THE EFFECT OF CO2 AND 02 CONTENT OF THE BLOOD ONTHE FREEZING POINT OF THE PLASMA.1 By GIACOMOMESCHIA 2 and DONALD H. BARRON. From the Yale UniversitySchool of Medicine, New Haven, Connecticut.

(Received for publication 318t October 1955)

INTRODUCTION

DURING the course of an investigation on the freezing point of theplasma of the foetal rabbit in comparison with that of the maternal,we observed that, in the maternal circulation, the freezing point ofthe arterial plasma was higher than that of the venous (inferior vena cavadistal to the renal veins), the difference being about 0*003° C. Thelower freezing point of the venous blood appears to be an effect of theexchange of substances that takes place in the capillaries between bloodand tissues and, in our particular case, between fcetal and maternalbloods in the uterus.

Of all the substances exchanged between mother and foetus, 02 andCO2 would appear to be quantitatively the most important. Ninety-fiveto 97 per cent of the 02 iS carried by the blood combined with thehamoglobin and it is therefore osmotically inactive. The CO2 is carriedby the blood mainly as bicarbonate, i.e. as an osmotically active sub-stance. This simple consideration suggests that when the blood loses02 and takes up C02, the net effect would be an increase of concentrationof the osmotically active substances of the blood. Passing throughthe tissues the blood usually gains from 1 to 2 millimols of CO2 per kg.of water and loses about the same amount of 02. One millimol ofideal solute in 1 kg. of water lowers the freezing point by 0-00186.Assuming (i) that the loss of 02 does not change the osmotic pressureof the blood, (ii) that all the CO2 gained is osmotically active, it followsthat the freezing point of the venous blood ought to be 0.00190 to0.0037° C. lower than that of the arterial blood. This lowering corre-sponds in order of magnitude to the difference found experimentallybetween arterial and venous plasma.

1 Aided by grants from the Division of Research Grants and Fellowships,National Institutes of Health, U.S. Public Service, and the Medical Fluid ResearchFund of Yale University.

2 Toscanini Fellow. Present address: Laboratorio di Fisiologia, via Mangalli 32,Milano, Italy.

180

C02 and 02 of Blood and Freezing Point of Plasma

That the addition of CO2 to blood increases its osmotic pressurehas been demonstrated by Margaria [1931], who measured the vapourpressure of samples of blood exposed to different C02 pressures. Theactual arterial-venous difference in osmolality and the effects thatoxygenation and reduction may have on the freezing point of theplasma have not previously been measured, as far as we know. There-fore it seemed worth while to investigate the subject further in viewof the possible physiological implications, and accordingly some experi-ments were performed in vitro and in vivo in order to estimate:

(i) The effects that changes in C02 and 02 content of the bloodhave on the freezing point of the plasma.

(ii) The order of magnitude of the difference in freezing point betweenarterial and venous plasma.

In the present paper an account is given of the experiments performedin vitro, together with a discussion of the biochemical implications.An account of the experiments in vivo and a discussion of the physio-logical implications will be given in a following paper.

The osmotic pressure is expressed in osmolalities m'. The relation-ship between osmolality and freezing point depression, At, is defined bythe following equation:

m' =At/1.858. . . . . . (1)To avoid the use of many decimals, the osmotic pressure will be givenalso in terms of milliosmols per kg. of water. A solution having anosmolality of one has one thousand milliosmols per kg. of water.

EFFECT OF C02 CONTENT OF BLOOD ON FREEZING POINTOF PLASMA

The choice of variables has been made so that the results will havethe broadest possible meaning. If a sample of blood were taken andthe osmolality of plasma studied as a function of the C02 concentrationof the plasma itself, the relationship between the two variables wouldbe precise only for the given set of circumstances, for the C02 concentra-tion of the plasma depends upon the Donnan distribution of the diffusibleions across the red cell membrane, which in turn depends upon thepH and the haemoglobin, K and Na concentrations [Van Slyke, Wu andMcLean, 1923]. The decision to study the osmolality of plasma as afunction of the C02 content per kg. of water of the blood was basedon the consideration that a simple relationship might exist betweenthe two variables which is independent, within physiological limits,of the values of other variables such as pH, heemoglobin, K and Naconcentrations. This relationship would then be of a more generalapp[ication.

