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BUFFER SYSTEMS OF BLOOD SERUM.
BY EDWARD A. DOISY, EMILY I’. EATON, AND Ii. S. CHOUKE.
(From the Laboratories of Biological Chemistry, Washington University School of Medicine, St. Louis.)
(Received for publication, May 2, 1022.)
Loaaed and Self-Possessed Serum Buffers.
In a previous paper (Doisy and Eaton, 1921), we have shown that the loss of hydrochloric acid from blood serum, by its migra- tion into the cells, approximately accounts for the increase in the binding power of the serum for carbon dioxide when blood is exposed to increased tensions of that gas (Reaction 1). We considered that other acid radicals probably shift in a similar manner, but were unable to obtain any evidence of the passage of base across the cell membrane. Our work was incomplete in that we measured only the buffers loaned to the serum by the cells, and did not attempt to measure the “self-possessed” buffer value of the serum. Of the two types of reaction (Van Slyke, 1921),
(1) BA + H2C03+BHC03 + (HA)-+cells. (‘L) B protein + H&03-+BHC03 + H protein.
our earlier experiments furnished data only for the first. In order to evaluabe the relative importance of each of these buffer reac- tions, we have extended our work by a further study of the blood of four dogs and of two men.
As was pointed out in our previous paper, the corpuscles of blood occupy a larger volume as the tension of carbon dioxide in the gas with which they arc in equilibrium is increased. Although this phenomenon was noted some time ago (von Lim- beck, 1894-95), and its importance emphasized in our former paper,
61
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62 Buffer Systems of Blood Serum
it has not been considered in other recent investigations1 For this reason it seemed probable t,o us that other estimates (Van Slyke and Cullen, 1917; Fridericia, 1920) of the extent of the migration of hydrochloric acid into the cells were too low. The use of anticoagulants is still another reason for thinking that the values previously reported for chloride shift were too low. Our work has made it seem probable that all of the potential acids of the serum take part in the migration, and that they enter into this reaction to a degree which is dependent upon their concentration and possibly upon their relative acid dissociation constants. Samples of oxalated blood generally show that less than 50 per cent of the increase of bicarbonate of serum (on treatment of oxalatcd whole blood with increasing tensions of CO,) is due to a loss of chloride, whereas our results on defibrinated blood showed the chloride shift to account much more closely for the gain in bicarbonate. It therefore seems fair to suppose that a shift of oxalic acid explains the difference in behavior, and if this is true it becomes important to avoid all such additions to blood in such studies of acid-base equilibria.
In the experiments here reported we have attempted to analyze the sources of alkali by which the blood combines with increasing amounts of COZ and to estimate bhe quantitative contribution of each. Within a given range of pH, how much of the base for the increase in sermn bicarbonate is made available by the migration of acids into the cells (rcact.ion of Type 1, above)? Is the migra- tion of HCI alone sufficient to account for this quota, or do the acids of other salts also migrate? How much base is furnished from the self-possessed, non-migrating buffers of the serum, and to what relative extents do the serum proteins and the serum phosphates participate?
The plan of our experiments was to determine the gain in serum bicarbonate on equilibrating fully oxygenated, defibrinated blood
1 L. J. Henderson has discussed this point rather briefly in some of his recent papers (McLean, F. C., Murray, H. A., Jr., andHenderson, L. J., Proc. Sot. Exp. Biol. and Med., 1919-20, svii, 181; Henderson L. J., J. Biol. Chem., 1920, xii, 427), but apparently has not attempted to make the corrections for change in corpuscle volume as described in this paper. The mechanical models of Henderson and Spiro (Spiro, I<., and Henderson, L. J., Biochem. Z., 1909, xv, 114) are very illuminating.
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Doisy, Eaton, and Chouke 63
with 20, 40, and 60 mm. tension of COZ. By determining also the gain in bicarbonate of separated sera similarly equilibrated with increasing tensions of CO,, there is obtained the effect of the self- possessed serum buffers (Type 2). As indicated by Van Slyke in his treatment of the data on Joffe’s blood, the difference between the two sets of values (for true serum and separated serum) represents the buffer action of Type 1, which is due to the pres- ence of the corpuscles. The determination of chlorides in the true sera shows the amount of base liberated from NaCl by the migra- tion of HCl. Since the earlier experiments gave no evidence of migration of base, by a comparison of the molar gain of bicarbon- ate due to ‘Lloaned buffer” with the molar loss of chloride in true sera, we attempt to determine the extent to which other acids migrate and thus cont,ribute to the loaned buffer action. For one blood we determined also the hemoglobin and inorganic phosphates of the cells, which data allow the calculation of the relat,ive amount of base furnished by each to the migrating acids.
