid possible methemoglobin formation, water loss, action of light, and other nonspecific uncertainties in prolonged equilibration, the closed system was devised in which measurements could be performed at elevated pressure. Solutions of hemoglobin completely saturated with oxygen or carbon monoxide were used to determine the time required to establish equilibrium in this system. Samples (5 ml.) of COHb or O2Hb (about 9mM/L. total hemoglobin) were placed in the stainless steel cylinders and pressurized to approximately 3 atmospheres absolute with a gas con-

4 60 RODKEY, O NEAL, COLLISON taming per cent CO in air. The cylinders were submerged in a water bath and rotated in the horizontal position. Samples of the gas phase were removed at intervals for CO analysis. At room temperature the exchange of CO between solution and gas phases was about 85 per cent complete in 30 minutes and 98 per cent complete in 1 hour. Equilibration at 37#{176}was more rapid. The gas exchange was 96 per cent complete in 30 minutes and above 99 per cent after 1 hour at this temperature. In every case the CO concentration obtained after 90 minutes or more of equilibration remained constant within the error of the analytical procedure ( about 2 per cent of the actual value). This was true whether the original liquid contained 100 per cent COHb or O2Hb or a mixture of the two forms. To insure equilibrium we have used a 2 hour equilibration time for all closed system experiments and we have maintained the partial pressures of oxygen and CO sufficiently high to saturate all active hemoglobin present. For all subsequent experiments the COHb and OHb of the equilibrated liquid samples were experimentally measured in addition to the CO concentration in the gas phase. The oxygen concentration of the equilibrium gas phase was taken to be the analyzed concentration in the original gas. This assumption is most valid at higher pressures. However, even at the lowest pressures used, about 3 atmospheres total pressure of 0.1 per cent CO in air, the relation of tonometer volume of total hemoglobin present was such that 100 per cent change in bound oxygen ( O2Hb ) could produce a change of less than 0.3 per cent in the total oxygen present in the gas phase. Conversion of about half of the total hemoglobin, as normally occurred when either O2Hb or COHb was present in the original solution, could change, respectively, the original oxygen percentage (20.92) to or The accuracy of the remaining analytical procedures does not justify the application of any correction for such a smail change in the gas oxygen concentration. Effect of temperature Portions of O2Hb and COHb solutions were equilibrated at both 22#{176} and 37#{176}inthe closed system. These experiments allow comparison of the closed system technic with the open system equilibrations at atmospheric pressure as well as estimation of the effect of temperature and ph on the relative affinity constant. The data obtained are summarized in Table 2. Total pressure recorded in Table 2 is that to which the cylinders were initially pressurized and the ph values were determined after equilibration at the temperature of equilibration. The same equilibrium state was attained at a given temperature irrespective of ph or the form of active hemoglobin in the original solution. Values of K determined at 37#{176} were the same as those observed in the open system at atmospheric pressure. The temperature of equilibration has a significant effect on the equilibrium distribution of oxygen and CO. Table 2 indicates an increase in the value of K of 4.6 units for a decrease in temperature of 1 C. These data are compatible with the data of Hartridge who showed a decrease of COHb saturation of 0.5 per cent for each degree rise in temperature.

5 OXYGEN AND CARBON MONOXIDE EQUILIBRIA 61 Table 2.-Relative Affinity Constant of Adult Human Hemoglobin at 22#{176} and 37#{176} C. Temp C Initial Tonometer pressure Atm. Abs. solution Equilibrium values Hb Hb ph COHb COHb total fonu + mm/l. Hb 2 mm/l. Po, -i- ICO K O2Hb COHb O2Hb COHb O2Hb COHb O2Hb COHb O2Hb COHb Avg. 210 Avg (Atm. Table 3.- Relative Affinity Constant of Human Hemoglobin a t 37#{176}with Different Total Pressures of 0.1% CO in Air Total Pressure Abs.) Avg a Samples equilibrated with a mixture of 0.1 per cent CO in air ( 3 atm. ) plus helium ( 18.4 Atm.) Effect of Elevated Pressure of Inert Gas The relative affinity constant was measured at 37#{176} in a series of hemoglobin solutions prepared from ACD blood. A mixture of 0.1 per cent CO in air was used at total pressures ranging from 2.7 to 21.4 atmospheres. As shown in Table 3, the value of K and the standard deviation for 25 such determinations, K=210±5, were the same as those obtained at atmospheric pressure. No alteration in K was observed when solutions of hemoglobin were equilibrated with the air-co mixture at 3 atmospheres followed by addition of helium to 21.4 atm. These data support the view that the equilibrium distribution of CO and oxygen bound to hemoglobin is determined only by the ratio of oxygen and CO in the gas phase and is independent of the total pressure or the nature of the inert gases present. Variation in Normal Individual9 The extent of variation in K among normal persons was estimated by measurements made on 15 individual samples of whole blood from 13 men

