A PHYSICOCHEMICAL STUDY OF BLOOD SERA. 111
CASES COLLOIDOSMOTICPRESSURE. AN ANALYSISOF ONE HUNDRED JOSI? ZOZAYA Gladwyne Research Laboratorzes, Gladuyne, Pennsylvania Received February 5, 1938
The importance of the colloid osmotic pressure of the blood proteins in the interchange of fluids in the body is generally rccognized. Since the work of Starling (14) a great many investigators have studied the colloid osmotic pressure of blood serum or plasma. These investigators have used different techniques, with variations in the conditions observed, such as temperature, concentration of serum, pH, different bathing fluids, and many kinds of membranes, all of TT hich influence the final result. Few of these TT orkers have used a theoretical colloid osmotic pressure value which could be calculated from the molecular weight of the serum proteins by the formula R T C I M , where C is the protein concentration and M the molecular weight of the protein. The literature on the determination of the colloid osmotic pressure of human serum is large. For our present consideration we are interested mainly in the actua! values obtained by different workers, so n e have used the table given by Drinker and Field (4) and have determined the aaerage values from the list given by them. These will be discussed later. The bibliography is given by Drinker. Colloid osmotic pressure measurements M ere used in the first determinations of the molecular weight of serum albumin in a nell-defined composition by Sorensen (13), who obtained the value of 45,000. By a similar method hdair (2) found the value to be 62,000, but he made some modifications in the calculations. Svedberg and Sjogren (15), using the ultracentrifuge, found the molecular weight of serum albumin to be 68,000 and that of globulin 169,000. In further work with the colloid osmotic pressure method, Adair (3) found the ne\T values 72,000 for serum albumin and 170,000 for globulin These last figures are the ones used in our calculations. I t is of interest to note the close agreement between the values obtained by the dificrent methods There are many factors which influence the colloid osmotic pressure of protein systems, such as the Donnan effect, the pressure due t o the diffusible ion, etc , but they become of greater interest in academic studies with 657
perfectly controlled systems. The important physiological consideration is the constant and accurate measurement of the colloid osmotic pressure of the blood serum as is, for it is in this form that the important physiological effect is obtained In this work we shall not discuss the probable factors influencing our determinations other than those mentioned, for they already take into consideration the optimum conditions for eliminating some of the effects Adair (3) studied the serum albumin and globulin separately and in the a hole serum, and he concluded that their behavior, so far as colloid osmotic pressure is concerned, follon s Dalton’s law of partial pressures and that it appears that serum protein is not a compound of albumin and globulins, ,He further believes that the state of aggregation of the proteins in the untreated serum appear.. to be the same as their state of aggregation in the purified proteins K i t h these experimental facts, we studied one hundred samples of human serum (from mental and nervous cases), only one showing a marked alteration (This was sample S o . 88, from a case with granuloma inguinale, which had a total protein of 10.75 and an A / C of 0 77 The colloid osmotic pressures determined experimentally a e r e compared n i t h the theoretical values calculated on the basis of the added effects which the amount of serum albumin and globulin produced, their moleciilar weights in an ideal solution being considered. METHODS
The general technique n hich T T folloned ~ is essentially that described by Adair (1) in his studies on the molecular weight of hemoglobulin. A detailed description of the method is given below.
Collodzoiz membranes A 2 per cent parlodion (Pyroxylin Purified, Xallinckrodt) solution n as made with equal parts of ether (distilled over sodium) and absolute alcohol To this solution was added sufficient ethylene glycol to make a final concentration of 2 per cent. The percentage of ethylene glycol affects the permeability of the memhrane, 11hich incrdases n i t h the percentage of the glycol. In our experience I\ e found that a 2 per cent concentration gave the ideal permeability It is best to let the solution of collodion rest for at least two neeks before using it. On a revolving glass tube, 1 1 ern in diameter, arranged in a rotary mechanicnl device, the collodion was poured on at least 4 em of the length of the tube, including the end. After 1.5 ininutes a second coating of collodion nas applied. This procedure n a s repeated four times n i t h 15-minute intervals betneen each pouring. After the fourth coating, 6 minutes mere alloned to pass before shutting off the motion of the tube and the heat. Then the tube was allowed t o dry oyrrnight before the membrane n a s removed. An important provision i q t o have a heater t n o feet in front of the tube, while the
PHYSICOCHEMICAL STUDY OF BLOOD SERA.
