SOLUBILITY OF CARBON MONOXIDE IN SERUM ...

SOLUBILITY

OF BY

(From

CARBON MONOXIDE PLASMA.*

H. R. O’BRIEN?

the Department

AND

of the Interior,

(Received for publication,

IN

SERUM

W. L. PARKER.1 Bureau of Mines, November

AND

Pittsburgh.)

12, 1921.)

INTRODUCTION.

* Published by permission of the Director of the United States Bureau of Mines, Washington. t Assistant Surgeon (Reserve), United States Public Health Service. $ Junior Chemist, United States Bureau of Mines. 1 Bohr (4), p. 62. 289

Downloaded from http://www.jbc.org/ by guest on August 21, 2015

In the course of the work of the writers on a method of determination of carbon monoxide in blood, the question arose as to how much of the total gas united with the hemoglobin, and how much merely dissolved in the serum. With the known strong affinity of carbon monoxide for hemoglobin (220 to 300 times as strong as that of oxygen) (1) it would be expected that by far the largest percentage would enter into combination with the hemoglobin, but it seemed of value to investigate just what percentage could be accounted for as being in simple solution. This was especially the case with the Van Slyke method, where the total amount of carbon monoxide in the blood is measured, and not the amount of carbon monoxide hemoglobin. Van Slyke (2) passes over the subject in the case of carbon monoxide; but when dealing with oxygen in the blood (3) has a table of deductions and corrections. These are estimated on the basis of Bohr’s recommendation that the solubility of air in serum is roughly nine-tenths that in water at the corresponding temperature. On this subject of the solubility of gasesin serum and blood, Bohr goes quite into detail (4). He states1 that the absorption coefficients of oxygen and carbon dioxide in whole blood and that of carbon dioxide in plasma cannot be obtained directly,

290

Carbon

Monoxide

in Serum and Plasma

Coe.$icient

of Abswption

in

Cc. of Gas

02 15” _____~---

Blood Whole Blood

plasma. . .. blood.. corpuscles..

0.033 0.031 0.025

(at 0’ and

760 Mna.)

N%

CO2

38”

15”

38”

15”

38”

0.023 0.022 0.019

0.017 0.016 0.014

0.012 0.011 0.010

0.994 0.937 0.825

0.541 0.511 0.450

In the case of carbon monoxide, he says2 simply that the plasma absorbs the gas physically, but proportionately to the tension, and in a slightly lesser amount than would be absorbed by the same volume of water. It seemed worth while, therefore, to make direct determinations instead of relying on estimates. During t,he progress of this work, conducted at the Pittsburgh station of the Bureau of Mines, some other data were obtained which resulted in certain important conclusions on the use of a table of deductions to correct for the amount of carbon monoxide in the blood, uncombined with the hemoglobin. Method of Obtaining Serum and Plasma. As a medium of investigation, beef serum was selected as the one most readily obtainable in quantity; furthermore, most of the related work was being done on beef blood. To supplement this the results were checked on sheep and human sera. Van Slyke and others have assumed that the solubility of carbon monoxide is the same in serum and in plasma. This was also checked 2Bohr

(5),

p. 122.


because there is also a chemical reaction taking place. But the percentage of deduction (or relation of the coefficient in plasma to that in water) of various gases, when they do not react, is about the same (5). Therefore, when he made determinations and found that the absorption coefficients of oxygen and nitrogen in plasma are 97.5 per cent of those in water, and the absorption coefficient of hydrogen in whole blood is 92 per cent of the figures in water, he felt safe in making the generalization, For later comparison his figures are given; they are calculated by the aid of his deductions.

