gasometric determination of oxygen and carbon

GASOMETRIC DETERMINATION OF OXYGEN AND CARBON MONOXIDE IN BLOOD.

BY JULIUS SENDROY, JR., AND S. H. LIU.*

(From the Hospital of The Rockefeller Institute for Medical Research, New York.)

(Received for publication, July 21, 1930,)

Van Slyke and Neil1 (l), in their original description of the manometric blood gas apparatus, described a method for the determination of O2 and CO which was sufficiently exact for most purposes. However, estimation of the CO was less exact than that of 02. While O2 was determined by specific absorption with NalSzOI solution, the CO had to be estimated by subtracting from the residual CO + Nz a value of 1.2 or 1.4 volume per cent corresponding to the mean Nz content of blood. CO could not be determined by absorption, because no suitable absorbing solution was available which did not form an unmanageable clot when mixed with the blood in the extraction chamber. The procedure by which CO is estimated by subtraction of the mean Nz content of blood from the CO + Nt, is definitely less exact than one in which CO could be determined by direct absorption. Further- more, the estimation by subtraction of the mean Nz is not valid for blood saturat’ed under experimental conditions with inert gases other than air at atmospheric pressure.

A technique which made possible the precise determination of CO by direct absorption was later devised by Van Slyke and Rob- scheit-Robbins (2) who used the Harington-Van Slyke (3) modi- fication of the extraction chamber of the manometric apparatus. At the bottom of this extraction chamber there is an added cock, by means of which the chamber and the gases in it can be washed with successive portions of cleaning and absorbing solutions. The blood could be washed out and the CO measured by absorp-

* On leave of absence from the Department of Medicine, Peking Union Medical College, Peiping, China.

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134 0, and CO in Blood

tion with cuprous chloride solution. The results were highly exact, but the numerous washings made the procedure rather long, 40 to 50 minutes being required for an analysis. Also, an incon- venience was introduced in this analysis, with the necessity for using the special Harington-Van Slyke extraction chamber, while all other analyses described for the manometric apparatus can be carried out with the simpler Van Slyke-Neil1 chamber.

In the present paper a procedure is described in which determination of CO by absorption is accomplished in analyses made with the Van Slyke-Neil1 chamber. The mixture of 02 + CO + Nz extracted from blood is removed to a micro-Hempel pipette, where the 0, is absorbed, by a technique similar to that employed previously for manometric determination of amino nitrogen (4). The extraction chamber is then washed free of blood, and the gases are returned for completion of the analysis. The procedure equals in accuracy the Van Slyke-Robscheit-Robbins method, is less laborious, and can be carried through in 25 to 30 minutes.

Description of Method.

Reagents.

Acid Ferricyanide Reagent.-This is prepared for use each day by mixing equal parts of the following two solutions; (a) 32 gm. of potassium ferricyanide and 8 gm. of saponin dissolved in water to make 1 liter of solution, (b) 8 cc. of concentrated lactic acid (sp. gr. 1.20), diluted to 1 liter.

Alkaline Pyrogallate.-15 gm. of pyrogallic acid in 100 cc. of a saturated solution of KOH (sp. gr. about 1.55). This absorbent is kept under paraffin oil in a stoppered bottle and is not used until 3 weeks after preparation. If kept confined under oil in the modified Hempel pipette used for this work, one portion of pyrogallol solution may be used for 30 to 40 analyses.

Air-Free N Sodium Hydroxide.-Approximately 40 gm. of NaOH per liter solution. This is extracted air-free for use daily, and kept under oil in a calcium chloride tube ((1) p. 534).

Glycerol-Salt Solution.-One volume of glycerol is mixed with 3 volumes of saturated NaCl solution.

Winkler’s Cuprous Chloride Solution.-200 gm. of CuCl, 250 gm. of NH&I, and 750 cc. of water. The addition of a few gm. of


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J. Sendroy, Jr., and S. H. Liu 135

pure metallic copper serves to keep the CuCl in reduced state. This solution is freed of air, kept under a layer of paraffin oil, and should be used within 4 hours after having been rendered air-free.

Cuprylic Alcohol.-This is used to prevent foaming.

Procedure.

The analysis consists of the following steps. 1. The gases, COZ, Or, CO, and Nz are extracted from the blood

sample in the chamber of the Van Slyke-Neil1 apparatus. 2. COz is absorbed by the addition of N NaOH. 3. The mixture of residual gases, 02, CO, and Nz is transferred to

the Hempel pipette containing alkaline pyrogallate, which ab- sorbs the oxygen.