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collodion is being poured, to hasten drying. Before use all membranes were submitted to a pressure of 2.5 atmospheres with a mercury pump, to test their permeability and strength.
Osmometer
A heavy glass tube with a thin capillary opening graduated in millimeters was used as the osmometer (Westergren sedimentation tube). The details of the arrangement are best seen in figure 1, where A is the vacuum of a thermos bottle, B the collodion sac of 2-cc. capacity, which is fitted tightly to a rubber stopper D by means of a fine rubber thread C. Inserted through the stopper is a thin glass tube E, which by means of a
FIG.1. Osmometer apparatus in thermos
rubber tube G connects the osmometer I with the collodion bag. The bag containing the serum is immersed in a large test tube through a stopper, which has a small glass tube inserted on the side to enable it to touch the surface of the bathing liquid. Both should be at the level of the zero reading of the osmometer. The tube prevents pressure on the liquid when the stopper is tightly fitted to the tube. The whole apparatus is immersed in a thermos bottle which has previously been filled with finely cracked ice, a space being formed in the ice for the tube to fit into it. To keep a constant temperature of 0°C. the bottle was refilled with ice every other day, for low temperature is the most important factor in insuring chemical stability.
660
JOS6 ZOZAY.4
Bathing fluid The bathing fluid used in these experiments was an equal mixture of Sorensen’s disodium hydrogen phosphate and potassium dihydrogen phosphate in M / 3 0 concentration. K i t h the use of this low concentration of salts, the Donnan effect is negligible and no correction is made in our calculations. Further experiments are being made to measure the partial osmotic pressure of the protein ions. The p H a t room temperature (18°C.) was 6.81 and at 0°C. was 6.91. The solution in the tube was changed daily at the time of the reading of the manometer. A constant p H of the fluid in colloid osmotic pressure determinations is of importance, for we know that it has an effect on the colloid osmotic pressure of proteins.
Calculatzons s p . G. R8) -13.6 RI = the scale reading of the meniscus on the osmometer, R 2 - Rs = the difference in the meniscus reading n hen the tube is dipped in the tube of the inner liquid (capillarity), and Sp G = the specific gravity of the serum Readings of the manometer nere made every day until an identical reading nas observed for t x o or three consecuti\r days, when it \$as thought that equilibrium had been reached. A11 results are expressed in millimeters of mercury and a t 0°C The theoretical calculation of colloid osmotic prcssure n as dcteimined by the formula RT( l o c o ) RT( 10 C,) p = -_____ + 1 M , M, p = R1 - (Rz
760 X 22.412 = 17,033 at 0°C , molecular n eight of serum albuiiiiii (42,000), molecular xeight of serum globulin (170,000), Concentration of albuniin in the serum (per rcnt), and roncentration of globulin in the ieiuiii (per cult) The Concentration of the proteins is multiplied by 10 t o mahe the relation in terms of parts per thousand The theoretical colloid osmotic pressure per gram of proteln is deiiotcd by no in comparison t o n, which is the relation of the co!lod osmotic pres5 u r ~determined per gram of protein The value of no \!as cxpcrimcntally determined by Adair (3) as 2 36 far albumin and 1 00 for globulin He d s o determined it in unfractionated serum and found it t o be 1 90 f 0 2 Using the data published hy 1erney 116)’ >Jay IO), and Lhrrack and iien:t+ (Y), and iecalculatlng thnir x d u e s to m lnletrrs 07 mercury and 10 0°C idair found 1 8 + 3 ‘3 $0 -k 0 2 and 1 0 :-0 2 , r C i p c t i r eAy where R?’
=
Xcz= Y, = C, = C, =
~
PHYSICOCHEMICAL STUDY O F BLOOD SERA.
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The determined value for noin the serum studied by Adair was 1.94, which agrees with the theoretical one within the limits of experimental error. From the values obtained for albumin and globulin one can calculate the theoretical value for no for any given serum by taking into consideration the relative percentages of each fraction in the whole serum. The ratio p/RTCp is the relation of the observed colloid osmotic pressure and the calculated one.
Other measurements The determination of the specific gravity and the relative viscosity as well as the fractionation of the proteins have already been described in a previous paper (18).