H. R. O’Brien

and W. L. Parker

Introduction

of Carbon Monoxide

into the Liquid,

The carbon monoxide used was made by dropping formic acid into concentrated sulfuric acid at 150°C. The evolved gas was washed through potassium hydroxide and stored in a gasometer over water, whence it was delivered as needed, by water displacement. The specimen of serum was allowed to reach thermal equilibrium within a thermostat adjusted to the desired temperature. The carbon monoxide reached this same temperature by being forced through a glass spiral also placed within the thermostat. From here it passed through a bubbler into the serum. 15 minutes were thought sufficient for the saturation of the sample. Frothing was prevented within the specimen tube by the addition of a drop of caprylic alcohol. The effluent gas passed off through a tube into a hood; the serum was thus saturated at atmospheric pressure. The analysis of the gases dissolved in the serum was performed on the Van Slyke apparatus. All sera saturated below room temperature were kept in ice water, to prevent loss of gas, pending the time when analysis could be made. The method of analysis employed was a modification of that used by Van Slyke in his


The serum in each case was gotten by allowing on beef plasma. the blood gathered in the slaughter house to clot quietly in sterile containers, pouring off the serum, and removing remaining corpuscles with a high speed centrifuge running for 10 to 20 minutes. With Beef serum has a golden tinge; that of sheep blood is grayer. human blood, considerable trouble arose from a tendency on the part of the corpuscles to hemolyee; but a few specimens of good serum were obtained. Beef plasma was gotten from blood caught in a sterile container as it poured from the vessels of a freshly killed animal. This was at once poured into 100 cc. bottles containing as a preservative, 0.2 gm. of sodium oxalate, The blood 0.3 gm. of fluoride, and 0.4 gm. of citrate, well mixed. was centrifuged for 3 to 4 hours; the supernatant plasma then pipetted off and further purified by half an hour more in the cenThe oxalated blood separates most readily; best results trifuge. are gotten by centrifuging within 2 or 3 days after it is drawn from the animal.

292

Carbon

Monoxide

in Serum and Plasma

determination of oxygen, hemoglobin, and carbon monoxide in blood. It consists in general of drawing off the gases under a vacuum produced by a mercury pump, and of analysis of the evolved substances. The exact technique used in the Bureau laboratory is as follows. Technique of the Analysis Van

of Gases Dissolved in Blood Serum by the Slyke Apparatus.

3 120 gm. of KOH are added to 80 cc. of water, and 50 gm. of pyrogallic acid to 150 cc. of water; 300 cc. of the alkali are then mixed with 40 cc. of the acid solution.


Before each analysis the apparatus is washed out first with a solution of concentrated ammonia diluted with 3 volumes of water and then twice with distilled water. 5 cc. of serum are measured by pipette into the cup. The end of the pipette is held below the surface of the liquid after starting the delivery, so as to reduce as much as possible exposure of the liquid surface to air. 2 drops of caprylic alcohol are added to the serum, the liquid is drawn into the burette, and the capillary above the stop-cock is sealedwith mercury. The apparatus is evacuated and shaken for 2 minutes. The serum is drawn down into the lower bulb and the extracted gases are measured over mercury at atmospheric pressure as described by Van Slyke. The serum is run back into the extraction chamber and shaken further for 1 minute under the same conditions, then the gas volume is read as before. The extraction is repeated until the volume is constant. It is recorded as total gases. The volume contracts a little with standing only a few seconds, as COz dissolved in the small amount of serum on top of the mercury. Oxygen, CO, and Nz are completely given off- by 1 minute shaking in vacuum; the COz comes off much more slowly, requiring usually about 2 minutes. 0.5 cc. of 10 per cent KOH is added to the cup, and is carefully drawn into the pipette, the mercury in the leveling bulb being held slightly below that in the burette. The COz is quickly absorbed. The volume of contraction is noted and the KOH is drawn down into the lower bulb with the serum. About 5 cc. of potassium pyrogallate3 solution are next put into the cup and a drop of straw oil quickly added on top of the pyro

H. R. O’Brien

and W. L. Parker

293

Volume 1st extraction.

of total

2nd extraction.

Volume

gas. 3rd extraction.

KOH

after

absorption

Pyre.

by CUZCIZ

cc.

cc.

cc.

cc.

cc.

cc.