4, The blood is removed from the extraction chamber and replaced by air-free glycerol-salt solution.

5. The mixture of residual gases, CO and NP, is returned to the chamber of the apparatus.

6. CO is absorbed by the addition of Winkler’s reagent. The details of the successive steps are given below. Since much

of the technique has already been described in papers on other manometric analyses, it would be advantageous for the reader not already familiar with the manometric apparatus to consult previous papers referred to (14) for more complete explanations of general details and precautions.

The directions below apply when 2 cc. samples are used. The procedure for 1 cc. samples is given in a later section.

1. Extraction of Gases from Blood Sample.-From the cup of the Van Slyke-Neil1 apparatus, 2 drops of caprylic alcohol are admitted into the extraction chamber, followed by 8 cc. of the acid ferricyanide reagent. The stop-cock is sealed with mercury, and the chamber is evacuated and shaken for 2 minutes. The extracted air is ejected ((4) p. 428) and 4 cc. of the air-free reagent are allowed to run up into the cup. The blood sample is de- livered from a 2 cc. rubber-tipped, stop-cock pipette ((1) p. 531). Traces of blood remaining in the cup are washed into the chamber with 1 cc. of the reagent, and the stop-cock is sea,led. The chamber is evacuated and shaken for 3 minutes to extract the blood gases.

2. Absorption of CO2 with NaOH.-Mercury is readmitted to


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O2 and CO in Blood

the chamber unt,il the level of the liquid above comes to within a few cc. of the 2.0 cc. mark. 2 cc. of air-free N NaOH are placed in the cup, of which 1 cc. is slowly admitted into the chamber ((1) p. 545). The stop-cock is sealed and pl, representing the total pressure of the gases 02, CO, and Nz, is observed with the solution level at the 2.0 cc. mark.l

3. Transfer of Gases to Hempel Pipette and Absorption of Oxygen. -The Hempel pipette ((4) p. 437) contains alkaline pyrogallate protected by a layer of oil in the upper bulb. A little of the solution is run out to clear the stop-tiock a (Fig. 1) of any air that may be present, then the capillary limbs, I and r, are filled with mercury from the cup G above. 1 cc. of mercury is poured into the cup k of the Van Slyke-Neil1 apparatus, and all air is dislodged from the capillary leading down from the cup to the chamber.

The stop-cock of the manomet.ric apparatus which admits mercury from the leveling bulb to the extraction chamber is opened, and the leveling bulb is raised to such a height, (this will have to be determined by the analyst) that t.he extracted gases will be compressed into a bubble at the top of the chamber at slight posi- tive pressure. The stop-cock is closed, and the leveling bulb set at rest in the uppermost ring, above the chamber.

The free end of the Hempel pipette, with mercury flowing through 1 from the cup c above, is thrust firmly down into the cup k so that the rubber tip fits snugly. Stop-cock a is opened in the position indicated (Fig. 1). Stop-cock b is then opened. At this point, if the internal pressure of the gas bubble has been correctly fixed, a small amount of the gas should run into the capillary limb of the Hempel pipette under its own pressure. The rest of the gas, followed by the blood solution, is forced up into the Hempel pipette by admitting mercury slowly from the leveling bulb into the extraction chamber. As soon as the blood solution has passed slightly beyond the stop-cock a, the latter is turned in a clockwise

1 If the precipitate formed by the interaction of the blood with the acid reagent obscures the meniscus, gentle movement of the chamber by hand will facilitate the solution of the proteins in the added alkali. Reading pr may then be taken over a clear solution. When dealing with darkly colored solutions, a source of light placed in back of the chamber has been found to be of great help in the adjustment of the meniscus to the volume mark. The light should only be used momentarily at the time of adjustment, so that no increase in temperature of the jacket and chamber may occur.


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direction to the closed position shown in Fig. 1, position a, and the Hempel pipette is withdrawn.2

The free arm 1 is cleared of blood solution by the admission of mercury from cup c, and the capillary r, by continued turning of a in a clockwise direction, is likewise cleared of blood solution and gas. The pipette is set aside for the absorption of oxygen. Oc- casional gentle movement of the gas bubble to and fro, or in a horizontal rotatory manner facilitates the absorption, which is complete in 3 to 4 minutes.

Hg. leveling bulb

wagent solution

Position .3 Position b

FIG. 1. Arrangement of apparatus for different stages of transfer of gas between extraction chamber and Hempel pipette.