The sera were also analyzed for inorganic phosphates and from this one may calculate the relative amount of base furnished by phosphate and protein buffers, the self-possessed buffers of the serum.
EXPERIMENTAL PROCEDURE.
The dog’s blood for our experiments was drawn from the femoral artery while blood from the arm vein was used in t’he case of the two men. In each experiment it was defibrinated by stirring or shaking with beads. The fibrin was removed by filtering through gauze. Approximately one-half of the blood drawn was divided into three samples and equilibrated for 25 minutes at 38°C. with previously prepared gas mixtures containing about 20, 40, and 60 mm. of carbon dioxide. The other half was centrifuged to obtain “separated” serum, separate portions of which were simi- larly equilibrated with gas mixt.ures of the same composition as used with whole blood.
After equilibration of the whole blood, the samples were trans- ferred to ordinary centrifuge and hematocrit tubes containing paraffin oil. The transfer was accomplished without contact with any other gas. On the completion of centrifugation the corpuscle volume was det.ermined, and from the larger tubes serum was obtained for the chloride and carbon dioxide analyses. All
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64 Buffer Systems of Blood Serum
possible precautions were taken to avoid or minimize loss of CO2 from samples of blood and serum from the completion of equili- bration to the transfer to the apparatus for COP determination. After equilibration of the “separated” serum, the samples were transferred under oil to tubes suitable for withdrawing 1 CC.
portions for carbon dioxide analyses. Carbon dioxide analyses were carried out by transferring 1
cc. of serum directly from the tubes to the cup of the Van Slykc macro apparatus (1917). The calculations were made according to the details suggested by Van Slyke and Stadie (1921). Chlo- ride determinations were made on picric acid filtrates using the volumetric principles of the McLean-Van Slyke (1915) titration. In our hands the end-point appeared a bit sharper when the buffer and starch were used in separate solutions. In this case the starch solution was prepared fresh every few days. We also found that picric acid filtrates gave a sharper end-point under any conditions than the trichloroacetic acid filtrates used in our previous work.
After equilibration was completed, the gas mixtures in our saturators were analyzed with a Haldane burette. Phosphate analyses were made by the calorimetric method described by Bell and Doisy (1920).
Correction jar Change in Corpuscle Volume.
The increase of corpuscular volume at the expense of water from the serum, which results from absorption of COZ causes an increase in the concentration of all substances in serum and the amount of this increase must be deducted before calculating loss of chloride and gain of bicarbonate caused’by increasing CO, tension.
In order that we might plot our values of chloride and bicar- bonate and thereby secure the advantages of the graphic method, we have modified the method of correction for change in the vol- ume of the corpuscles. In our first paper, the correction was ap- plied by calculating the concentration of chloride and bicarbonate that would exist due to the change in volume of corpuscles if no migration qf ions had occurred. For example, in Experiment 1, we have the data given in Table I.
As corrected formerly, the values were obtained in the following way :
56.2 55.6 X 0.112 = 0.1134 M NaCl 56.2 5io X 0.0164 = 0.0166~ NaHCO.,
I .
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Doisy, Eaton, and Chouke 65
This method is undesirable in that it leaves different basic values with which to compare each value obtained at higher tensions of COZ, and such results for three tensions cannot be plotted on the same curve. It is preferable to correct all observed concentra- tions to one basic volume taking for this purpose the volume of the serum at the lowest carbon dioxide tension. The values for bicar- bonate and chloride at the higher CO2 tensions are merely multi- plied by the necessary factor to give the respective concentrations that would exist in the serum had its volume not changed due to the taking up of water by the corpuscles.
TABLE I.
Experiment 1.
COZ, mm. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.6 Serum, per cent of total blood volume. . . . 56.2 NaHC03, molar concentration. . . . 0.0164 NaCI, molar concentration. . . . 0.1122
TABLE II.
39.3 61.6 55.6 55.0
0.0219 0.0257 0.1097 0.1085
Correction for Change of Corpuscle Volume.
CO% mm.. . . . . .
NaCl-loss, mols.. . . . . NaHCOa-gain, mols. . Percentage to loss of
chloride. . . . , . . .
