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Nomogram for calculation of buffer line shift due to reduction of haemoglobin at constant PCO2
CLINICA
CHIMICA
ACTA
277
NOMOGRAM
FOR
REDUCTION
OF HAEMOGLOBIN
CALCULATION
OF BUFFER
LINE
AT CONSTANT
SHIFT
DUE
TO
Pco,
G. R. ICELMAN
Department (Received
of Physiology, May
University
of Aberdeen
(U.K.)
I I, 1968)
SUMMARY
Oxyhaemoglobin
is a stronger
acid than reduced
haemoglobin
and the buffer
line of reduced blood therefore lies to the right of that of oxygenated blood on Siggaard-Andersen nomogram. This shift of buffer line may cause errors in determination of bicarbonate concentrations and carbon dioxide tensions when Astrup interpolation technique is used with desaturated blood. A nomogram been constructed, based buffer line position.
on experimental
Since oxyhaemoglobin
is a stronger
findings,
for the calculation
acid than reduced haemoglobin,
the the the has
of the true
the buffer
line for oxygenated blood lies to the left of that of reduced blood on the SiggaardAndersen nomogram’. This shift of buffer line may cause inaccuracies when the interpolation technique is used for the determination of bicarbonate concentrations and carbon dioxide tensions. With the Astrupz technique, blood is equilibrated with mixtures of carbon dioxide in oxygen and the haemoglobin perforce becomes fully saturated. The pH which is used in the interpolation, however, is the actual pH of the blood before equilibration; thus, if the blood is venous, or arterial from a hypoxaemic patient, errors will occur. In order to minimize these errors, a nomogram has been constructed for the calculation of the position of the true buffer line. The results of most previous investigations have expressed the buffer line shift in terms of the amount of base liberated per unit mass of haemoglobin reduced. Since there are some discrepancies between the results of different workers, it was decided to measure the buffer line shift directly in terms of dpH at constant Pco*, and to base the nomogram on these experimentally determined values. This approach avoids uncertainties about the precise value of pK’ which is needed to convert d base into dpH at a given Pco,.(It was not .possible to use the results of a similar study by Rossi-Bernardi and Roughtons because they did not report explicitly the variation of dpH with Pco,.)
Clin. Chim.
Acta,
22 (1968) 227-280
278
KELMAN
METHODS
Aliquots of the same blood were equilibrated with approximately 37: and 8 “,b CO, in either 0, or N, in two Radiometer microtonometer units (type AMT I). The shift of buffer line was then determined by measurement, in each case, of the pH at the two different CO, tensions. The measurements were made on fresh human blood withdrawn from an antecubital vein and rendered incoagulable with heparin (50 units heparin injection B.P./ml). The haemoglobin concentration of the blood, measured by the cyanmethaemoglobin method, was 16.0 g/100 ml. Two Radiometer microtonometer units were connected in parallel to the same water bath and maintained at the same temperature (37 f 0.1’) as each other and as a glass capillary pH electrode (Radiometer type E 5021). The CO, concentration of the gas mixtures was measured with a Lloyd-Haldane apparatus and converted into tension in the usual way. The blood and gas were shaken for 3 min with the sample size, shake amplitude and gas flow rate found to be optimal by Kelman et ~1.~.The pH was measured in the manner recommended by these workers. The pH meter (Radiometer type PHM 27b) was checked frequently against phosphate buffers of pH 6.841 and 7.383 at 37’. The horizontal position of the buffer line was calculated on a digital computer (PDP-8/S, Digital Equipment Corporation) as the pH at a PCO, of 60 mm Hg (pH,,) and the pH at a PCO, of 20 mm Hg (pH2,,). Th’IS calculation was based on the properties of similar triangle9. The buffer line shift was determined on three different bloods which had respectively a metabolic acidosis (mean standard bicarbonate = 14.8 mequiv/l), a normal metabolic acid-base state (mean standard bicarbonate = 22.8 mequiv/l) or a metabolic alkalosis (mean standard bicarbonate = 37.0 mequiv/l). These mixtures were prepared by diluting blood to twice its original volume with either isotonic sodium chloride solution (154 mequiv/l) or with one of two isotonic solutions containing both sodium chloride and sodium bicarbonate (NaCl 130 mequiv/l, NaHCO, 24 mequiv/l; NaCl106 mequiv/l, NaHCO, 48 mequiv/l). The haemoglobin concentration of each of these three bloods was 8.0 g/roe ml.