4. Replacement of Blood Solution by Air-Free Glycerol-Salt Solu- tion in Extraction Chamber.--In the meanwhile, the blood solution is removed from the chamber, which is then flushed two or three times with water. 5 cc. of glycerol-salt solution are then admitted into the apparatus and rendered air-free by shaking the evacuated

z In order to minimize the contamination of the absorbent by the blood solution, which precipitates in the pyrogallate and may even serve to trap gas, it is important to allow as little blood solution to pass into the capillary limb r as possible.


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chamber for 2 minutes. The extracted air is expelled, and 1.5 cc. of the glycerol-salt solution are admitted into the cup k, 3.5 cc. remaining in the chamber.

5. Transfer of CO and Nz from Hempel Pipette to Extraction Chamber.-1 cc. of mercury is poured into the cup 7c, stop-cock a is turned counter clockwise, and the Hempel pipette is placed in position while mercury is flowing from cup c into cup k (Fig. 1, position b). Stop-cock a is then turned counter clockwise to the position shown in Fig. 1. The mercury leveling bulb is placed in its lower position and stop-cock b is opened. The stop-cock connecting leveling bulb and extraction chamber is carefully opened, and, by withdrawal of mercury from the bottom of the chamber, the gas bubble from the Hempel pipette is slowly drawn into the top of the chamber. The minimum possible amount of pyrogallate solution is allowed to flow past the stop- cock a, which is then again turned back to the position indicated in Fig. 1, position b. By careful manipulation, with alternate opening and closing of the stop-cock a, the last portion of gas (and a slight amount of pyrogallate) is completely driven down, followed by mercury from cup c into the chamber, which is then sealed through stop-cock b.

The glycerol-salt solution level is lowered slightly below, then allowed to come to rest at the 2.0 cc. mark where a reading is taken. Due to the slow drainage of the viscous solution, two or three successive readings may be necessary to obtain the constant final reading, to be denoted as p,. When CO is not present in too great amount, greater accuracy in its measurement is gained by obtaining a new base line reading, denoted by ~‘2, at the 0.5 cc. mark. In this work, most of the CO pressure differences have been read at the 0.5 cc. mark.

6. Absorption of CO by Winkler’s Solution.-6 cc. of the air- free Winkler’s reagent are placed in cup Ic. Of this, 5 cc. are slowly admitted into the chamber at slight negative pressure (leveling bulb at height corresponding to the bottom of the chamber ((3) p. 581)). Due to the trace of pyrogallate which has followed t,he gas from the Hempel pipette into the chamber, the introduction of the first few drops of CO absorbent causes a precipitate to form. This, however, upon further addition of the reagent, drops to the bottom of the liquid, leaving the top with a clear meniscus. Absorp-


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J, Sendroy, Jr., and S. H. Liu

tion of CO is complete in 2 minutes. The solution is gently lowered to the 0.5 or 2.0 cc. mark, and the pressure p3 is observed (see Van Slyke and Robscheit-Robbins (2) for precautions).

Determination of Corrections cl and cz.-Before calculating the amounts of oxygen and carbon monoxide from the pressure differences pl - p2 and p’, - p3 (or p2 - &, respectively, it is necessary to apply a correction in each case. Such corrections take into account whatever amounts of air may have been introduced wit.h the reagent and not extracted the first time, and differences in pressure readings which result when either the vapor tension or the volume of the liquid above the mercury is altered.

To obtain the .first correction cl, the procedure previously out- lined is followed. 2 drops of octyl alcohol and 8 cc. of the acidified ferricyanide reagent, are extracted for 2 minutes, the extracted air is ejected, and 4 cc. of the solution are admitted into the cup k. 3 cc. are readmitted into the chamber and the extraction is repeated for 3 minutes. I, cc. of air-free N NaOH is added and the reading p2 made at the 2.0 cc. mark.

The chamber is cleaned and 5 cc. of glycerol-salt solution are extracted for 2 minutes. Following the eject,ion of extracted air, 3.5 cc. of the solution are left in the chamber. Reading pz is then made at the 2.0 cc. mark.

The difference in pressure pl - p2 = cl and represents the correction to be applied to the pl - p2 difference obtained in the analysis of the blood. In this work, the reading at 2.0 cc., over 3.5 cc. of glycerol-salt solution, has been found to be consistently between 1 and 2 mm. lower than the same reading over 8 cc. of the ferricyanide-NaOH mixture. This correction is largely the alge- braic sum of two factors, namely, the increase in 132 over p1 due to the decrease in volume from 8 cc. to 3.5 cc., and the decrease in p2 from pl due to the lower vapor tension of the glycerol-salt solution as compared with the tension of the ferricyanide-NaOH mixture.