Former method. New method.
17.6 to 39.3 17.6to61 6 17 6 to39.3 17.6 to 61 6
0.0037 0.0062 0.0036 0.0060 0.0053 0.0090 0.0053 0.0088
7o I 6g I 681 68
From the data just given, the correction by this method is:
55.6 55.6 562 X 0.1097 = 0.1086 M NaCl 56.2 X 0.0219 = 0.0217 M NaHCOa
The values for the highest tension of CO2 are calculated in the same 55.0
way using in this case the factor ~ and the concentrations found 56.2
by analysis. As shown by comparison in Table II, the numerical values obtained by the two methods are substantially the same. The new procedure has the advantage that the results may be plotted on a single graph.
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66 Buffer Systems of Blood Serum
RESULTS.
The experimental data from the different samples of blood are given in detail in the protocols. The values for serum bicarbonate and chloride are corrected for change in serum volume as above described, and are brought together in Table III. In order to
TABLE III
Loaned Buffer; Loss oj Chloride and Gain of Bicarhorlate.*
Dog 1.
Dog 3.
Dog 3.
Dog 4.
Man 1.
Man 2.
---
co2 PH NaHCOa
mm.
17.6 39.3 61.6
7.57 7.34 7.22
mols
0.0164 0.0217 0.0252
18.5 7.51 0.0152 42.5 7.30 0.0212 66.0 7.18 0.0244
18.5 7.53 0.0159 37.3 7.34 0.0204 74.8 7.12 0.0243
20.0 7.50 0.0159 44.3 7.27 0.0207 76.0 7.11 0.0237
23.3 7.47 0.0175 43.0 7.33 0.0230 72.5 7.17 0,0”67
16.7 35.2 63.8
7.65 7.44 7.27
0.0189 0.0241 0.0290 / 0.0101
-
i -
Differenre n NaHCO,.
mob
0.0053 0.0088
0.0060 0.0092
0.0045 0.0084
0.0048 0.0078
0.0055 0.0092
0.00.x
iiaC1
mozs
0.1122 0.1086 0.1062
0.1122 0.1079 0 1042
0.1110 0.1076 0.1042
0.1182 0.1142 0.1091
0.1110 0.1071 0.1043
0.1103 0.1065 0.1020
Difierrnre in X&I.
mols
0.0036 0.0060
0.0043 0.0080
0.0039 0 0067
0.0038 0.0083
* Bicarbonate and chloride values have been corwcted for change in corpuscular volume.
compare the relative participation of the various buffer factors it is necessary to choose an arbitrary pH range, and to calculate the effect of each factor within this range. Without change in hydrogen ion concentration, the effect of the self-possessed serum buffers is, of course, zero, the buffer action being limited to that depending upon migration of acid to and from the cells. We have chosen the range of pH 7.45 to 7.25 as the basis of our calculations,
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Doisy, Eaton, and Chouke
and in doing so it may be noted that we undoubtedly exaggerate the relative participation of the self-possessed buffers, which as pointed out by Van Slyke (1921) play less and less of a role under physiological conditions the smaller the difference in pH between arterial and venous blood. This range was chosen to encompass the varying values which appear to occur naturally, if not physio- logically, and especially in order to minimize the errors in the de- terminat,ions.
Since some of our experimental data commonly fall without this pH range, it is necessary to plot them in the form of curves and
LX ! J, I ,
71 I ! / i ! / 11
YI i i i i I / I / I I Ii .Oi7 OIB ,019 ,020 .02! ,022 .0,?3 .OQ+ .OU .026MNaHCOJ
SIZ .M A0 ,109 .I09 .I07 .IOOA4NaCI
FIG. 1. Data are taken from Experiment 1 which was conducted on dog’s blood. Chloride and bicarbonate values of true sera corrected for the change in volume of the corpuscles are plotted against pH.
obtain the desired values by interpolation. The most convenient method of treatment is to plot the molar concentrations of bi- carbonate and of chloride of the true sera (corrected for change in serum volume) and the molar concentrations of bicarbonate of the separated serum against the pH which is calculated from the ratio of free and combined CO,. The corrected data, given in Table III are plotted* in this way in Figs. 1, 2, and 3. From the
* Only three of the six charts are presented. The concavity to t.he abscissa is greatest in Fig. 2, while the convexity is greatest in Fig. 3. All the curves of t,he other experiments occupy intermediate positions. It should be noted that if the true serum bicarbonate curve is convex to the abscissa?, the chloride curve varies from a straight line in the same direction. Generally this has been true in all our experiments. At present, we do not understand the significance of these variations.