0.270

0.290

0.280

0.065

0.060

0.060

It was found that ammoniacal cuprous chloride does not give contraction in volume of the gases extracted from serum when CO gas is not present. 4 400 gm. of cuprous chloride and 500 gm. of NH&l are dissolved in 1,500 cc. of water. For use this is mixed with NH,OH (sp. gr. 0.90) in proportions of 3:l (Winkler, L. W., Handbook of technical gas analysis, London, 2nd English edition, 1902,73).


to exclude the oxygen of the air. The “pyre” is drawn into the burette, but not the straw oil, as the latter interferes with gas absorption by solutions, especially CO by Cu2C12. Oxygen absorption is slow, but is hastened by working the leveling bulb down and up to insure complete contact of gas with the ‘(pyre.” When the volume has become constant the ‘(pyre” is drawn down into the liquid in the lower bulb. The volume of gas is read as before. The cup is rinsed out with distilled water, as the pyrogallate left would form a precipitate with the next reagent to be used. A small dropping pipette has been found convenient for removing liquids from the cup. 0.5 cc. of ammoniacal cuprous chloride4 is now added and carefully drawn through the remaining gases. CO is quickly absorbed and a constant volume obtained almost immediately. The remaining gas is probably nitrogen, though its volume is sometimes higher than would be expected. A complete analysis is made by this method in less than 30 minutes. With practice, readings may easily be made to within 0.005 cc. In checking up the carbon monoxide determination by this method, the total gases extracted from untreated serum were analyzed as a blank. An analysis made on sheep serum will serve as an example of the results that were obtained.

TABLE

Solubility

of 100 Per

Cent

Temperature.

20

I.

CO in Serum and of Serum. Sheep

serum.

Plasma

Human

in

Cc. of Gas

wxum.

0.0210 0.0201

0.0203

0.0206

0.0185 0.0176

0.0183 0.0191

0.0181

0.0187

0.0157 0.0~66

0.0148 0.0148 0.0173

0.0183 0.0183

0.0173 0.0164

0.0161

0.0156

0.0183

0.0169

0.0150 0.0140

0.0150 0.0141 0.0158 6.0169 0.0150

0.0158

0.0147” 0.0147

0..0145

0.0153

0.0147

0.0136 0.0117 0.0136 0.0126

0.0153 0.0144 0.0135

0.0143 0.0142

0.0131 0.0150 0.0127 0.0131 0.0131

0.0129

0.0144

0.0142

0.0134

0.0198

0.0180

the method of calculating a 5 cc. sample, saturated

X 3oo 273 X 745 760 22 x n2 760

0.0181 0.0181 0.0181

37

* A sample is shown to illustrate 0.080 cc. of CO was gotten from and analyzed at 27°C. and 742 mm. 0.016 = CO per cc. of sample.

0.0203 0.0195

Table I. at 3O”C.,

= 0.0147

The first fraction corrects for temperature, the second for the partial pressure of the analyzed gas (the barometric pressure minus water vapor tension at 27”), and the third for the partial pressure under which the gas went into solution (barometric pressure less vapor tension of serum at 30”). There are no figures in the literature for this last, but it is taken as approximately equal to that of water. As a change of 10 mm. in pressure alters the result only 0.0002, which is less than the possible error in reading the apparatus, this approximation seems reasonable. 294


30

0.0209

Cc.

plasm.

0.0203 0.0203

25

0.016

Beef

per

H. R. O’Brien Determination As a satisfactory determinations was the four fluids; i.e., The figures obtained

and W. L. Parker

of Solubility

295

of Carbon Monoxide.

method has now been developed, a series of carried out at five different temperatures with beef, sheep, and human sera, and beef plasma. are shown in Table I.

cc.

0.022 Downloaded from http://www.jbc.org/ by guest on August 21, 2015

0.020 2 g 0.018 %8

L 0.016 s2 cl 0.014 0.012 "C.15

20

25 30 Temperature CHART

. 35

40

1.

DISCUSSION.