To obtain the second correction c2, a reading pf2 at the 0.5 cc. mark is taken with the same 3.5 cc. of glycerol-salt solution, im- mediately after the 2.0 cc. reading. After the addition of 5 cc. of air-free Winkler’s reagent, the reading p3 is taken at the 0.5 cc. mark. When readings for CO are made at the 2.0 cc. mark (which, however, will occur only in analyses of blood of high CO content), the ~‘2 reading is omitted, and pa is read at 2.0 cc., as in the analysis,


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The difference in pressure

P’a - PS (or PZ - ~3) = cz

and represents the correction to be applied to the p12 - 1)s (or p2-- p3) difference obtained in the analysis of the blood. At the 0.5 cc. mark the reading over 5 cc. of Winkler’s reagent has been found to range from 3 to 5 mg. lower than the same reading over 3.5 cc. of glycerol-salt solution. Here also, the correction is the resultant of two factors; namely, the decrease in p3 from ~‘2 due to increase in volume from 3.5 to 5.0 cc., and the increase in pa over p12 due to the higher vapor tension of the glycerol-salt- Winkler’s solution mixture as compared with the tension of the glycerol-salt solution alone.

The analyst should determine these two corrections for each day’s analyses. If a new lot of any reagent is introduced during a series of analyses, the c corrections are redetermined.

Calculations.

The pressure of O2 gas from the sample analyzed is calculated as

PO* = PI - pa - Cl

where the O2 content in terms of volumes per cent or millimols per liter is calculated as

02 content = poZ X factor.

The values of the appropriate factor are taken from Column 9, Table II or Table III, of Van Slyke and Neill’s paper (1) for a sample of 2 cc., S = 7 cc., a = 2.0 cc., and i = 1.0.

The pressure of CO gas from the sample analyzed is calculated as

PC0 = P’a - p3 - ca

where the CO content in terms of volumes per cent or millimols per liter is calculated as

CO content = pco X factor.

The values of the appropriate factor are taken from Column 8 of the same tables, Tables II and III (l), for a sample of 2 cc.,


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S = 7 cc., a = 0.5 cc., and i = 1.0. In some instances, when CO is present in large amounts, it may be necessary to take these readings (pi and p3) at the 2.0 cc. mark, in which case the factors used will be the same as those for oxygen given above.

Estimation of O2 and CO with I Cc. Blood Sample.

When the blood is about half saturated with O2 and half with CO, or when it is necessary to economize in the amount of material used, one may obtain accurate results with blood samples of 1 cc. When one or both gases are present in small amount, there is a decrease in the percentage accuracy with which the less abundant gas is determined, since the absolute error (about 0.001 cc. of gas) remains constant. Whether the resultant error is relatively too great to permit use of the 1 cc. sample depends upon the purpose of the analysis. The amounts of reagents required are less than when 2 cc. samples are used, all readings are made at 0.5 cc., and the c corrections are different.

Briefly, the changes from the technique described for 2 cc. samples are as follows. Instead of 8 cc. of acid ferricyanide, only 5.5 cc. are rendered air-free. Of this, 4 cc. are run up into the cup k, and the 1 cc. sample is introduced into the chamber. This is then followed by 1 cc. of the reagent, thus making 3.5 cc. of liquid to be shaken. Of the N NaOH used to absorb the COZ, only 0.5 cc. is introduced into the chamber. Reading pl is made at 0.5 cc. After the return of the gas from the Hempel pipette, reading p2 is taken over 3.5 cc. of air-free glycerol-salt solution at the 0.5 cc. mark. For the CO absorption, 3.5 cc. of air-free Winkler’s solution are introduced into the cup, of which 2.5 cc. are used for absorption. Reading p3 is also taken at the 0.5 cc. mark. The cl and c2 corrections are detexmined in the manner described in the previous sectibn, the appropriate am0unt.s of reagents being used.

The gas contents are calculated from

02 content = (pl - p2 - cl) X factor

and CO content = (pz - pa - c2) X factor

where the factor to be applied is obtained from Column 6, Table II of Van Slyke and Neill’s tables of factors (1) for sample = 1 cc., S = 3.5 cc., a = 0.5 cc., and i = 1.0.


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Determination of Blood 02 or CO Alone.