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68 Buffer Systems of Blood Serum
.018 .Uf9 .020 .02 2 423 024 .o CO3 Sll If0 .I09 .I08 JO7 IO6 .fO.TM NaCI
FIG. 2. Data are taken from Experiment 5 which was conducted on human’blood. Chloride and bicarbonate values of true sera corrected for the change in volume of the corpuscles are plotted against pH.
.Of9 .020 ,021 .022 ,023 .024 .025 .026 .0,?7 ,028 .029M NaffC0~ .M MO IO9 .lOB IQ7 IO6 /OS JO4 .103M NaN
FIG. 3. Data are taken from Experiment 6 which was conducted on re- duced human blood. Chloride and bicarbonate values of true sera CCC- rected for the change in volume of the corpuscles are plotted against pH.
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TABL
E IV
.
Seru
m R
ugkr
s, pH
7.
45 to
7.2
5.
True
se
rum
. Se
parat
ed
seru
m.
Molar
Na
HCOs
. Mo
lar
NaHC
Os
pil..
......
....
7 45
7.2
5 Ga
in.
7.45
7.25
Gain.
~-
~ -~
Dog
l.....
. 0.
0188
0.
0242
0.
0054
0.
0195
0.
0201
0.
0006
(‘
2 . .
. . .
. O
.OIF
9 0.
0225
0.
005G
O
.OlG
3 0.
0168
0.
0005
“
3 . .
. . .
. 0.
0181
0.
0222
0.
0041
0.
0154
O
.OlG
l 0.
0007
“
4 . .
. . .
. O
.OIG
'Lj 0
.021
1 0.
0042
0.
0225
0.
0233
0.
0008
M
anl..
...
0.01
87
0.02
50
0.00
63
0.02
45
0.02
57
0.00
12
(( 2
. . .
. .
0.02
38
0.02
96
0.00
58
0.02
53
0.02
66
0.00
13
Rle
an...
......
......
......
......
......
......
......
......
....
True
se
rum
. Pe
rcenta
ge
of ba
se
supp
lied
from
:
hlolnr
Ns
CI.
7.45
7.x
Loss
. ~-
0.11
05
0.10
69
0.00
36
0.11
12
0.10
66
0.00
46
0.10
95
0.10
61
0.00
34
0.11
75
0.11
38
0.00
37
0.11
03
0.10
56
0.00
46
0.10
67
0.10
16
0.00
51
HCI
migr
ation
.
11
67
9 a2
17
83
19
88
19
75
23
88
. . . .
. . .
. . .
. . .
. . .
. . .
. . .
. 16
80
.5
0 b
-7 6
0 -1
1 g
3+
.>
. 71
,,
>.
--I
- .-
-
- .-
-
-.
- -
,.%
>
-
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Buffer Systems of Blood Serum
curves in these figures, we read off the changes in concentration corresponding to the change in pH from 7.45 to 7.25 and these values are given in Table IV. Taking the data from Experiment 1 as an example, it will be seen (Table III) that on passing from a pH of 7.45 to 7.25, the true serum gained 0.0054 mols of bicarbon- ate, of which only 0.0006 mols or 11 per cent is due to the buffers of the separated serum (the self-possessed buffers), the remaining 0.0048 mols or 89 per cent being due to the presence of corpuscles (the loaned buffers). Of the latter, the disappearance of NaCl (by migration of HCl into the cells), 0.0036 mols, accounts for three-fourths, or for 67 per cent of the total gain. The remaining 22 per cent of the tot’al gain (0.0012 mols) which is not accounted for in this experiment is perhaps due to the migration of other acids from serum salts into the cells. All of the other experiments show somewhat smaller fractions to be unaccounted for (from + 0.0005 to -0.0006 molar). The difficulty in the accurate meas- urement of small differences between much larger quantities is such that the variations between extrcmcs is no greater than might be expected. The effect of small errors in individual values is considerable, as may be illustrated by the following calculations on the serum of Dog 1, Experiment 1.