As would be expected, the figures for the three different sera are practically identical. It is especially interesting to find the results the same with serum and plasma. The accompanying comparison with the table of solubility of carbon monoxide in wat’pr (Winkler) gives figures of the same magnitude, but not

296

Carbon

Monoxide

in Serum and Plasma

In fact, quite agreeing with Bohr’s hypothesis of a 9: 10 relation. the ratio swings from 8: 10 to 7: 10 as the temperature rises. As a check on our method, the same procedure was used in making a determination of the solubility of carbon monoxide in water. Before being used, the water was doubly distilled, lhen freshly boiled. It was saturated with CO, and then analyzed in the way described above for serum. The results obtained (shown in Table II) are quite in agreement with the figures of Winkler. Solubility

of 100 per cent CO in beef serum

2

1

Temperature. co

‘II.

per cc. of serum.

“C.

cc.

15 20 25 30 35 37 40

0.0203 0.0181 0.0161 0.0145

1

co

per

3

and

water.

l-4

1 cc. water.

Winkler.’

Author’s,

cc.

cc.

0.02543 0.02319 0.02142 0.01998 0.01877

0.0129

Column 1 f Column 3.

,

0.0253 0.0236 0.0213 0.0198

0.80 0.77 0.76 0.73

0.0179

0.72

0.01775 I

* Solubility xxxiv, 1408;

of CO 2. physik.

in water (Winkler, L. Chem., 1892, ix, 171.

W.,

Ber.

them.

Ges.,

1901,

The application of these figures of solubility to the calculation of results from the Van Slyke method for the determination of CO and hemoglobin jn blood presented some difficulty. As carbon monoxide passes from air into blood, the reaction may be pictured somewhat as follows: CO

(in air)

dc”, (d’ssolved) 1

When the hemoglobin serum is also, and the from the total results. saturated with carbon

e

CO-hemoglobin

is saturated it might be assumed that the figures of solubility might be subtracted But when the hemoglobin is not entirely monoxide, is a saturated condition of the


TABLE

H. R. O’Brien

and W. L. Parker

Saturation

of Serum with Carbon Monoxide Mixed with Air.

To ascertain whether this expectation is realized in the case of carbon monoxide in serum, beef serum was saturated at 15 and 37”C., with air which contained 1.13 per cent carbon monoxide (analyses by Bureau gas laboratory), with the results shown in Table III. The results indicate clearly that the usual laws of partial pressures in gaseous mixtures apply in the case of carbon monoxide in serum, and, therefore, in plasma. The volume of the gas dissolved in 5 cc. of serum, even in as rich a mixture as 10 per cent, is so slight (0.010 cc.) that the experimental error in handling the Van Slyke apparatus with a blood sample would equal or exceed the figure. There is, therefore, in ordinary conditions of CO poisoning, no advantage to be gained in accuracy by subtracting from the total result any figure allowed for carbon monoxide dissolved in serum. The values obtained are also of importance in their bearing on another question. The figures given by Bohr, cited earlier in this paper in regard to the solubility of gases in plasma and in water, are fundamental in the calculations of hydrogen ion concentrations. In the case of carbon monoxide WC have found absorption coefficients whose ratios differ sharply from the 9:iO one Bohr assumed. It seems possible, therefore, that t,he resuhs of this work may be found to have some influpncc on tjhc basic figures for hydrogen ion determinations.


serum to be expected? In view of the strong affinity of the gas for hemoglobin, it would be safe to assume that the tendency in the equation above is to the right. Other and similar questions arise. It may be mentioned that the air in cases of fatal carbon monoxide poisoning rarely contains more than 5 to 10 per cent of the gas (frequently not more than 1 to 2 per cent). From the laws of partial pressure of gases in mixtures, it would be expected that the volume of any one constituent in solution would be proportional to its tension and solubility coefficient in the gaseous mixture with which the solution is saturated.

298

Carbon

Monoxide

in Serum and Plasma TABLE

Serum saturated at temperature.

Total

III.

co2

gas.

02

“C.

cc.

cc.

cc.

cc.

15

0.135 0.120 0.250 0.195

0.040 0.035 0.175 0.125

0.035 0.020 0.025 0.020

0.060 0.055 0.050 0.050

37

was repeated

with

TABLE

air having

9.8 per

cent

cc.

carbon

mon-

IV.