Time of observation I n the present series the average time for a serum to come to equilibrium was 10 days, the shortest time being 6 days, and the longest 32 days. (This extreme was a serum with a total protein of 10.75 and an A / G of 0.77.) RESULTS
For lack of space the complete data on the one hundred cases cannot be published, so they have been tabulated in the form of general averages (table I), showing the maximum and the minimum in each measurement to give an idea of the range of variation. Our first consideration was to compare the theoretical and observed colloid osmotic pressures. I n the total averages we find a difference of 1.24 mm. of mercury (1.78 cm. of water), or a difference of 9.4 per cent. Besides this discrepancy the colloid osmotic pressure reading has to account for the corrections in the probable error in the determination of the protein fractions, etc. We made only one determination of each one of the sera. One must consider also the correction of the volume occupied by the proteins, ion-pressure differences in non-ideal solutions, the volume occupied by the protein molecules, that is, the b in van der Kaals equation, and other technical corrections, which were not taken into consideration. So we consider that the agreement between these two values is very good. The ratio p/RTCp in the general average is 0.906. The minimum 0.70 was from a serum with a total protein of 10.75 and anA/G of 0.77, a specific gravity of 1.0352, and a viscosity of 2.59, a definitely abnormal serum. The maximum, 1% hich should be 1.00, was found t o be 1.07 in a serum with a total protein of 6.04 per cent and an A / G of 2.45, probably opposite to the one above. I t may be suggested that probably the general average would be higher if some of these abnormal sera had been eliminated from our general consideration. But it is of importance to know how these kinds of sera behave. T H E JOURNAL OF PHYNCAL CHEMISTRY. VOL.
42, NO. 5
662
JOS6 ZOZAYA
The literature on colloid osmotic pressure determinations on human serum is very extensive, as has already been noted in the introduction, so the results of our averages were determined from Drinker's table. The colloid osmotic pressure was 33.25 cm. of water or 24.44 mm. of mercury. No correction was made for the temperature. The total protein in the average was 7.76 per cent, and the number of cases studied was three hundred and ninety-four. The value of n would be 3.02 mm. of mercury, compared to 1.702 in our cases. We are not able to explain these differences, the figures given by Drinker and Field being so much higher, nor can we correlate their findings with the theoretical expectation when taking into consideration the molecular weights of serum albumin and globulin. These high values for the colloid osmotic pressure of human serum as found by other investigators have made the understanding of the water TABLE 1 Total average with maxima and minima o j the various calculations on one hundred sera
-*p =
observed colloid osmotic pressure in millimeters of mercury. RTCp = calculated colloid osmotic pressure from formula 1. n = colloid osmotic pressure per gram of protein (determined). no = colloid osmotic pressure per gram of protein (theoretical).
interchange rather difficult, and several hypotheses have been advanced t o explain this fact, mainly those of Krogh (7), Schade (12), and Landis (8). Krogh and his coworkers could not detect any accuniulation of fluid in the tissues until the obstructing pressure exceeded 15 mm. of mercury. They concluded that this represented a critical pressure below which there was no gross disturbance of the fluid balance between blood and tissues. It is of interest to note that our highest colloid osmotic pressure observed was 13.54 mm. of mercury, which is below the critical pressure observed by them. I n another paper we shall discuss this point further. Govaerts (6), who studied the colloid osmotic pressure of human serum extensively and whose results are generally quoted in the literature, found that 1 g. of serum albumin ezerted a pressure of 5.5 mm. of mercury and I g. of globulin a pressure of 1.4 mm. of mercury. From these findings
PHYSICOCHEMICAL STUDY OF BLOOD SERA.
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he suggested the calculation of the colloid osmotic pressure of a serum from the A / G , and he thinks that there is an approximation of within 10 per cent. We have plotted the A / G and our value n in figure 2. We can see the wide distribution of our findings away from the theoretical line. So it is difficult to imagine any close agreement by calculating colloid osmotic pressure from these data. At the same time it would be inadvisable to calculate any colloid osmotic pressure of any serum from standardized formulas, for one would not be able t o detect those sera which TABLE 2 Group of cases showing the same values of A / G and no, with different values of n and p / R T C p n
3.
,
,
,,,,, , , ,
4.. ,,, ,,, , ,, 19. . . . . . . . . . . . 20. . . . . . . . . . . . 25. . . . . . . . . . . . 31. . . . . . . . . . . . 39. . . . . . . . . . . . 62. . , . , , , . . , . . 73. , . . , . . , , , , . 84. . . . . . . . . . . . ,
1.0274 1.0254 1.0277 1.0273 1.0258 1.0246 1.0265 1.0271 1.0261 1.0289
1.75 1.65 1.79 1.75 1.61 1.64 1.69 1.73 1.68 1.90
7.58 6.81 7.03 7.03 6.59 5,83 6.16 6.59 6.37 8.23
2.05 2.02 2.00 2.00 2.03 2.00 2.05 2.03 2.05 2.02
no
PIRTCP
____
~
1.91 1.91 1.91 1.91 1.91 1.91 1.91 1.91 1.91 1.91
1.68 1.80 1.92 1.80 1.85 1.72 1.73 1.56 1.92 1.51
0.88 0.94 1.00 0.94 0.97 0.90 0.91 0.94
1 .oo 0.79
TABLE 3 Group o j cases showing a value f o r n of 1.80 CASE N O .