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The assumptions that must be made in order to obtain a simplerelationship between freezing point of the plasma and total C02 of theblood are:

I. The osmotic pressures inside and outside the red cells are equal.The protein concentration of the red cells is much larger than that

of the plasma. If the cell membrane were impermeable to the proteinsbut permeable to all other ions, the unequal distribution of ions due toDonnan equilibrium would cause the osmotic pressure inside the redcells to be far greater than that outside the cells, so much so, in fact,as to rupture the cells. It is the impermeability of the red cell membraneto Na and K, a steady state maintained by the metabolic activity ofthe red cells, which prevents the establishment of a higher internalosmotic pressure [Wu, 1926]. The water within the cells has thesame thermodynamic activity as that in the plasma, and the two phasesare at the same hydrostatic pressure.

II. The addition of C02 to the blood, within the limited range of pHconsidered, from 7 to 8, does not affect the osmolality of the othermolecules present in the blood. It is assumed, in other words, thatthe addition of CO2 does not cause any association or dissociation ofother molecules present in solution, nor does it affect appreciably thevalue of any osmotic coefficient. The hydrogen ion concentration isincreased by the addition of C02, but the order of magnitude being10-7 to 10-8 mols per litre, any change in it does not affect the osmolalityof the blood in a measurable amount.

III. The osmotic coefficient, i, of C02 is equal inside and outside thered cells. In the case of electrolytes, the osmotic coefficient is theratio between osmolality (m') and the product of the molal concentration(m) times the number of cations and anions produced by completedissociation of one molecule of electrolyte, n:

=M'/n.m. . . . (2)For highly diluted solutions the value of f tends to be one, whereasfor a moderately concentrated solution of electrolytes, like the blood,we can reasonably expect, from what is known about the physico-chemical properties of electrolytic solutions, to find it less than one,since 90 to 95 per cent of the C02 present in the blood is present asbicarbonate ion, i.e. as a monovalent electrolyte. From the C02molecule n is equal to one and is therefore omitted in the followingequations. The symbols used are defined below:

a=that part of the osmolality (m') due to substances otherthan C02,

(CO2) =mols of C02 per kg. of water,and subscripts c, p and b meaning in cells, plasma and blood

respectively.

182

CO2 and 02 of Blood and Freezing Point of Plasma

By definition:mir=ac + b(CO2), * (3)Mt =aV + O(CO2) VI . (4)mIb =ab + (CC2)b- (5)

From the second assumption:ab = constant. . . . . . (6)

From the first, second and third assumptions:m',= constant + k(CO2)b. (7)

It appears that, as long as b is constant, the osmolality of the plasmashould be a linear function of the C02 content per kg. of water of theblood.

METHOD

The blood of a ram was used for all the experiments, with theexception of one case in which the measurements were performed onthe blood of a goat. In four of five experiments the blood was mixedwith a solution of sodium fluoride and potassium oxalate (NaF 6.8 g.and K2C204 .H20 22 g. in 1 litre of distilled water), in the proportionof one part of the solution to four parts of the blood, in order to reducethe rate of glycolysis during the time the blood was kept at 380 C.,because it was thought that a too active glycolysis might change theosmolality of the blood. However, it was later found that the bloodcould be kept for two hours at 380 C. without any appreciable changeof the osmolality, despite a change in pH from 7-47 to 7 40, whichcorresponds, by a rough estimation of the buffering power of the blood,to the production of about two milliequivalents of acid per litre. Thelast experiment, therefore, was performed on heparinized blood.

For each experiment four tonometers (capacity 250 millilitres) werefilled with mixtures of air and C02 at different pressures (c. 0, 20, 40and 60 mm. Hg respectively), and then 12 millilitres of blood wereintroduced into each tonometer. The tonometers were rotated in awater bath at 380 C. for 40 minutes. After equilibration the bloodsamples were withdrawn into syringes under oil and divided into twoparts: about 3 millilitres of blood were kept in the syringes, while therest was transferred under oil into centrifuge tubes and centrifugedat 380 C. After separation of the cells from the plasma, the plasma wascollected under oil in syringes and stored at 40 C. On the whole bloodthe C02 content was measured by the Van Slyke-Neill apparatus, andthe water content by desiccation at 1150 C. to constant weight. Thefreezing point of the plasma was measured with the Fiske osmometer.1With this apparatus the determination of the freezing point is carried

1 Built by the Fiske Associates, Inc., 44 Bromfield Street, Boston 8, Mass. Theapparatus was purchased with a grant from the New Haven Heart Association.

