We determined normal human blood p50 at various pH values (range 7.0 to 7.6) as a function of the proportion of carboxyhemoglobin (COHb) in total hemoglobin, from 0 to 23%. The d(log p50)/d[COHb] coefficient is 0.00848, independent of pH and 2,3-diphosphoglycerate. The derived equation allows the calculation of p50 as a function of COHb with an approximation of +/- 0.54 mmHg (about 72 Pa), and can be combined with other calculations (Clin Chem 27:1856-1861, 1981; Clin Chem 29:110-114, 1983) to predict p50 under any condition of pH within the range 7.0-7.6, ratio of [2,3-diphosphoglycerate] to [total hemoglobin] (range 0.3-2.5), pCO2 (range 20-90 mmHg), temperature (range 19-43 degrees C), and COHb (range 0-23%)

E. Rovida

M. Niggeler

M. Samaja

S. Carlone

AIR Universita degli studi di Milano

0 10 20 30CLIN. CHEM. 30/7, 1250-1251 (1984)1250 CLINICAL CHEMISTRY, Vol. 30, No. 7, 1984Carboxyhemoglobinand Oxygen Affinityof Human BloodErmanna Rovlda,”4 Mlchela Nlggeler,2 Stefano Carlone,3 and Michele Samaja4We determined normal human blood p at various pH values(range 7.0 to 7.6) as a function of the proportion of carboxy-hemoglobin (COHb) in total hemoglobin, from 01023%. Thed(log )/d[COHb] coefficient is 0.00848, independent of pHand 2,3-diphosphoglycerate. The derived equation allows thecalculation of p as a function of COHb with an approxima-tion of ±0.54 mmHg (about 72 Pa), and can be combinedwith othercalculations(C/inChem 27:1856-1861, 1981; C/inChem 29:110-114, 1983) to predict p under any conditionof pH within the range 7.0-7.6, ratio of [2,3-diphosphoglycer-ate] to [total hemoglobin] (range 0.3-2.5), p (range 20-90mmHg), temperature (range 19-4.3 #{176}C),and CbHb(range0-23%).Exact relationships between human blood Pso (the P02 atwhich hemoglobin is half.saturated with oxygen) and themain factorsaffectinghemoglobin function [pH, Pco2, theratio between concentrations of 2,3-diphosphoglycerate (2,3-DPG) and hemoglobin, temperature, and glycosylated he-moglobins] have been recently defined (1-3), allowing theprediction of p within ±0.39 mmHg (SD), with two limita-tions: the presence of hemoglobins with altered affinities foroxygen, and the presence of carboxyhemoglobin (COHb),which is known to affect the oxygen affinity in blood (4)#{149}5The purpose of the present work is to extend the reportedequations to include the alterations of the oxygen affinity inblood related to the presence of clinically relevant (up to23%) proportions of COHb in blood, as has been reported,e.g., in heavy smokers (5) andin some patients with chroniclung diseases (6).Materials and MethodsBlood samples. Fresh blood from a nonsmoking donor(M.S.) whose oxygen affinity parameters were within thenormal range (1, 2) was collected in sodium heparin andstored in ice. All experiments were performed within thenext 8 h. The blood was partly saturated with CO bytonometry for 10 mm with 1 to 3 mL of gaseous C0,introduced via a gas-tight Hamilton syringe, in a 100-mLsealed flask containing 5 mL of oxygenated blood at icetemperature. The flask was then opened and the blood wasequilibrated with air for about 30 miii to remove excess CO.The total hemoglobin concentration and the concentrationsof COHb and methemoglobin (metHb) were determinedspectrophotometrically, with use of previously reported ab-sorptivity coefficients (7). We measured the 2,3-DPG concen-tration with kits provided by Boehringer Biochemia.Hill coefficient. For each of the five COHb proportionsconsidered (range, 0 to 33%), we filled as many as four‘Centro Studi di Fisiologia del Lavoro Muscolare del C.N.R.2Ospedale San Raffaele, Milan.33a Cattedra di Semeiotica Medica, University ofRoma.