Obviously, the procedure described with 1 or 2 cc. blood samples may be used when either O2 or CO content alone is the aim of the analyst. While this method offers no advantage over the original Van Slyke-Neil1 technique for blood 02, for CO it is less subject to the possible errors pointed out by those authors ((1) pp. 563 and 564).

When CO content alone is desired, the use of N NaOH is omitted. The gases liberated by the acid ferricyanide are transferred directly to the Hempel pipette, where COz and O2 are absorbed by the pyrogallol solution. The base line pressure reading pl, with 3.5 cc. of air-free glycerol-salt solution, is made at either the 0.5 or 2.0 cc. mark. The final reading p2, after the CO absorption by Wink- ler’s reagent, is taken at the same volume as that at which pl was read. The appropriate c corrections in each of the above cases are determined as before.

EXPERIMENTAL.

The present method has been rigorously tested and compared with four other different techniques for the analysis of oxygen or carbon monoxide or a mixture of both, in blood. The results given in the following serve to indicate both the relative accuracy of the various methods, and the absolute accuracy of the newly modified procedure. The data presented are representative but not selected results. Of all the analyses done in preliminary tests of the method less than 30 per cent would have to be discarded because of inaccuracies outside the limit of experimental error.

Analysis of Blood for 02 Content Only.

In order to t,est the accuracy of the gas bubble transfer from the extraction chamber to the Hempel pipette and back again, results of blood oxygen content analyses by the new technique were compared with values obtained by the method of Van Slyke and Neill. Table I is indicative of the good agreement obtained.

Analysis of Blood for CO Content Oniy.

For the purpose of comparing results with the Van Slyke- Robscheit-Robbins (2) technique, the experiments grouped in Table II were performed. They indicate good agreement. Even


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when CO is present to the extent of only 4.5 volumes per cent, the comparison of results from 2 cc. samples used in the new technique, with results from 5 cc. samples by the Van Slyke-Rob-

TABLE I.

Results of Analyses of Blood for Oxygen Content Only.

I Volumes per cent 0s by method of:

Experiment No.

-

4

Experiment No.

Van Slyke-Neill.

20.40

24.40 24.35

20.19 20.28 20.33

21.99 21.93

- Average. AVerapt%

20.40

24.38

20.27

21.96

TABLE II.

T

-

Authors.

20.34 20.32

24.28 24.34

20.25

21.97 21.95

20.33

24.31

20.25

21.96

Results of Analyses of Blood for CO Content Only.

Volumes per cent CO by method of:

Van SlykeRobscheit-Robbins.

Average.

8.24

11.59

4.64

Authors.

Average.

8.07 8.07 8.07

11.56 11.56 11.56 4.75 4.72 4.73

scheit-Robbins method, shows no appreciable difference between the two sets of determinations.

The reliability of the method for CO absorption was tested in


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O2 and CO in Blood

another way. A portion of fresh ox blood was placed in the double tonometer system used in this laboratory (5). The system was evacuated and refilled with hydrogen three times, then equilibrated by rotation for 20 minutes at room temperature. The process of blood reduct,ion and elimination of oxygen was repeated. Following this, a calculated amount of pure CO gas made from formic acid was added to the system. A low tension of 10 or 12 mm. was sufficient to saturate the blood thoroughly with CO while the amount physically dissolved at this pressure was so small (0.02 to 0.03 volume per cent) as to be negligible. The blood and gas phase were again allowed to come to equilibrium, after which the blood was analyzed for CO according to directions given in preceding sections.

Another portion of blood was evacuated and equilibrated the same number of times as the first, except that air was used as the gas phase. The 02 capacity was finally estimated by analyzing for oxygen according to Van Slyke and Neil1 (1) and subtracting the physically dissolved O2 according to the equation

(1) B-W Dissolved 02 (vol. per cent) = 760 X 20.9 X ao2 X 0.84

where B - W represents the barometric pressure minus the water vapor tension at the temperature of saturation and a!oZ is the Bunsen solubility coefficient at the same temperature, for oxygen in water. The numerical constants 20.9 and 0.84 are respectively the percentage of oxygen in air, and t,he approximate water content of blood.

The combining capacity of the blood for O2 and CO should be the same. Table III shows the results of two such experiments. In Experiment 1, it may be added, further analyses indicated an oxygen content of 0.25 volume per cent, incomplete reduction of the sample saturated with CO thus accounting for the sliahtlv lower CO results.