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out on a 2 ml. sample, contained in a test-tube immersed in a thermo-static bath kept at - 5° C. Temperature is measured by means of athermistor, and freezing is induced by vibrating a steel rod when thesolution has reached a fixed temperature of supercooling. Since thetemperature measured is not the exact cryoscopic point, the apparatusis standardized with solutions of NaCl of known freezing point. Oncethe sample is frozen, it can be thawed and frozen again, so that on asingle 2 ml. sample many measurements can be carried out to obtainan average figure that is reliable within ±0.5 milliosmols per kg. ofwater. The standard deviation of the mean value of successive measure-ments carried out on the same sample is ±0 25 milliosmols/kg. of water.

During the measurement of the freezing point the samples of plasmawere exposed to the air, and therefore there was a loss of CO2 from thesamples equilibrated with higher pressures of CO2. To minimize thisloss, the samples of plasma were cooled to about 40 C. and transferreddirectly from the syringe to the bottom of the test-tube through along needle and immediately thereafter cooled to the freezing point.To estimate the quantity of CO2 lost, the CO2 contents of the plasmabefore and after exposure to the air were measured with the Van Slyke-Neill apparatus. It was assumed that the loss of x millimols of C02from 1 litre of plasma would decrease the osmolality of plasma ofx milliosmols per kg. of water, and therefore from the amount of CO2lost during the exposure the original value of osmolality was calculated.

Suppose that the osmolality of plasma given by the osmometerwas 300 0 milliosmols per kg. of water, and that the plasma had lost0-5 millimols per litre: we assume that the osmolality of the unexposedsample was 300 0 + 0 5 = 300 5 milliosmols per kg. of water. Thecalculation is somewhat arbitrary, since we do not know the exactvalue of the osmotic coefficient of the CO2 lost, and some of the CO2could have been lost in removing the solution from the test-tube afterthe freezing point determination; but fortunately the quantity of C02lost never exceeded 1 1 millimols per litre (see Table II), so that evenan error of 50 per cent in the estimate does not affect the reliability ofthe results.

RESULTS

The results are tabulated in Table I. The osmolality of bloodtreated with NaF and oxalate is greater than that of the heparinizedblood, because the NaF-oxalate solution used was hypertonic. Whenthe change in osmolality given by a change in C02 content of the bloodis considered, the results appear to be fairly consistent. To obtain arepresentative value for the osmotic coefficient of CO2 out of all theexperimental data, the change in CO2 content expressed in mM per kg.of water of the blood, A(CO2) - 103 has been plotted against the change

184

CO2 and 02 of Blood and Freezing Point of Plasma 185

Animal Expt.

Ram 18.10.54

Ram 19.10.54

Ram 21.10.54

Goat 22.10.54

Ram 2.2.55

12 -

10

8-

1')0 6

E 4

2

TABLE I

Blood, OsoaiyPlasma, smalt (0)A'02 capacity02 OsmoraditY C2 lOSt Osrmrlatd 10 . 10C volume

mM/kg. H20 raig / cretd 10 0 per cent

12-5 *3633 0-0 *3633 0-0 0.0 13*219-7 *3692 0-3 *3695 7'2 6-222*1 *3717 0-6 *3723 9*6 9-024'9 *3732 1-1 *3743 12'4 11-0

12-8 *3649 0.0 *3649 0-0 0.0 12-516*6 *3687 0.0 *3687 3-8 3-819*9 *3717 0.1 *3718 7*1 6-926-2 *3765 0-6 *3771 13-4 12*2

9.7 *3674 0-0 *3674 0-0 0.0 12-814*7 *3714 0.0 *3714 5-0 4-019'7 *3765 0-0 *3765 10'0 9.123-8 *3787 0-9 *3796 14*1 12*2

11-5 *3572 0-0 *3572 0-0 0-0 12*616-5 *3617 0*1 *3618 5*0 4*620-0 *3643 0-6 *3649 8-5 7-723-3 *3669 0-8 '3677 11-8 10-5