0.06
. r = -043
_,
._#
I
000
7.0
7.2
74
Fig. I. Relationship
Clin. Chim. Acta,
7.6 pli 60
74
between pH and actual (oxygenated)
22 (1968) 277-280
PH. Pcoz = 60 mm Hg.
BUFFER
LINE
SHIFT
NOMOGRAM
279
RESULTS
For convenience, the change of buffer line position between reduced and oxygenated blood will be expressed by the symbol dpH, while the level of CO, tension at which the measurement was made will be indicated by a subscript. Thus dpH,, will refer to the difference, at a PCO, of 20 mm Hg, between the pH of fully reduced and of fully oxygenated blood. The relationship between flpH 6,, and dpH,, and the actual pH of oxygenated blood is shown in Figs. I and 2. In both cases the value of flpH decreased markedly with increasing metabolic alkalosis. The linear correlation coefficients between ApH and actual pH were -0.634 and -0.915 respectively, both values being significantly different from zero (P < 0.001). The regression coefficient of dpH on actual oxygenated pH was markedly greater at the lower CO, tension (-0.083 (SE & 0.007) cf. -0.046 (SE & 0.011)). These two regression coefficients are significantly different from each other, i.e. the slope of the relationship between dpH and pH depends on the CO, tension. The nomogram (Fig. 3) This is based on the linear relationships pH shown in Figs.
I
and
2.
The two regression
between dpH and actual (oxygenated) equations
are:
dpH,,
= 0.0107
-
0.0103
(pH -
7.0) per g reduced Hb
dpH,,
= 0.0053
-
0.0057
(pH -
7.0) per g reduced
Hb
The assumption is made that the amount of base which is liberated is linearly related to the quantity of haemoglobin which is reduced, i.e. is proportional to the product of the total haemoglobin concentration x the fractional desaturation. The nomogram is used by drawing the oxygenated blood buffer line on the Siggaard-Andersen nomogram in the usual way and reading off the oxygenated pH at PCO~= 20 and Pcot = 60 mm Hg. The values of dpH for each of these pH values are then read off from the central scale of Fig. 3, and added to the uncorrected values to give corrected pH, from which the true position of the buffer line can be drawn.
PI
72
74
7-6
78
&O
PI-! 20
Fig.
2. Relationship
between
pH and actual
(oxygenated)
pH.
Pcoz
=
20 mm Hg.
Clin. Chinz. Acta,
22 (1968) 277-280
KELMAN
10
a
6
4
2 reduced
Fig.
0 lib
2
4
6
6
18
g/lOOml
3. The nomogram.
Exanzple of use pH values at PCO, of 60 and
20
mm Hg on the fully oxygenated
buffer line
7.20 and 7.47, respectively. Actual measured pH 7.24. Haemoglobin concentration 15 g/100 ml. Oxyhaemoglobin saturation 70%. Concentration of reduced haemoglobin is therefore 4.5 g/roe ml. Running down the line from pH 7.20 on the PCO, = 60 mm Hg (i.e. right hand) side of the nomogram until it intercepts a vertical line through 4.5 g on the right hand reduced
Hb scale gives a point opposite
a dpH value, on the central
scale, of
0.018. Similarly, the value for ilpH,, is 0.026. These therefore give corrected pH values of 7.218 and 7.496 respectively, and on the new buffer line, the actual pH value corresponds to a PCO, of 54.8 as against 51.0 mm Hg on the line for fully oxygenated blood. The bicarbonate concentration at a PCO%of 40 mm Hg, on the new buffer line, is 20.0 mequiv/l, whereas the corresponding bicarbonate concentration at a PCO, of 40 mm Hg on the fully oxygenated line-the standard bicarbonateis 19.0 mequiv/l. REFERENCES I 0. SIGGAARD-ANDERSEN, P. 2 0. 12 3 L. 4 G.
56.
Clin.
Chim.
SIGGAARD-ANDERSEN, (rg6o)
The Acid-Base
K.ENCEL,
Status
K.J~RGENSEN
of the Blood,
AND P.
Munksgaard,
Copenhagen,
ASTRUP,Scand.J.Clin. Lab.Invest.,
I.
ROSSI-BERNARDI AND J. J. W. ROUGHTON,]. Physiol., 189 (1967) I. R. KELMAN, A. J. COLEMAN AND J. F. NuNN,J. AppZ.PhysioZ., 21 (1966) 5 G. R. KELMAN, Rap. Physiol., I (1966) 335. Acta,
22 (1964)
277-280
1964,
1103.