Assuming that the basic value 0.1122 M NnCl is correct, let us see what effect an error of -0.0005 M or 0.5 per cent products in our estimation of the loaned buffer value. The corrected value
at 39.3 mm. of CO2 (0.1097 - 0.0005 = 0.1092. 0.1092 X E
= 0.1080) becomes 0.1080 instead of 0.1086 and the loss of chlo- ride is now 0.0042 instead of 0.003G. This account,s for
0.0042 x 100 =
0.0053 ) 79 per cent of the total gain of bicarbonate in-
stead of the 68 per cent actually found. Viewed in another way, an error of 0.5 per cent in the determination of one value produces n difference of about 12 per cent in the result. It is thus apparent that too much must nqt be expcctcd from individual det,ermina- tions. We, therefore, deem it more desirable to consider only t,he mean values. According to the average of all of our data, the buffer value of serum in the pH range of 7.45 to 7.25 is loaned to it to the extent of 80f per cent, by a migrnt.ion of hydrochloric acid into the corpuscles; while the effect of t,he non-migrating self-
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Doisy, Eaton, and Chouke 71
possessed buffers amounts to 16 per cent. This leaves 3 per cent unaccounted for and this is probably due to a migration of acids other than hydrochloric. The evidence presented by de Boer (1917) on the migration of SOa--, our results concerning phos- phates, the behavior of oxalated blood already referred to, and particularly the fact that a large migration of H2C0, into the corpuscles takes place, would seem to indicate that such a con- clusion is justified.
We can feel fairly certain that we know the nature of all of the buffer reactions of the serum when the state of oxidation of the hemoglobin remains unchanged. In a recent paper, L. J. Hen- derson and coworkers (1919-20) state that when the oxygenated
TABLE V.
Alkali Furnished by Phosphate Bu,ffer System.
Experiment
Dog 1. ‘( 2. “ 3. “ 4.
hlan 1. (6 9 -.
Total gain of NaHCOa in
eparsted mm.
pH 7.45 to 7.25.
molar
0.0006
0.0005 0.0007 0.000s 0.0012 0.0013
Total inorganic P
of sera.
molar
0.00129 0.00174 0.00084 0.00119 0.00087 0.0010
-
b ,H 7.45 to 7.25.
-
Base yielded by NazHPO,
m&r
0.0001
0.00014 0.00007 0.0001 0.00007 0.00008
- I Percentage of total alkali
supplied by NalHPO,.
Separated 86x&.
-
. _ True wra.
per cent per cent 17 1.9 28 2.5 10 1.7 12 2.4 F 1.1 G 1.4
Mean. . . . . . . I Il.8 13
blood is reduced isohydrically about 60 per cent of the increased capacity of serum to bind COP, is clue %o a migration of chloride. Although this might seem to indicate some other loaned buffer mechanism, we shall attempt to show in a later paper that at least 90 per cent of the increased capacit,y is actually due to a shift of HCl. The lower estimate is due to neglecting t,he change in concentrations produced by the swelling of the corpuscles.
By means of our inorganic phosphate determinations we may calculate the participation of the serum phosphate in the self- possessed buffer action. By using the Hasselbalch equation,
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72 Buffer Systems of Blood Serum
we can solve for the ratio of disodium to dihydrogen phosphate at pH 7.45 and 7.25 and from our data on serum inorganic phosphate calculate how much of the separated serum buffer value is due to the phosphate system. The fraction which this represents of the total base supplied by all buffers of separated serum shows the relative participation of the phosphates.
The data on this point are given in Table V. According to these calculations from 6 to 28 per cent of the self-possessed buffer action may be due to the phosphates; the remainder being due to salts of proteins and amino- and other organic acids. The serum phosphates appear to supply only from 1 to 3 per cent of the base contributed by the total serum buffers.
SUMMARY.
A series of experiments on samples of blood of four dogs and two men show that between the pH range af 7.45 and 7.25, the base furnished for the increase of bicarbonate in serum comes from the sources indicated (mean results) :
(a) “Self-possessed”, non-migrating serum buffers: 16 per cent. Of this, phosphates supply 1 to 3 per cent.
(b) “Loaned” buffer, due to presence of corpuscles: 84 per cent. Of this, migration of HCl into corpuscles liberates 80 per cent. The remaining 3 per cent is probably liberated from the salts of other acids, by migration of the latter into the corpuscles.
Protocols.
The blood was defibrinated by one of the usual procedures. Each gas- equilibrating mixture consisted of air plus COz (except Experiment 6). With the exception of Experiment 4, no hemolysis occurred, and in this case it was very faint.