Finally, the CO content of a sample of blood thoroughly saturated with that gas was confirmed in still another way. The blood was equilibrated with an atmosphere of almost pure CO, then put aside in a closed vessel over mercury. 2 cc. samples were withdrawn for analysis according to the method described in this paper. After analysis, the amount of CO mesh-acted in the course


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of the analysis was calculated by multiplying the total CO by the appropriate factor for unextracted gas obtained from the equa- tions developed by Van Slyke and Stadie (6). At equilibrium,

TABLE III.

Comparison of 02 and CO Blood Capacities.

Volumes per cent gas by method of:

Van Slyke-Neill. I Authors.

Experiment No.

Average.

1 22.36 22.12 22.32 22.18 22.15

2 19.67 19.81 19.71 19.71

TABLE IV.

Determination of CO Extracted from Blood in Van Slyke-Neil1 Apparatus.

Volumea per cent co.

Total by authors’ method. Calculated. Extracted, analysis by 1206 method.

j Average. 1 Unextraoted. 1 Extracted. 1 1 Average.

yl 22.60 / 0.09 / 22.51 1 ii::; / 22.77

the ratio of unextracted gas to the total gas present is defined by the equation

(2)

A cd A-S

A a

l+A-S

where A = volume of gas phase, S = volume of liquid phase, and CY ’ = the Ostwald solubility coefficient at the prevailing temperature. To determine the accuracy of the results, several other samples were extracted in the evacuated chamber of the Van Slyke


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apparatus. In each case, after extraction, the stop-cock b (see Fig. 1) of the chamber was opened to dilute the extracted CO with outside air. The gas in the chamber was then completely transferred through the capillary side arm S of the apparatus through stop-cock b, by displacement with mercury, into an 800 cc. partially evacuated tonometer. Air was admitted into the tonometer to atmospheric pressure. Following this, the gas was displaced by glycerol-salt solution and passed over hot 1205. The iodine liberated by the CO was absorbed in KI solution and

TABLE V.

Comparison of Results of Combined Analysis of Blood for Both On and CO by Method of Van Slyke and Robscheit-Robbins and by That of the Authors.

“::2- NO.

1

2

3

4

5


Van Slyke-Robscheit-Robbins.

01

11.22

10.78 10.74

5.71

15.40 15.54

5.85 5.74

-

-

i-

i-

bverage.

11.22

10.76 5.71

15.47

5.80

co

12.14

11.22 11.45

6.21 6.16 5.42 5.46 6.05 6.20

-

_

-

Average.

12.14

11.34

6.19

5.44

6.13

Authors.

02

P -

11.37 11.27 10.54 10.56

5.61 5.63

15.56 15.47

5.65 5.56

-

I iverage --

11.32

10.55

5.62

15.51

5.60

CO

12.02 11.94 11.42 11.32

6.32 6.36 5.46 5.48 6.26 6.31

iverage.

11.98

11.37

6.34

5.47

6.29

then titrated with sodium thiosulfate. More complete details as to the procedure employed will be given in a later publication from this laboratory. Table IV shows that CO, thus determined by an entirely independent method of measurement, agrees very well with the results obtained by the manometric technique.

Combined Analysis of Blood for 02 and CO Conhat.

The accuracy of the procedure having been tested against other methods with respect to one or the other of the two gases, experiments were performed for the purpose of confirming results by the


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combined technique. The first of several series of such experiments is recorded in Table V, where comparative results with respect to the Van Slyke-Robscheit-Robbins method are given. In view of the fact that the latter method was not designed for oxygen analysis, and its use for that purpose introduces several

TABLE VI.

Comparison of Results of Combined Analyses of Blood for Both 01 and CO by Several Different Methods.

“El? No.

1

2

3

Blood 8ZilI&.

02 + co

02

1A:lBt

02 + co

02

1A:lBt

02

co 1A:lBt


-

Ot 02 co ---

0.41 18.98 0.32 18.97

18.97 19.08

0.26 22.64 0.20 22.69

22.64 22.50

23.79 23.71

11.85 12.54 11.93 12.55

02

11.3 11.31

11.81 11.8!