SPECIFIC QRAYlTY
4.......... 20 . . . . . . . . . . 34 . . . . . . . . . . 47, . . . . . . . . . 48 . . . . . . . . . . 70. . . . . . . . . . 72. , , . , , , , . . 81. . , . . , . . , .
1,0254 1,0273 1.0247 1,0251 1,0273 1,0260 1,0248 1,0260
=/ 1.65 1.75 1.64 1.69 1.74 1.68 1.65 1.69
6.81 7.03 6.59 6.59 6.92 6.70 6.05 6.92
QLORULIQ
no
2.02 2.00 1.71 1.31 2.54 1.78 2.71 2.10
1.91 1.91 1.85 1.79 1.97 1.87 1.99 1.92
0.94 0.94 0.97 1.01 0.91 0.96 0.81 0.94
behave differently owing to radical changes in the behavior of the molecules because of some disease condition. To study further differences in sera besides the A/G, we selected a group of cases which had the same total protein per cent (table 4) and observed the variations in the other measurements. The value of A / G ranged from 1.31 to 2.46, the value of n from 1.56 to 1.96, and the ratio p / R T C p from 0.85 to 1.01, showing that the concentration of the protein in itself had no direct effect on the value of n.
664
JOS6 ZOZAYA
We then studied the group of cases with a p/RTCp of less than 0.85 (table 5 ) , and noticed that the total concentration of the protein ranged from 6.05 per cent to 10.75 per cent, the A/G from 0.77 to 2.81, and the value of n from 1.09 to 1.67, with nothing characteristic to explain the low ratio. Then we selected a group of cases which showed a value for
'I r-.
FIG.2. Relation of albumin-globulin ratio to osmotic pressure per gram of protein TABLE 4 Group of cases showing the same total protein and different ualues of
~~
~~~
n and
p/RTCp
Vl8COBITY
TOTAL PROTEIN
ALBUMIN QLOBULIN
1L
no
PIRTCP
1.69 1.62 1.67 1.72 1.68 1.73 1.77 1.73 1.61 1.67 1.78 1.68
6.59 6.59 6.59 6.59 6.59 6.59 6.59 6.59 6.59 6.59 6.59 6.59
1.31 1.43 1.86 1.86 1.86 1.86 1.86 2.03 2.03 2.24 2.46 2.46
1.80 1.74 1.69 1.61 1.79 1.88 1.78 1.56 1.85 1.85 1.91 1.96
1.79 1.80 1.88 1.88 1.88 1.88 1.88 1.91 1.91 1.94 1.96 1.96
1.01 0.96 0.90 0.85 0.95 1.oo 0.95 0.94 0.97 0.95 0.97 1.oo
~
47.. . . . . . . . . . . 43. . , . . , , . , , 21. . . . . . . . . . . 6... . . . . . . . . . 28. . . . . . . . . . . . 11. . . . . . . . . . . 80. . . . . . . . . . 62. . . . . . . . . . 25. . . . . . . . . . . 45. , . . , . . , . , 91. . . . . . . . . . . 41, . . . . . . . . . . ,
,
,
1.0251 1.0255 1.0255 1.0277 1.0261 1,0264 1.0271 1.0271 1.0258 1.0262 1.0275 1.0260
-
-
p / l Z T C p of 1.00 or higher (table 6), and here again we noticed the variation in the total protein from 6.04 to 7.47, in the A / G from 1.31 t o 2.45, and in the value of 7~ from 1.76 to 2.11. The value of 1.07 is not explained by us, except as an error in the determination of the total protein. Pauli (11) discusses the relation between viscosity and colloid osmotic
PHYSICOCHEMICAL STUDY O F BLOOD S E R A
.
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TABLE 5 Group of cases with a value f o r plRTCp of less than 0.86 CARE NO
.