CHIMICA
ACTA
277
NOMOGRAM
FOR
REDUCTION
OF HAEMOGLOBIN
CALCULATION
OF BUFFER
LINE
AT CONSTANT
SHIFT
DUE
TO
Pco,
G. R. ICELMAN
Department (Received
of Physiology, May
University
of Aberdeen
(U.K.)
I I, 1968)
SUMMARY
Oxyhaemoglobin
is a stronger
acid than reduced
haemoglobin
and the buffer
line of reduced blood therefore lies to the right of that of oxygenated blood on Siggaard-Andersen nomogram. This shift of buffer line may cause errors in determination of bicarbonate concentrations and carbon dioxide tensions when Astrup interpolation technique is used with desaturated blood. A nomogram been constructed, based buffer line position.
on experimental
Since oxyhaemoglobin
is a stronger
findings,
for the calculation
acid than reduced haemoglobin,
the the the has
of the true
the buffer
line for oxygenated blood lies to the left of that of reduced blood on the SiggaardAndersen nomogram’. This shift of buffer line may cause inaccuracies when the interpolation technique is used for the determination of bicarbonate concentrations and carbon dioxide tensions. With the Astrupz technique, blood is equilibrated with mixtures of carbon dioxide in oxygen and the haemoglobin perforce becomes fully saturated. The pH which is used in the interpolation, however, is the actual pH of the blood before equilibration; thus, if the blood is venous, or arterial from a hypoxaemic patient, errors will occur. In order to minimize these errors, a nomogram has been constructed for the calculation of the position of the true buffer line. The results of most previous investigations have expressed the buffer line shift in terms of the amount of base liberated per unit mass of haemoglobin reduced. Since there are some discrepancies between the results of different workers, it was decided to measure the buffer line shift directly in terms of dpH at constant Pco*, and to base the nomogram on these experimentally determined values. This approach avoids uncertainties about the precise value of pK’ which is needed to convert d base into dpH at a given Pco,.(It was not .possible to use the results of a similar study by Rossi-Bernardi and Roughtons because they did not report explicitly the variation of dpH with Pco,.)
Clin. Chim.
Acta,
22 (1968) 227-280
278
KELMAN
METHODS
Aliquots of the same blood were equilibrated with approximately 37: and 8 “,b CO, in either 0, or N, in two Radiometer microtonometer units (type AMT I). The shift of buffer line was then determined by measurement, in each case, of the pH at the two different CO, tensions. The measurements were made on fresh human blood withdrawn from an antecubital vein and rendered incoagulable with heparin (50 units heparin injection B.P./ml). The haemoglobin concentration of the blood, measured by the cyanmethaemoglobin method, was 16.0 g/100 ml. Two Radiometer microtonometer units were connected in parallel to the same water bath and maintained at the same temperature (37 f 0.1’) as each other and as a glass capillary pH electrode (Radiometer type E 5021). The CO, concentration of the gas mixtures was measured with a Lloyd-Haldane apparatus and converted into tension in the usual way. The blood and gas were shaken for 3 min with the sample size, shake amplitude and gas flow rate found to be optimal by Kelman et ~1.~.The pH was measured in the manner recommended by these workers. The pH meter (Radiometer type PHM 27b) was checked frequently against phosphate buffers of pH 6.841 and 7.383 at 37’. The horizontal position of the buffer line was calculated on a digital computer (PDP-8/S, Digital Equipment Corporation) as the pH at a PCO, of 60 mm Hg (pH,,) and the pH at a PCO, of 20 mm Hg (pH2,,). Th’IS calculation was based on the properties of similar triangle9. The buffer line shift was determined on three different bloods which had respectively a metabolic acidosis (mean standard bicarbonate = 14.8 mequiv/l), a normal metabolic acid-base state (mean standard bicarbonate = 22.8 mequiv/l) or a metabolic alkalosis (mean standard bicarbonate = 37.0 mequiv/l). These mixtures were prepared by diluting blood to twice its original volume with either isotonic sodium chloride solution (154 mequiv/l) or with one of two isotonic solutions containing both sodium chloride and sodium bicarbonate (NaCl 130 mequiv/l, NaHCO, 24 mequiv/l; NaCl106 mequiv/l, NaHCO, 48 mequiv/l). The haemoglobin concentration of each of these three bloods was 8.0 g/roe ml.