6 62 RODKEY, O NEAL, COLLISON Table 4.-Relative Affinity Constant Determined at 37#{176} on Whole Blood and Hemoglobin Solutions from Normal Adults Initial Solution Subject Sample Hb Total mm/l. Equilibrium values ph COHb + O,Hb mm/l. COHb OHb Pu. K 1 2 blood Hb.Sol. blood Hb. Sol blood Hb. Sol blood Hb. Sol blood Hb. Sol blood Hb. Sol : blood Hb. Sol blood Hb.Sol. blood Hb. Sol blood Hb. Sol blood Hb. Hb. Sol. Sol blood0 blood #{176} Avg Female subjects. and 2 women. Analyses were repeated on 10 of the samples by use of a hemoglobin solution prepared from the same blood. The data presented in Table 4 for the entire series show a mean and standard deviation of K = 218±8. The average value of K determined on whole blood is identical to that derived from hemoglobin solutions. Only one measurement deviates by more than 5 per cent from the mean value. Since the experimental error for estimating K is approximately ±3 per cent for a given sample, these data indicate essentially no individual variation in K between normal adults. The value of K at 37#{176} reported here, 218±8, is only 4 per cent higher than that observed by Sendroy et al.,2 at 38#{176}. Correction of their value to 37#{176} by means of the temperature variation in K reported here gives a value of 215, well within the experimental error of the procedures. The value of K = 210 obtained for hemoglobin solutions prepared from a limited number of ACD blood samples is not significantly different from that obtained with fresh blood.

7 OXYGEN AND CARBON MONOXIDE EQUILIBRIA lpco og- Fig. 1.-Relation of bound COHb/O2Hb to the equilibrium P0/PO2 of the gas phase. Experimental points ( #{149} ) are compared with the solid line representing a relative affinity constant of 218. Measurements at Different Ratk s of COHb/O2Hb The above estimations of K have all been made with whole blood or concentrated hemoglobin solutions in order to provide the greatest accuracy for the gasometric measurements of COHb and O2Hb. Values of K at widely different ratios of oxygen to CO in the gas phase have also been determined. For these experiments the COHb content of the liquid phase was measured by gas chromatography.16 The O2Hb content was taken as the difference between the active hemoglobin content and the COHb content. Equilibralions were performed in air to which varying amounts of CO were added. The range of COHb saturation was from 5 to 90 per cent. Results of these measurements are plotted in Figure 1 as the log COHb/O2Hb versus log P0/ P02 to conveniently cover the nearly three hundred fold range of ratios determined. Experimental points obtained are in close agreement with the solid line for the theoretical value of K = 218. The deviation observed with the very low COHb/O2Hb ratio is probably not outside the experimental errors for measurement of the CO content of the gas and liquid phases at these very low levels. The data suggest that at least over the range of 5 to 90 per cent COHb saturation the value of K remains constant. This would imply that at least over this range the oxygen and carbon monoxide dis-