1. . . . . . . . . . 13. . . . . . . . . . 16 . . . . . . . . . . 40 . . . . . . . . . . 66 . . . . . . . . . . 72 . . . . . . . . . . 77. . . . . . . . . . 79. . . . . . . . . . 84 . . . . . . . . . . 85. . . . . . . . . . 88 . . . . . . . . . . 95 . . . . . . . . . . 98 . . . . . . . . . . 105. . . . . . . . . .
SPECIFIC QRAVITY
VISCOSITY
1.0268 1.0270 1.0263 1.0287 1.0267 1.0248 1.0270 1.0271 1 .0289 1.0325 1.0352 1.0272 1.0290 1.0298
1.74 1.72 1.68 1.85 1.71 1.65 1.74 1.78 1.90 2.32 2.59 1.82 1.93 1.96
TOTAL PROTEIN
ALBUMIN
n
QLOBULIN
no
PIRTCP
1.87 1.73 1.98 1.94 1.88 1.99 1.81 1.89 1.91 1.60 1.58 1.68 1.81 2.00
0.81 0.81 0.84 0.81 0.82 0.81 0.84 0.80 0.79 0.83 0.69 0.74 0.79 0.83
. . . 8.34 7.69 6.70 7.91 6.37 6.05 7.14 7.14 8.23 9.11 10.75 6.92 8.02 7.47
1.55 1.41 1:65 1.57 1.54 1.80 1.52 1.52 1.51 1.33 1.09 1.23 1.43 1.67
1.80 1.17 2.58 2.29 1.91 2.71 1.50 1.91 2.02 0.78 0.77 1.00 1.51 2.81
Group of cases showing values f o r plRTCp of 1.00 or over CABE NO
.
11. . . . . . . . . . . 19 . . . . . . . . . . . 22. . . . . . . . . . . 26 . . . . . . . . . . . 33 . . . . . . . . . . . 38 . . . . . . . . . . . 47 . . . . . . . . . . . 55 . . . . . . . . . . 73 . . . . . . . . . . . 59 . . . . . . . . . . .
8 PE CI F IC QRAVITY
VIWOSITY
1.0264 1.0277 1.0275 1.0258 1.0263 1.0282 1.0251 1.0268 1.0261 1 .0286
1.73 1.79 1.72 1.64 1.71 1.82 1.69 1.81 1.68 1.83
ALBUMIN
TOTAL PROTEIN
QLOBULIN
6.59 7.03 7.47 6.70 6.37 7.69 6.59 6.04 6.37 7.36
1.86 2.00 1.16 1.78 2.26 1.09 1.31 2.45 2.06 1.50
n
no
. . .. 1.88 1.91 1.73 1.87 1.94 1.71
1.88 1.91 1.72 1.85 1.94 1.76 1.80 2.11 1.92 1.81
plRTCp
1.00 1.00 1.00 1.00 1.00 1.03
TABLE 7 Cases showing viscosity of 1.64 to 1.66 CASE NO
.
4. . . . . . . . . 15. . . . . . . . . 17.,. . . . . . .
~~~~~y
1.0254 1.0257 1.0255
1
ALBUMIN
VISCOSITY
TOTAL PROTEIN
6.81 7.02 6.70
2.02 2.02 2.33
1.80 1.72 1.83
(ILOBULIN
21 . . . . . . . .
1.0255
6.59
1.86
1.69
26 . . . . . . . .
1.0258 1.0247 1.0257 1.0248 1.0253
6.70 6.59 7.03 6.05 7.14
1.78 1.71 1.84 2.71 1.76
1.85 1.80 I .72 1.80 1.78
34 . . . . . . . . .
35 . . . . . . . . . 72 . . . . . . . . . 102. . . . . . . .
I
no
1.91 1.89 1.95 1.88 1.87 1.85 1.88 1.99 1.86
0.94 0.89 0.93 0.90 1.00 0.97 0.92 0.81 0.95
666
JOS6 ZOZ.4YA
pressure in albumin solutions, showing the parallelism between them. In table 7 we selected a group of cases with a viscosity of 1.64 to 1.65. We noted that the protein concentration varied between 6.05 and 7.14, the A / G between 1.71 and 2.71, the value of n between 1.69 and 1.85, and the value of p/RTCp between 0.81 and 1.00. If viscosity has any direct relation to colloid osmotic pressure we are not able t o show it in our cases. It may well be that there are too many other factors which work in different directions t o be able t o show it here. Finally we studied a group of cases with st specific gravity of 1.0272 to 1.0273 (table 8), and we noted that the protein concentration varied between 6.70 and 7.36, the -4/G between 1.50 and 2.54, the value of n between 1.51 and 1.88, and the p/RTCp between 0.79 and 0.97, showing that there is no general effect. TABLE 8 Cases showing specific gravity of 1.0272 to 1.0373 ALRUMiN
____ 8 . .. . . . . . . . 1.78 20... . . . . . . . 1.75 2 3 . .. . . . . . . . 1.75 24.,. . . . . . . . 1.72 1.74 27. . . . . . . . . . 48.. . . . . . . . . 1.74 5 3 . .. . . . . . . . 1.74 1.74 5 8 . .. . . . . . . . 1.87 82.. . . . . . . . .