15'5 *2889 0-2 *2891 0-0 0.0 14'420-0 *2929 0-5 *2934 4'5 4-324-4 *2962 0-8 '2970 8-9 7.927*7 *2988 1.0 *2998 12*2 10*7

0

0 2 4 6 8 1o 12 14A (CO2) * 103

FiG. 1.-The relationship between the change in the C02 content of the blood,A(CO2) * 10', expressed in mM/kg. H20, and the change in millOSmolality Am'. 103l

Meschia and Barron

in milliosmolality, Am' - 103, as shown in fig. 1. The slope of the linedrawn through the experimental points has been calculated accordingto the statistical formula:

= EA(CO2) x 'AM'Z( (CO2))2 (9)

The formula gives the best fitting line for cases in which it may beassumed that the relation between the two variables is linear, thatthe straight line must pass through the origin, and that m' alone isliable to error [Worthing and Geffner, 1943]. The value of b is 0 9,which means that for each millimol of CO2 added to 1 kg. ofwater of the blood, the osmotic pressure of the plasma rises by0 9 milliosmolalities.

DIscuSSION

The osmotic coefficient for solutions of monovalent electrolyteshaving an ionic strength approximately equal to that of the blood isabout 09. A solution of NaCl for instance, at ionic strength 016, hasan osmotic coefficient equal to 0-925 [International Critical Tables,1928]. The value of 0 9, that we have found for the CO2 in the blood,seems therefore to find its explanation in the ionic strength of theblood and in the fact that CO2 is mainly present in it as a monovalentelectrolyte. The assumption that despite the great complexity of thesystem, the effect of the CO2 content of the blood on the freezing pointof the plasma can be explained in rather simple terms, within the limitsof pH and CO2 concentration considered in this paper, appears to beconfirmed by the experimental results. It has to be recalled, however,that in this series of experiments the oxygenation of the heemoglobinwas kept constant. In the organism CO2 uptake and 02 delivery takeplace simultaneously most of the time, so that in order to have a completepicture of what happens in vivo, we must know the effects that oxygena-tion and reduction may have on the freezing point of the plasma.

EFFECT OF OXYGEN CONTENT OF BLOOD ON FREEZING POINTOF PLASMA

Oxygenation and reduction of heemoglobin affect the bufferingpower of the blood. In a solution of horse haemoglobin, for instance,it has been found [Hastings, Van Slyke, Neill, Heidelberger andHarington, 1924] that at pH 7-3 the change of one mol 1 of oxyhoemo-globin to reduced haemoglobin enables the haemoglobin solution totake up about 0 7 mols of CO2 without change of pH. This reversible

1 Molecular weight 17,000. This is the molecular weight for gram-atom of iron,i.e. one-quarter of the molecular weight obtained by physico-chemical methods.

186

CO2 and 02 of Blood and Freezing Point of Plasma

change in buffering power is produced by some mechanism which hasstill to be clearly established. Henderson [1920] advanced the hypo-thesis that the oxygenation of the hwmoglobin affects the dissociationconstant of an acid group of the hawmoglobin, so that the oxyhaemoglobinbecomes a stronger acid than the reduced one. One implication ofthis hypothesis is that the enhanced buffering power of the reducedhaemoglobin is not limited specifically to the 002, but extends to otheracids as well, since the effect of oxygenation and reduction would beprimarily a change in the hydrogen ion concentration. This hypothesiswas supported by the titration of oxygenated and reduced haemoglobinwith HCI [Hastings, Sendroy, Murray and Heidelberger, 1924], for theadditional quantity of HCI that the reduced hwsmoglobin took up atconstant pH was equal to the quantity calculated from the results ofthe C02 titration experiments.

A second implication is that the oxygenation and reduction of thehaemoglobin should affect only the thermodynamic activity of thehydrogen ions, leaving unchanged that of other ions, for instancebicarbonate and chloride ions. We are not aware of any researchattempting to measure the activity of the chloride ions in presence ofoxygenated and reduced haemoglobin, but some evidence exists thatthe oxygenation and reduction of the haemoglobin affect the physico-chemical status of C02 in the blood. A detailed account of this evidencehas been given by Roughton [1935] and only a brief outline of it needbe given here.