11.3! 11.52

12.5: 12.5t

Calculated.*

01 co

9.6E 9.49

ll.K 11.33

11.88

* 02 values calculated from Van Slyke-Neil1 and Van Slyke-Robscheit- Robbins analyses. CO values calculated from Van Slyke-Robscheit- Robbins analyses.

t 1 part A + 1 part B.

extra steps in the published procedure, the agreement in values is quite satisfactory.3

8 The additional steps involved in analyzing blood for oxygen by the Van Slyke-Robscheit-Robbins method were the following. After the extraction of the blood gases, 2 cc. of air-free N NaOH were added to absorb


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Table VI gives comparative results, analyzed and calculated, for experiments with mixtures of ox blood saturated with CO and with 02, the bloods having been analyzed for CO or 02 before and after the mixture was prepared. From the preliminary analyses by the Van Slyke-Neil1 and Van Slyke-Robscheit-Robbins methods, values of O2 and CO for the mixture were calculated. The agreement of the analytical data with the calculated values is as good as could be expected considering the number of steps involved in preparing the final mixtures.

The most rigorous test of the accuracy of results by the combined method is that based on the principle of the identity of the oxygen- and carbon monoxide-combining power of a given sample of blood. When blood is equilibrated with an atmosphere containing either O2 or CO at a tension sufficient to have all of the available hemoglobin combined with gas, the amount of 02 and CO combined in either case will be identical. The amounts of dissolved 02 or CO will depend on the tension of the respective gases, as indicated by Equation 1 where CO may be substituted for 02. As shown by Sendroy, Liu, and Van Slyke (7) the tension of CO

required to saturate blood with that gas will be but k0 part of the

tension of O2 required to combine all the blood hemoglobin with oxygen. In the one case, therefore, one may have the blood fully combined with CO, with a negligible amount of dissolved CO present, while in the other, when the blood is fully combined with 02, a correction must be made for dissolved gas.

Table VII indicates the results obtained by analysis of mixtures of blood, the amounts of O2 or CO in which could be calculated from O2 or CO capacity data. Thus, for instance, in Experiment 1 (Table VII), a certain portion of blood (Sample A) was saturated with air, and analyzed for O2 as in the regular 02 capacity method

COz, and the blood solution was removed from the Harington-Van Slyke chamber through the lower stop-cock. The chamber was washed once with air-free glycerol-salt solution. This was rejected and then replaced by another 5 cc. over which the reading at the 2.0 cc. mark was taken. The difference between this reading and the subsequent one following the absorption of oxygen by pyrogallate gave the pressure due to OZ. A new table of factors similar to that given by Van Slyke and Robscheit-Robbins was calculated for use in oxygen analyses.


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TABLE VII.

Comparison of Results of Combined Analyses of Blood for CO and 01 Compared with Values Calculated from 02 Capacity Results.

Experi merit

NO. Blood sample.

A, ,saturated with air.

B, saturated with CO at 10

C z?parts A + 1 part B.

A, saturated with air.

13, saturated with CO at 10

C z? part A + 1 part B.



C rf? parts A + 1 part B. D = 5 parts A + 2 parts B.



C “?5 parts A + 2 parts B. D=5partsA+lpartB.


B, saturated with air containing CO.

C= lpartA+5partsB.


B, saturated with CO at 10 mm.

C= lpartA+2partsB. D = 1 part A + 5 parts B.

- I Volumes per cent g&a by

method of:

‘an Slyke Neill.

02

20.3& 19.84c

20.34t 19.82c

20.65t 20.16~

21.76t 21.26~

22.59t 22.09c

19.45t 18.98c

Authors.

02 co --

19.9:

15.06 5.0:

19.8:

10.19 9.9;

17.20 3.4: 14.79 5.71

19.10 2.51 17.90 3.6:

0.33 21.6!

4.21 17.9f

6.59 12.5: 3.26 15.5!

-

Calculated:

0% co

15.25

19.84

4.96

19.82

9.92

17.21 14.75

20.16

3.36 5.76

19.20 18.13

21.26

2.50 3.55

18.06

18.98

6.49 12.65 3.24 15.81


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TABLE VII-concluded.

“;:z? NO.

co I I 01 CO 02 -/- l-

7 A, saturated with air. 22.07t 21.57~

Blood sanple.

B, saturated with CO at 10 21.57t

C?partsA + 1 part B. 14.54 7.1: 14.71

t = total gas. c = combined gas. *02 values calculated from total 02 according to Van Slyke-Neil1

method _ CO values calculated from combined 01 values.

7.19


JanSlyke- Hi&r. Authora.

-

Calculated.

co

of Van Slyke and Neill. The total 02, by analysis, was 20.36 volumes per cent. The calculated dissolved 02 was 0.52 volumes per cent, thus making the OS-combining power of the sample equal to 19.84 volumes per cent. Hence, the CO-combining power of the same blood, when exposed to an atmosphere containing CO at sufficient tension, should be identical with this value.