QLOBULIZI
7.14 7.03 7.25 6.70 7.03 6.92 6.93 6.70 7.36
1.87 2.00 2 37 2 13 2.41 2.54 1 63 1.50 1 11
1.51 1.80 1.57 1.88 1.77 1.80 1.79 1.71 1.65
1.88 1.91 1,95 1.92 1.96 1.97 1.84 1.81 1.71
0.79 0.94 0.91 0.97 0.90 0.91 0.97 0.94 0 97
From these observations we can come t o the general conclusion that the colloid osmotic pressure of human serum can not be predicted from a generalized formula, for there are too many factors which influence it besides the A / G , the protein concentration, the specific gravity or the viscosity. There is no doubt an intrinsic individuality in the physicochemical conditions that are found in each serum, besides the many effects such as the electrolytic antagonism of ions, Na, K against Ca, Mg, as well as the cholesterol-phospholipin equilibrium which von Farkas (17) has shown affects the colloid osmotic pressure of sera. Fishberg (5) finds that the colloid osmotic pressure per gram of protein is higher in lipaemic bloods. The comparison of the measured with the theoretical colloid osmotic pressure is an important criterion by which t o judge the results obtained as well as the deviation from the expected value which pathological sera may have. I t will also help t o direct further research to elucidate other
PHYSICOCHEMICAL STUDY O F BLOOD S E R A .
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factors that affect the measurement. R e can at the same time mistrust obtained values which deviate very far from the theoretical expectation.
I wish to acknowledge to Prof. G. S. Adair the kindness and personal instruction as to the use of this method and t o thank Dr. S. D e w . Ludlum for the interest and facilities given by him for the accomplishment of this work. REFERENCES (1) (2) (3) (4) (5) (6)
(7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18)
ADAIR, G. S.: Proc. Roy. SOC.(London) A108, 627 (1925). ADAIR, G. S.: Skand. Arch. Physiol. 49, 76 (1926). ADAIR, G. S., AND ROBINSON, M. E.: Biochem. J. 24, 1864 (1930). DRINKER,C. K., AND FIELD,IM.E.: Lymphatics, Lymph and Tissue Fluid. Williams & Wilkins Co., Baltimore (1933). FISHBERG, E. H.: J. Biol. Chem. 81, 205 (1929). GOVAERTS, P . : Compt. rend. sac. biol. 89, 678 (1923); 93, 441 (1925); 96, 724 (1926); Bull. acad. roy. m6d. Belg. 7, 356 (1927). A. H.: J. Clin. Investigation 11, KROGH,A,, LANDIS,E. M., AND TURNER, 63 (1932). LANDIS,E. M.: Heart 16, 209 (1929-30); Am. J. Physiol. 93, 353 (1930). MARRACK, J., AND HEWITT,L. F . : Biochem. J. 21, 1129 (1927). MAYRS,E. B.: Quart. J. Med. 19, 273 (1926). PAULI,W . : in Colloid Chemistry, edited by J. Alexander, Val. 11, p. 223. The Chemical Catalog Co., Inc., New York (1928). SCHADE, H., AND CLAUSSEN, F.: Z. klin. Med. 100, 363 (1924). SORENSEN, S.: The Proteins. The Fleischmann Co., Kew York (1925); Compt. rend. lab. Carlsberg 13, 1 (1917). STARLING, E. H.: J. Physiol. 19, 312 (1895-96). SVEDBERG, T., AND SJOGREN,B.: J. Am. Chem. SOC.60,3318 (1928); 62, 2855 (1930). VERNEY, E. B.: J. Physiol. 81, 319 (1926). VON FARKAS, G.: Z. ges. exptl. Med. 63, 666 (1926). ZOZAYA,J.: J. Biol. Chem. 110, 599 (1935).