The apparent first dissociation constant, pK',, of the carbonic acid,obtained by measuring the total C02 concentration, the C02 dissolvedand the pH, has been found to be lower in a solution of reduced hsemo-globin than in a solution of oxyhwmoglobin [Stadie and Hawes, 1928;Margaria and Green, 1933]. Stadie and Hawes [1928], from theirmeasurements on solutions of hwemoglobin, calculate the pK'1 in thered cells to be 5-98 and 5-87 for the oxygenated and the reduced staterespectively. According to Margaria and Green [1933], the lower pK',of carbonic acid in solutions of reduced hwmoglobin is due to theenhanced ability of reduced heamoglobin to combine chemically withC02. Ferguson and Roughton [1934] have shown that, by applyingthe chemical method of Faurholt [1925] for the determination of carb-amino compounds to solutions of hawmoglobin and C02, it is possible todemonstrate that the reduced hwmoglobin binds more C02 than theoxygenated. If the evidence for the formation of a CO2-hwemoglobincompound following the reduction of hwmoglobin is accepted, it remainsto be explained why the buffering power of reduced hemoglobin isequal for C02 and hydrochloric acid and not greater for C02. Toovercome this difficulty, it has been assumed that the carbonic acidbound to the heemoglobin has still an acid function, or that the reducedhwemoglobin is able to bind in larger quantity than oxyhaemoglobin not

VOL. XLI, No. 2.-1956 13

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Meschia and Barron

only CO2 but chloride ions as well [Roughton, 1935]. The measure-ment of the osmolality of the plasma of reduced and oxygenated bloodoffers another way to test the validity of the Henderson hypothesis.If the oxygenation and reduction of hemoglobin affect only the thermo-dynamic activity of the hydrogen ions, leaving that of the other ionsunchanged, since the concentration of hydrogen ions is of the order of10-7 mols per litre, we would expect oxygenation and reduction to haveno effect on the osmolality of the blood. The oxygen, per se, has anegligible osmotic effect, since only 0-1 to 0-2 millimols of oxygen aredissolved in one litre of blood and the remainder is chemically boundto the haemoglobin. If, on the contrary, the oxygenation and reductionof the haemoglobin have a measurable effect on the thermodynamicactivity of other molecules, either by changing the value of the osmoticcoefficient or by formation of a chemical bond, as seems to be the casefor C02, we should be able to demonstrate that the oxygenation andreduction of the heemoglobin affect the osmotic pressure of the blood.

METHOD

The blood was taken from the same ram that was used for theexperiments of the preceding section. One part of the fluoride-oxalatesolution was added to four parts of blood. For each experiment fourtonometers were filled with air plus CO2 at the approximate pressuresof 0, 20, 40, 60 mm. Hg respectively, and four other tonometers werefilled with nitrogen plus CO2 at the approximate pressures of 0, 20, 40,60 mm. Hg. In each tonometer 12 millilitres of blood were equilibratedwith the gas mixture for 40 minutes and then analysed for water,C02, 02 content of the whole blood, CO2 content and freezing point ofthe plasma according to the technical procedure previously described.The CO2 pressure with which each sample of blood was equilibratedwas obtained by measuring the percentage of CO2 in the gas mixturewith the Haldane apparatus and the total pressure of the gases. ThepH of the plasma was calculated by assuming the pK'2 of the carbonicacid in the plasma at 380 C. to be equal to 6*10.

RESIULTS

The results are given in Table II. The osmotic pressure is givencorrected for the CO2 lost by the plasma during the analysis (see Methodin preceding section). Since samples of plasma having approximatelythe same pressure of CO2 lost approximately the same amount of C02,the correction does not affect the results as far as the comparison ofoxygenated and reduced blood is concerned. Figs. 2 and 3 are drawnaccording to the data of experiment 18.2.55. In fig. 2 the milliosmolality

188

C02 and 02 of Blood and Freezing Point of Plasma

TABLE IIBlood

Experiment N2fkg. H20 CO2fkg. H20mM mM

9.2.55 12 11*81.1 17-90.5 22.30*6 25-07-8 11-07*5 15*07-5 19*97*3 23-2

15.2.55 1.0 13-41-2 20*11.1 24-40*9 27*17-2 12*27-6 18-27-2 22-87-6 25-7

18.2.55 1.0 13.71.0 19.30-7 24-00-6 26*46-5 12-96-8 16-76-3 22-06*0 25-2

36o0

0

cn0

E

3554

350-

PlasmaOsmolality

*3470*3529

*3587*3475*3515*3564*3585*3490*3544*3589*3611*3500*3546*3586*3618*3489*3537*3579*3596*3498*3535*3580*3602