‘Dipartimento di Scienze e Tecnologie Biomediche, do OspedaleSan Raffaele, Via Olgettina 60, 20132 Milano, Italy. (Use thisaddressfor correspondence.)Nonstandard abbreviations: Hb, hemoglobin;COHb, carboxyhe-moglobin; metHb, methemoglobin; and 2,3-DPG, 2,3-diphosphoglycerate.ReceivedFebruary 7, 1984; acceptedApril 4, 1984.tonometry flasks (1) with gas at an appropriate Po (so thatthe measured oxygen saturation would fall within tbe range0.3 to 0.7), a Pco, of 45 miriHg, and the rest N2. Thehemoglobin saturation with oxygen was determined after25mm of tonometry at 37#{176}C,according to the method de-scribed (8). Blood sample pH and [2,3-DPG]/[Hb] ratio wereleft unchanged. We calculated the Hill coefficient, n, as theslope of the regression line for the experimental data plottedas proposed by Hill (9).Determination of the p value. The p value was deter-mined as described previously (1), at five COHb proportions(range 0 to 23%) and at least four pH values in the range 7.0to 7.6 for each COHb value. 2,3-DPG was kept at the valuenormally found in vivo and tonometry temperature waskept constant at 37.3 #{176}C.To measure pH, we used an IL1302 gas analyzer (Instrumentation Laboratory, PadernoDugnano, Milano, Italy). By introducing appropriate Hill nvalues and hemoglobin concentrations in equations 1 andA4, respectively, we changed our previous calculations (1) toaccount for the presence of COHb in the blood sample.ResultsThe Hill n value decreases with increasing COHb (Figure1). The plots of log p vs pH-i.e., the Bohr effect-wereconsistently linear under all the conditions studied (r rangesfrom 0.980 to 0.996). Plots of log p vs COHb at various pHvalues were linear, with a mean constant slope of -0.00848(SD 0.0011) (Figure 2). Therefore, because the dependence ofp on CO is known to be unaffected by 2,3-DPG (10), andbecause CO2affects oxygen affinity only at po2 values muchlower than p (10), the following equation represents theCCOHb (%)Fig. 1.Hilln value vs% COHb (data fitted to an empiricalsecond-orderpolynomial:y= (7.32 X i0 X )) -(5.19 X 10-2 Xx) + 2.735;RMSerror = 8.4 x 1020 10 20COHb (%)Fig.2. Unearrelationshipbetweenlog and% COHbat various pHvaluesReferences1. Samaja M, Mosca A,LuzzanaM, et al. Equationsandnomogramfor the relationshipof human bloodp to 2,3-diphoephoglycerate,32 36 C02, and H. Clin Chem 27, 1856-1861 (1981).2. Samaja M, Melotti D, Rovida E, Roesi-Bernardi L Effect ofvalue and that temperature on thep value for human blood.Clin Chem 29, 110-114 (1983).log 5o, = log Pso, - (0.00848 x x) (1)CLINICAL CHEMISTRY, Vol.30, No. 7, 1984 125116 20 24 28EXPERIMENTAL P50Fig. 3. Correlation between the expenmentalcalculatedfrom equation 1effect of COHb on human blood p at 37#{176}C:where x is the percentage of COHb in the sample and p isthe Pscvalue of a sample containing no COHb.Figure 3 shows the correlation between predicted (calcu-lated from equation 1) and experimentally measured pvalues. The p value normalized with respect to COHb canbe predicted with an SD of 0.54 mmHg.The proportion of metHb never exceeded 1% in any of thestudies.DiscussionThe correction