8 64 RODKEY, O XEAL, COLLISON sociation curves of hemoglobin are identical when the scale for Pco is 1/218 that for P02. These in vitro data support the study of Caristen et al.17 who demonstrated experimentally the relationship between alveolar P0 and blood COHb saturation in vivo. AntoninP8 19 has stated that any modification of the hemoglobin molecule which produces a change in the equilibrium curve for oxygen will produce a parallel change in that for CO with no change in the relative affinity constant. The present data support this view over the ph range from 6.8 to 8.8. No change in K was observed whether the ph was altered with buffer solutions or by addition of CO2 to the gas phase. The discrepancy between these data on whole blood and hemoglobin solutions in vitro cannot at present be reconcued with the experiments of Allen and Root4 who reported more than 20 per cent change in K between ph 7.1 and 7.6. Measurements at different temperatures indicate a significant increase in K at lower temperature, but the value observed at any specific temperature is independent of ph. This would suggest that possibly the conformational changes of the hemoglobin molecule on binding ligands have different temperature dependence for various ligands. SUMMARY Adult human hemoglobin has been shown to have an affinity for carbon monoxide 218 times that for oxygen at 37#{176}. There is no change in the relative affinity between ph 6.8 and 8.8. The same value was obtained with whole blood and with concentrated undialyzed hemoglobin solutions. \ leasurements made at atmospheric pressure were identical with those at increased total pressure up to 21.4 atmospheres absolute. Substitution of helium for nitrogen as inert gas did not alter the value. The relative affinity constant K is increased by 2 per cent by a decrease in temperature of 1 2 C. SUMMARIO IN INTERLINGUA A 37 C, hemoglobina human de typo adulte possede, il esseva trovate, tin affinitate pro monoxydo de carbon que es 218 vices plus forte que su affinitate pro oxygeslo. Ii occurre nulle alteration del affinitate relative inter ph 6,8 e ph 8,8. Le valores obtenite con sanguine total e con concentrate non dialysate solutiones de hemoglobina esseva identic. Le mesurationes effectuate sub pression atmospheric esseva identic con illos effectuate sub augmentate pressiones total usque ad 21, 4 atmospheras absolute. Le substitution (IC helium pro nitrogeno como gas inerte non alterava le resultato. Le constante de afflnitate relative es augmentate per 2 pro cento per un declino in le temperatura per 1 C. REFERENCES 1. Douglas, C. C., Haldane, J. S., and ide and oxygen in blood. Fed. Proc. 14:137, Haldane, J. B. S.: The laws of combination of hemoglobin with carbon monoxide and 4. Allen, T. A. and Root, W. S.: Partition oxygen. J. Physiol. 44:275, of carbon monoxide and oxygen between 2. Sendroy, J., Jr., Liu, S. Fl., and Van air and whole blood of rats, dogs, and men Slyke, D. D.: The gasometric estimation of as affected by plasma pfi. J. AppI. Physiol. the relative affinity constant for carbon 10:186, monoxide and oxygen in whole blood at 38#{176}. 5. Joels, N. and Pugh, L. C. C. E.: The Amer. J. Physiol. 90:511, carbon monoxide dissociation curve of hu- 3. Sendroy, J., Jr., and O Neal, J. D.: man blood. J. Physiol. 142:63, Relative affinity constant for carbon monox- 6. Lilienthal, J. L., Jr., Riley, R. L.,

9 OXYGEN AND CARBON MONOXIDE EQUILIBRIA 65 Proemmel, D. D., and Franke, R. E.: The relationship between carbon monoxide, oxygen, and hemoglobin in the blood of man at altitude. Amer. J. Physiol. 145:351, Berger, L. B., Curry, T. F., Watson, H. A., and Pearce, S. J.: Safe use of respiratory protective equipment in work in compressed air: Detection and physiological effects of gases encountered. R. L. Bur. Mines, Report of Investigations 6540, Laue, D.: Em neuses Tonometer zur raschen Aquilibrierung von Blut mit verschiedenen Gasdrucken. Pflugers Arch. Ges. Physiol. 254:142, Sendroy, J., Jr. and Liu, S. H.: Gasometric determination of oxygen and carbon monoxide in blood. J. Biol. Chem. 89:133, Sendroy, J., Jr., Dillon, R. T., and Van Slyke, D. D.: Studies of gas and electrolyte equilibria in blood. XIX. The solubility and physical state of uncombined oxygen in blood. J. Biol. Chem. 105:597, Drabkin, D. L. and Austin, J. H.: Spectrophotometric Studies II. Preparations from washed blood cells: Nitric oxide hemoglobin and sulfhemoglobin. J. Biol. Chem. 112:51, Rodkey, F. L.: Kinetic aspects of cyanmethemoglobin formation from carboxyhemoglobin. Clin. Chem. 13:2, Henderson, Y.: Applications of gas analysis: IV. The Haldane gas analyzer. J. Biol. Chem. 33:31, Porter, K. and Volman, D. H.: Flame ionization detection of carbon monoxide for gas chromatographic analysis. Anal. Chem. 34:748, Hartridge, H.: the action of various conditions on carbon monoxide hemoglobin. J. Physiol. 44:22, Collison, H. A., Rodkey, F. L., and O Neal, J. D.: Determination of carbon monoxide in blood by gas chromotography. Clin. Chem. 14:162, Carlsten, A., Holmgren, A., Linroth, K., Sj#{246}strand, T., and Strom, C.: Relationship between low values of alveolar carbon monoxide concentration and carboxyhemoglobin percentage in human blood. Acta Physiol. Scand. 31:62, Antonini, E.: Hemoglobin and its reaction with ligands. Science 158:1417, Antonini, E.: Interrelationship between structure and function in hemoglobin and myoglobmn. Physiol. Rev. 45:123, 1965.

10 : Oxygen and Carbon Monoxide Equilibria of Human Adult Hemoglobin at Atmospheric and Elevated Pressure F. LEE RODKEY, JOHN D. O'NEAL and HAROLD A. COLLISON Updated information and services can be found at: Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: Information about ordering reprints may be found online at: Information about subscriptions and ASH membership may be found online at: Blood (print ISSN , online ISSN ), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC Copyright 2011 by The American Society of Hematology; all rights reserved.