C0211itremM11-1

20:623-110*915-619-621-812-318-522-524-411-717-021-624*712-718-021-924-812-216-221.124-0

CO2 pressuremm. Hg

6*319*535*652*15.3

15-735.351-46*5

21 138-554.78-0

21-743-662-46-5

20-440-458-17.9

18-840-961-4

pH

7-86

7.347-237.937*607.337*217-897.557*367-247-787-507*297*187*907.557.337-227-807.547-317*17

A Hb =5.4 mM/Kg water

G) Reduced blood0 Ox yenafed blood

I -I I I10 15 20 25 30

CO2 (m M per Kg of wa+er)FIG. 2.-The relationship between the CO2 concentration, mM/kg' H20,

and the milliosmolhlity of plasma in oxygenated and reduced blood.

189

190 Meschia and Barron

of plasma is plotted against the C02 concentration, expressed in millimolsper kg. of water of the blood. The points representing the samples ofoxygenated blood are disposed along a straight line having a slope of0.9, in agreement with the preceding series of experiments. The pointsrepresenting samples of reduced blood are located along a straightline having the same slope but shifted to the right by about 1-9 millimols

25-

-s20

015

)

E

O @ Reduced blood* Oxygenca+ed blood

0 10 20 30 40 50 60 70

pCO2 (mm Hg)FIG. 3a.-The CO2 concentrations in oxygenated and reduced blood are

plotted against the CO2 pressure of equilibration.

360 -

+' 355._

03 5

0

345-E

3 Reduced blood* Oxygernafed blood

0 l0 20 30 40 5o 60

pCO2(rnrnnH8)FIG. 3b.-Osmolality of oxygenated' and reduced blood is related to the

C02 pressure of equilibration.

CO2 and 02 of Blood and Freezing Point of Plasma

of CO2 per kg. of water. The meaning of the graph can be summarizedby saying that the reduced blood was able to take up 1-9 millimols ofCO2 per kg. of water more than the oxygenated one without changeof the osmolality. On the average, the oxygenated blood contained5-4 millimols more oxygen per kg. of water bound to the haemoglobinthan the reduced. In this case, therefore, the loss of 1 millimol of 02enables the blood to take up 035 millimol of CO2 without changeof the osmolality. In the other two experiments the quantity of CO2taken up at constant osmolality was estimated to be 035 and 0*26respectively for each millimol of 02 lost. In fig. 3a the CO2 per kg. ofwater in the blood is plotted against the CO2 pressure, and in fig. 3b theosmolality of blood is plotted against the CO2 pressure. It appearsthat in fig. 3b the points of reduced and oxygenated blood fall approxi-mately on the same curve. According to figs. 2 and 3a and b, therefore,the reduction of hoemoglobin enables the blood to take up an additionalquantity of CO2 at constant osmolality and constant CO2 pressure,and the two quantities, within physiological limits, are approximatelythe same.

DISCuSSION

We have seen that, as long as the oxygenation of blood is keptconstant, the addition of CO2 to the blood increases the osmolality ofplasma, but that the reduction of the haemoglobin has the peculiareffect of permitting more CO2 to enter the blood without a correspondingincrease of the osmolality. This result cannot be accounted for bythe theory that the primary effect of reduction is to change the dissocia-tion constant of an acid or basic group of the haemoglobin. If thattheory were correct, the additional quantity of CO2 taken up by thereduced blood would be as osmotically active as the rest of the CO2present in the blood, and the points corresponding to reduced andoxygenated blood in a diagram like that represented in fig. 2 wouldfall on the same straight line and not on two separated lines, as is actuallythe case. By the results of our measurements we are led to think ofa different mechanism to account for the higher buffering power of thereduced hemoglobin. Some clue as to what this mechanism can bemay be obtained by further analysis of the experimental data.