factor reported here, d(log p)/d[COHb] , ishigher than hitherto reported: 0.006(4,5) and 0.007(10,11).In this study, particular care was devoted to the preparationof partially CO-saturated blood samples. The describedmethod represents somewhat the “in vivo” situation, wherehemoglobin equilibrates with a low concentration of CO andthe ligand is distributed uniformly among the hemes in theerythrocytes. In separate experiments, fully CO-saturatedblood mixed with CO-free blood before tonometry yieldedessentially the same results (not shown), indicating that theredistribution of CO within and between cells occurswithinTable 1. Printout of a FORTRAN Subroutine toCalculate p5 from pH, PCO2, [2,3-DPG]/[Hb],Temperature, and COHbSUBROUTINE P50(PH,PCO2,DPG,T,COHB,P50)C NORMALIZE FOR PH, PCO2 AND [2,3-DPG]/[HB]G=ALOG1O(DPG)C FIND LOG P50 AT PH 7.0A=(_0.69117E_3*PCO2 + 0.3365)G +(O.3598E-3PC02 + 1.599)C FINDLOGP5OATPH7.6B=(-0.138E-2#{176}PCO2 + 0.36o7)*G + (O.9089E-3PCO2+ 1.36)C INTERPOLATE AT THE DESIRED PHP5OLOG=((PH_7.)*(B_A))/0.6 + AC NORMALIZE FOR TEMPERATURETK=T + 273. ! NEED ABSOLUTE TEMPERATUREP5OLOG=P5OLOG - 2149.#{176}((1./TK)- (1J310.))C NORMALIZE FOR COHB (%)P5OLOG=P5OLOG - 0.00848 #{149}COHBC END OF CALCULATIONSP50=1 0.**P5OLOGRETURNENDthe time required for tonometry (25 mm).At present, the p value can be either predicted orcompared with the experimental value, to learn whether anobservedapparently abnormal p is accounted for by abnor-mal values for pH, Pco, 2,3-DPG, temperature, and (or)COHb, or whether other factors such as hemoglobins withaltered oxygen affinity or other currently unknown cofactorsof hemoglobin oxygenation must be considered. Table 1showsthe printout of a FORTRAN IV subroutine summarizingthe equations reported here and in our previous papers (1,2). A similar program, which can be run on a Texas TI-59(210 program steps), is available upon request to the au-thors.3 Samaja M, Melotti D, Carenim A, Pozza G. Glycosylated haemo-globins and the oxygen affinity of whole blood. Dio.betologia 23,399-402 (1982).4. Roughton FJW, Darling RC. The effect of carbon monoxideonthe oxyhemoglobindissociation curve. Am J Physiol 141, 17-31(1944).5. Goldsmith JR, Landaw SA. Carbon monoxide and humanhealth. Science 162, 1352-1359 (1968).6. Hebbel EP, Eaton JW, Modler 5, Jacob HS. Extreme butasymptomatic carboxyhemoglobinemia and chronic lung disease. JAm Med Assoc 24, 2584-2586 (1978).7. Rossi-Bernardi L, Perrella M, Luzzana M, et al. Simultaneousdetermination of the hemoglobin derivatives, oxygencontent,andoxygensaturation in 10 zL ofwhole blood. Clin Chem 23, 1215-1225 (1977).8. Samaja M, Rovida E. A new method tomeasure the haemoglobinoxygensaturation bythe oxygenelectrode. JBiochemBiophys Meth7, 143-152 (1983).9. Hill AV. The possible effect of aggregation of the molecules ofhaemoglobin on its dissociation curve. J Physiol (London) 40, 4-7(1910).10. Okada Y, Tyuma I, Ueda Y, Sugiinoto T. Effect of carbonmonoxide on equilibrium between oxygenand hemoglobin.Am JPhysiol 230, 471-475 (1976).11. Hiastala MP, McKenna HP, Franada RL, Detter JC. Influenceof carbon monoxide on hemoglobin-oxygen binding. J Appi Physiol41, 893-899 (1976).

Carboxyhemoglobin and oxygen affinity of human blood

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Carboxyhemoglobin and oxygen affinity of human blood

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