Experhent I-Dog 1.
I True serum. Separnted serum. ,
CO,, mm ..................... 17.6 39.3 61.6 18.8 41.8 62.4 Total COz, ~01s. per cent ....... 37.9 51.9 62.0 44.5 47.9 51.6 Dissolved COZ, vols. per cent. 1.25 2.80 4.38 1.34 2.98 4.44 BHCOI, vok. per cent. ....... 36.6 49.1 57.6 43.2 44.9 47.2 pH ........................... 7.57 7.34 7.22 7.61 7.28 7.13 NaCl, mols ................... 0.1097 0.1085 Serum, per cent ............... 55.6 55.0 Inorganic P, mg .............. 4.0
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Doisy, Eaton, and Chouke 73
Experiment %--Dog 8.
COz, mm.. . . . . . . . . . . 18.5 66.0 19.7 39.8 65.0 Total CO*, vols. per cent.. 35.4 61.1 37.7 40.8 44.8 Dissolved COz, ~01s. per cenl.. . 1 32
I 3.03 4.70 1.40 2.83 4.63
BHC03, vols. per cent. . . . 34.1 48.4 56.4 36.3 38.0 40.2 pH........................... 7.51 7.30 7.18
.I 0.1122,40:099(4~:O% 7.51 7.23 7.04
KaCl, mols.. . . . . , . . . . Serum, per cent.. . . ,48.2 Inorganic P, mg.. . . . . . . . . .I
I ! 5.4
Experiment S-Dog S.
True serum. Separated serum.
co 2, mm..................... 18.5 37.3 74.8 Total CO,, ~01s. per cent.. 37.0 48.8 61.3 Dissolved Cot, ~01s. per cent. 1.32 2.66 5.32 BHCO,, ~01s. per cent. . . . 35.7 46.1 56.0 PH.. . . . . . . . . 7.53 7.34 7.12 NaCl, mols.. . _. _. 0.1110 0.1088 0.107: Serum, per cent.. . . . 56.3 55.6 54.6 Inorganic P, mg.. . . . . . . . .I
18.0 41.2 71.6 35.0 39.5 43.1
1.28 2.93 5.10 33.7 36.6 38.0
7.52 7.20 6.97
2.6 I I
Experiment &Dog 4.
True serum. Sepnrated tierurn.
NaCI, mols ................... Serum, per cent .............. ,150.T 55.8 ,54.3* Inorganic P, mg .............. .I I !
3.7
* Probably erroneous; change larger than expected.
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74 Buffer Systems of Blood Serum
Experiment b-Man 1.
CO2 , mm ................... 23.3 43.0 72.5 18.1 41.2 71.2 Total COZ, ~01s. per cent. ..... 40.9 55.4 66.4 52.6 58.7 G3.5 Dissolved COZ, vols. per c&t. 1.66 3.06 5.16 1.29 2.93 5.07 BHCOs, ~02s. per cent. ....... 39.2 ,52.3 61.2 51.3 55.8 58.4 pH ........................... 7.47 7.33 7.17 7.70 7.38 7.16 NaCl, mols ................... 0.1110 0.1087 0.1065 Serum, per cent ............... 54.1 53.3 53.0 Inorganic P, mg .............. .) 2.7
Experiment 6-iWan 2.
COP, mm.. . . . . . . . . . . . , . . . 16.7 35.2 63.8 15.6 44.7 82.0 Total COZ, ~01s. per cent.. . . . 43.5 57.1 71.3 55.3 61.0 68.6 Dissolved COz, ~01s. per cent. 1.19 2.50 4.54 1.11 3.18 5.83 BHCOJ, vols. per cent. . . . . . . . 42.3 54.6 66.8 54.2 57.8 62.8 pH........................... 7.65 7.44 7.27 7.79 7.36 7.13 NaCl,mols................... 0.1103 0.1076 0.1047 Serum, per cent.. . . . . . . . 56.5 55.9 55.0 Inorganic P, mg.. . . . . . 3.1
* The true serum of this blood was obtained by equilibration with Hz + cot. Approximately 95 per cent of the hemoglobin was in the reduced form.
BIBLIOGRAPHY.
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ChoukeEdward A. Doisy, Emily P. Eaton and K. S.
BUFFER SYSTEMS OF BLOOD SERUM
1922, 53:61-74.J. Biol. Chem.
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