0.06
. r = -043
_,
._#
I
000
7.0
7.2
74
Fig. I. Relationship
Clin. Chim. Acta,
7.6 pli 60
74
between pH and actual (oxygenated)
22 (1968) 277-280
PH. Pcoz = 60 mm Hg.
BUFFER
LINE
SHIFT
NOMOGRAM
279
RESULTS
For convenience, the change of buffer line position between reduced and oxygenated blood will be expressed by the symbol dpH, while the level of CO, tension at which the measurement was made will be indicated by a subscript. Thus dpH,, will refer to the difference, at a PCO, of 20 mm Hg, between the pH of fully reduced and of fully oxygenated blood. The relationship between flpH 6,, and dpH,, and the actual pH of oxygenated blood is shown in Figs. I and 2. In both cases the value of flpH decreased markedly with increasing metabolic alkalosis. The linear correlation coefficients between ApH and actual pH were -0.634 and -0.915 respectively, both values being significantly different from zero (P < 0.001). The regression coefficient of dpH on actual oxygenated pH was markedly greater at the lower CO, tension (-0.083 (SE & 0.007) cf. -0.046 (SE & 0.011)). These two regression coefficients are significantly different from each other, i.e. the slope of the relationship between dpH and pH depends on the CO, tension. The nomogram (Fig. 3) This is based on the linear relationships pH shown in Figs.
I
and
2.
The two regression
between dpH and actual (oxygenated) equations
are:
dpH,,
= 0.0107
-
0.0103
(pH -
7.0) per g reduced Hb
dpH,,
= 0.0053
-
0.0057
(pH -
7.0) per g reduced
Hb
The assumption is made that the amount of base which is liberated is linearly related to the quantity of haemoglobin which is reduced, i.e. is proportional to the product of the total haemoglobin concentration x the fractional desaturation. The nomogram is used by drawing the oxygenated blood buffer line on the Siggaard-Andersen nomogram in the usual way and reading off the oxygenated pH at PCO~= 20 and Pcot = 60 mm Hg. The values of dpH for each of these pH values are then read off from the central scale of Fig. 3, and added to the uncorrected values to give corrected pH, from which the true position of the buffer line can be drawn.
PI
72
74
7-6
78
&O
PI-! 20
Fig.
2. Relationship
between
pH and actual
(oxygenated)
pH.
Pcoz
=
20 mm Hg.
Clin. Chinz. Acta,
22 (1968) 277-280
KELMAN
10
a
6
4
2 reduced
Fig.
0 lib
2
4
6
6
18
g/lOOml
3. The nomogram.
Exanzple of use pH values at PCO, of 60 and
20
mm Hg on the fully oxygenated
buffer line
7.20 and 7.47, respectively. Actual measured pH 7.24. Haemoglobin concentration 15 g/100 ml. Oxyhaemoglobin saturation 70%. Concentration of reduced haemoglobin is therefore 4.5 g/roe ml. Running down the line from pH 7.20 on the PCO, = 60 mm Hg (i.e. right hand) side of the nomogram until it intercepts a vertical line through 4.5 g on the right hand reduced
Hb scale gives a point opposite
a dpH value, on the central
scale, of
0.018. Similarly, the value for ilpH,, is 0.026. These therefore give corrected pH values of 7.218 and 7.496 respectively, and on the new buffer line, the actual pH value corresponds to a PCO, of 54.8 as against 51.0 mm Hg on the line for fully oxygenated blood. The bicarbonate concentration at a PCO%of 40 mm Hg, on the new buffer line, is 20.0 mequiv/l, whereas the corresponding bicarbonate concentration at a PCO, of 40 mm Hg on the fully oxygenated line-the standard bicarbonateis 19.0 mequiv/l. REFERENCES I 0. SIGGAARD-ANDERSEN, P. 2 0. 12 3 L. 4 G.
56.
Clin.
Chim.
SIGGAARD-ANDERSEN, (rg6o)
The Acid-Base
K.ENCEL,
Status
K.J~RGENSEN
of the Blood,
AND P.
Munksgaard,
Copenhagen,
ASTRUP,Scand.J.Clin. Lab.Invest.,
I.
ROSSI-BERNARDI AND J. J. W. ROUGHTON,]. Physiol., 189 (1967) I. R. KELMAN, A. J. COLEMAN AND J. F. NuNN,J. AppZ.PhysioZ., 21 (1966) 5 G. R. KELMAN, Rap. Physiol., I (1966) 335. Acta,
22 (1964)
277-280
1964,
1103.