Serum

saturated

Total

at temperature.

gas.

co2

0%

N2

_-

co

T.

cc.

cc.

cc.

cc.

cc.

15

0.195 0.165 0.120 0.110

0.090 0.060 0.040 0.045

0.025 0.030 0.020 0.020

0.070 0.065 0.050 0.045

0.010 0.010 0.010 0.010

experiment

may

37

The results of the of the total gas (Table

second V).

TABLE

be expressed

in percentage

V. Per cent of tots1

gas* 8.9:

Specimen. CO2

Air. Serum

at 15”.

Serum

at 37”.

02

N2

18.9 23.8 28.6 25.0 26.7

71.3 66.7 61.9 62.5 60.0

co

9.8 9.5 9.8 12.5 13.8

* Carbon dioxide was disregarded in figuring percentages, as it was absent in the original air bubbled through the serum (although of course from the dissolved CO a small amount of free gas would be found above the solution).


The experiment oxide (Table IV).

co

Nz

H. R. O’Brien

and W. L. Parker

299

CONCLUSIONS.

In the course of the work the writers have received valuable suggestions from several laboratory and clinical men. They wish especially to express their appreciation for the assistance of Dr. R. R. Sayers, Chief Surgeon of the Bureau of Mines, and A. C. Fieldner, Superintendent of the Pittsburgh Experiment Station who supervised the work; Dr. N. R. Givens of the laboratory staff of the West Penn Hospital, Pittsburgh, for advice in purifying serum and plasma; Mr. Dan Monahan of the Pittsburgh Provision and Packing Company, who very kindly furnished beef and sheep blood for quantity work; Dr. J. C. Burt, who placed the facilities of the state clinic at the disposal of the writers in gathering specimens of human blood; Dr. Stegeman, Professor of Physical Chemistry at the University of Pittsburgh, for advice in connection with several points, and to Dr. D. D. Van Slyke of The Rockefeller Institute, and Dr. Yandell Henderson and Dr. H. W. Haggard of Yale University for suggestions and criticisms.


The writers have devised a method for the determination of carbon monoxide in serum and plasma. Beef, sheep, and human sera, and beef plasma were saturated with the gas at 15, 20, 25, 30, and 37°C. Compatible figures were obtained. These were of the same magnitude as those of the known solubility of carbon monoxide in water, but only about three-fourths as large as the latter. The method was checked by a determination of the solubility of carbon monoxide in distilled water. The solubility figures in serum and plasma are identical. Further work was done on the solubility of carbon monoxide by exposing the sera and plasma to mixtures (1 to 10 per cent) of carbon monoxide in air. The amount of this gas dissolved under those conditions was so very small that in calculating results in cases of poisoning under ordinary conditions, no allowance need be made for carbon monoxide dissolved in the serum. The results obtained may have some bearing on hydrogen ion calculations.

300

Carbon

Monoxide

in Serum and Plasma

BIBLIOGRAPHY.


1. Nicloux, M., L’instabilite de l’hemoglobine oxycarbon6e en presence d’oxygene. Son inter& au point de vue du traitemrnt de l’intoxicntion oxycarbonee, Pre.sse M&d., 1917, xxv, 153. of carbon 2. Van Slyke, D. D., and Salveson, H. A., The determination monoxide in blood, J. Biol. Chem., 1919, xl, 103. 3. Van Slyke, D. D., Gasometric determination of the oxygen and hemoglobin of blood, J. Biol. Chem., 1918, xxxiii, 127. 4. Bohr, C., Blutgase und respiratorischer Gaswecksel, in Nagel, W., Handbuch der Physiologie des Menschen, Brunswick, 1909, i, 54. 5. Bohr, C., Absorptions coefficienten des Blutes und des Blut Plasmas fiir Gase, Xkand. Arch., 1905, xvii, 104.

ARTICLE: SOLUBILITY OF CARBON MONOXIDE IN SERUM AND PLASMA H. R. O'Brien and W. L. Parker

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J. Biol. Chem. 1922, 50:289-300.