Accordingly, a second portion of blood (Sample B, Experiment 1) was saturated with an atmosphere containing enough CO to combine completely with the amount of blood present and to have in excess an amount which would make the CO tension at equilibrium 10 mm. of mercury. The dissolved CO was therefore negligible.

The saturation of the blood in each case was made at room temperature and repeated to make three saturations in all, according to the technique of Austin et al. (5). Hence, the concentration of the blood, due to the several evacuations of the tonometers, was the same in each.case.

Blood (Sample B, Experiment 1) was analyzed for CO according to the new technique and the results (19.93 volumes per cent) checked well with the calculated value (19.84 volumes per cent) obtained from the Van Slyke-Neil1 analysis for 02 capacity.

Definite volumes of blood (Samples A and B) were then accu- rately measured out and mixed, 3 parts by volume of Sample A to


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one of Sample B, and the analyses carried out by the combined technique. Again the results were compared with the calculated values.

In Table VII, there has been included, for convenience in pre- sentation, an experiment somewhat different from the preceding ones. In Experiment 7 the procedure employed was the reverse of that in the others, in that the CO capacity, as estimated by the

TABLE VIII.

Comparison of Combined Analyses of Blood for 02 and CO with 1 Cc. and 8 Cc. Samples.

Experi- ment

No.

T Volumes per cent.

02

1 cc. sample.

10.80 10.71 10.91

16.05 16.16

11.13 11.00

19.07 18.72 18.88

Average.

10.03

10.81

16.10

11.06

18.89

.-

-

2 cc. sample.

10.11

11.01 10.95

16.01 15.96

10.97 10.92

18.89 18.87

hversge.

10.11

10.98

15.99

10.95

18.88

T _I-

_-

-

co

1 CD. sample.

1

11.09

11.77 11.70 11.65

6.17 6.15

11.56 11.62

3.18 3.20 3.18

~vverage

11.09

11.71

6.16

11.59

3.19

_-

-

2 cc. sample.

11.01

11.71 11.73

6.18 6.19

11.76 11.78

3.12 3.15

- Lverage.

11 .Ol

11.72

6.18

11.77

3.14

method of Van Slyke and Hiller (S), was used as the basis of cal- culation of O2 values. Actually, blood Samples A and B were equilibrated as before, with air and with a low tension CO + Hz mixture. The blood mixture (Sample C) was prepared and kept over mercury. After analysis for O2 and CO by the combined technique, samples of the same mixture were employed for the estimation of the CO capacity within the apparatus. Experiment 7 thus constitutes another independent confirmation of the values


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0, and CO in Blood

given by the modified technique. The agreement of calculated and analyzed values in this table is within the limit of error to be expected in the preparation by volume of blood mixtures such as these.

Analysis of i Cc. Samples.

In order to determine the relative accuracy of results obtained by reducing the size of the blood sample to 1 cc., the results given in Table VIII were obtained. Apparently, for the mixtures here used, 1 cc. and 2 cc. samples give results agreeing within the limit of error of the method.

SUMMARY.

An improved technique is described for the determination of oxygen and carbon monoxide in a single blood sample by the use of the Van Slyke-Neil1 manometric apparatus.

BIBLIOGRAPHY.

1. Van Slyke, D.D., and Neill, J. M., J. Biol. Chem., 61, 523 (1924). 2. Van Slyke, D. D., and Robscheit-Robbins, F. S., J. Biol. Chem., 72, 39

(1927). 3. Harington, C. R., and Van Slyke, D. D., J. Bid. Chem., 61, 575 (1924). 4. Van Slyke, D. D., J. Biol. Chem., 83,425 (1929). 5. Austin, J. H., Cullen, G. E., Hastings, A. B., McLean, F. C., Peters,

J. P., and Van Slyke, D. D., J. Biol. Chem., 64, 121 (1922). 6. Van Slyke, D. D., and Stadie, W. C., J. BioZ. Chem., 49,1 (1921). 7. Sendroy, J., Jr., Liu, S. H., and Van Slyke, D. D., Am. J. Physiol., 90,

511 (1929). 8. Van Slyke, D. D., and Hiller, A., J. BioZ. Chem., 78,807 (1928).


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Julius Sendroy, Jr. and S. H. LiuBLOOD

OXYGEN AND CARBON MONOXIDE IN GASOMETRIC DETERMINATION OF

1930, 89:133-152.J. Biol. Chem.

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