If the CO2 content of the blood is maintained constant, the reductionof one millimol of hsemoglobin per kg. of water brings about a decreaseof the osmolality of the blood of about 0 3 milliosmol/kg. of water(see fig. 2). There are two different ways to achieve this result. Ifm molecules per kg. of water are present in the blood and m' = 0 x m ofit are osmotically active, a reduction of the osmolality can be achievedby lowering the value of b or by forming a chemical bond betweenmolecules so that the value ofm is decreased. As far as CO2 is concerned,

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Meschia and Barron

fig. 2 shows that, within the limits of experimental error, the two linesfor reduced and oxygenated blood run parallel to each other, i.e. thatthe osmotic coefficient of CO2 is the same in reduced and oxygenatedblood. A second alternative would seem, therefore, more in agreementwith the experimental data, i.e. that a chemical bond is formed betweenthe reduced hawmoglobin and some of the molecules present in solutionin the proportion of approximately 0 3 millimol for each millimol ofhaemoglobin. The nature of our measurements is such that no indicationis given about what kinds of molecules may be bound by the reducedhsemoglobin. The data of Margaria and Green [1933] and Fergusonand Roughton [1934] lead us to think that one of these molecules isprobably CO2 itself. According to these observations, the CO2 boundby one mol of heemoglobin passing from the oxygenated to the reducedform is about 0.1 to 0 5 mols, depending upon the values of pH, ionicstrength, CO2 pressure and concentration used by the different authors.There are, however, some indications that possibly other moleculesare bound by the reduced haemoglobin besides CO2. The practicalidentity of the titration curves of oxygenated and reduced hasmoglobinwith CO2 and HCI has been considered by Hawkins and Van Slyke[1930] as a proof that the Henderson hypothesis is correct and thatthere is no CO2 combined to the hsemoglobin in any appreciable amount,unless the hypothesis that the hsemoglobin is able to combine CO2 andHCI in roughly the same amount would be taken into consideration.At present, however, it seems fairly well established that the reducedhaemoglobin has the capacity to bind more CO2 than the oxygenatedand, according to the data presented in this paper, Henderson's hypo-thesis is unable to account for all the experimental facts. This beingthe case, it seems reasonable to consider again the hypothesis that thereduced hsemoglobin is able to bind some other molecules besides CO2,as for instance chloride ions.

To show that the formation of the CO2-hkmoglobin compound maynot be the only explanation of the effect that oxygenation and reductionhave on the osmolality of the plasma, we have reproduced in fig. 4 aplot of CO2 content per litre of plasma against the osmolality of plasmaitself, taken from the same experiment from which figs. 2 and 3a and bare taken. It appears that even for the plasma, and not only for thetotal blood, the two sets of points from reduced and oxygenated blooddo not fall on the same line. This fact means that the plasma separatedfrom reduced blood has more CO2 in it, for a given value of osmolality,than the plasma separated from oxygenated blood. We have to suppose,therefore, that in the plasma separated from reduced blood the CO2has replaced some other molecules that were made osmotically inactive,possibly by formation of a chemical bond with the reduced haemoglobin.However, this is indirect evidence, and some other more direct approachto the problem must be attempted before reaching any conclusion.

192

C02 and 02 of Blood and Freezing Point of Plasma

3 60

0°355-/Een .*00

- 350 /E

/ 0 Plasma of reduced blood* Plasma of oxygena+ed blood

10 I5 20 25

CO2 (rm M per Ii+re of plasma)FIG. 4.-A graph of the C02 content, mM per litre of plasma, against the

osmolality of that plasma.

SIUMMARY1. The effect of CO2 and 02 on the osmotic pressure of plasma has

been estimated by measuring the freezing point depression of plasmawhose blood had been equilibrated at 380 C. with gas mixtures ofdifferent composition.

2. At constant oxygenation, for each millimol of CO2 added toone kg. of water of the blood, the osmotic pressure of plasma rises by0 9 milliosmols per kg. of water.

3. If the CO2 content of blood is kept constant, the reduction ofone millimol of haemoglobin (m. wt. 17,000) per kg. of water lowers theosmotic pressure of blood by about 0x3 milliosmol per kg. of water.

4. The biochemical implications of these findings are discussed.

ACKNOWLEDGMENTWe wish to thank Dr. D. I. Hitchcock for reading the paper and for his

criticism.

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194 Meschia and Barron

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