172
D . H . DRUMMOND, J. P . TOLLMAN, A N D F. I,. RICHARDS
THE EFFECT OF
itoEwrC;m IRRADIATION UPON ERYTHROCYTES
D. H. DRUMMOND, J . P. TOLLMAN,
AND
F. L. RICHARD8
Department of Pathology, University of Nebraska Medical School, Omaha, Nebraska
Received May 10, 1939
It was suggested by Failla (2) that as a result of irradiation the proteins of the cells were broken down to compounds of lower molecular weight. This raised the intracellular osmotic pressure, causing entrance of water into the cell with swelling. I n order to test this theory Miss Woodard (7) studied the effects of irradiation with x-rays on the colloidal properties of sheep erythrocytes. Although red blood cells are quite radio-resistant, these were chosen since they presented a relatively simple single membrane equilibrium which would not be the case in nucleated cells. Miss Woodard found that cells which were suspended in hypertonic saline showed swelling after irradiation; those suspended in isotonic or hypotonic saline showed a decrease in volume after irradiation, as the result of the extensive hemolysis which occurred. Cells which had been kept in the ice-box for 6 or more days showed some spontaneous swelling and showed a decrease in volume after irradiation. This was considered to be due to a coagulating effect on the proteins. She felt that there was no primary damage to the cell membrane as a result of stretching in those cells which showed marked swelling. Since the effect of imbibition of water would raise the concentration of electrolytes in the suspending solution, it was felt that a study of the concentration changes in the saline would give more information on this problem. Also, injury to the cell membrane would cause an opposite effect by allowing diffusion of salt into the cell. I n the present work we studied these relationships by measuring the cell volume, the chloride concentration of the solution in which the cells were suspended, and the per cent of hemolysis. EXPERIMENTAL
Sheep blood was used for the work, since it is quite resistant to hemolysis. The blood was collected in a flask containing sufficient sodium citrate to prevent coagulation. The blood was then centrifuged to throw down the cells, and the serum was removed. The cells were then washed four times with saline of the concentration to be used in the experiment and finally suspended in saline, the volume being made up to a value somewhat above that of the original blood volume. The washed cells were kept in the ice-box. Before each series of experiments the cells were washed once more, and then 15-cc. samples of the suspension were removed and trans-
EFFECT OF ROENTGEN
IRRADIATION
ON ERYTHROCYTES
173
fcrred to Pyrex tcst tubes which were stoppered. These were then exposed for 45 min. to irradiation from a therapy tube at 22 cm. distance, using 200 kilovolts and 18 milliamperes and producing 365 r. per minute, giving a total irradiation of 16,425 r. The only filtration was by the wall of the Pyrex tube, which was about 1 mm. thick. At varying times after irradiation blood from one of the tubes was transferred to a hematocrit tube which was then centrifuged and the supernatant fluid was removed. After suitable dilution, to adjust the chloride-ion concentration to the correct range, the chloride content was determined by the method of Whitehorn for chlorides in urine (6). The same determinations were made on the control tubes. The cells in the control tubes were hemolyzed by adding distilled water and making up to a volume equal to the initial TABLE 1 Results of preliminary work with isotonic solutions
1
NO.
37.6 37.6
Cells equilibrated with 0.145 m N a C l . . . . , .
3.0
37.4(mean) Cells centrifuged and reequilibrated with 0.145 m NaCl
Cells irradiated after 15 hr. in ice-box
36.1 36.8 (mean)
1~ : I
22.0 18.0 18.0
3.1 51.5 39.0 41.0
volume of the suspension. This was designated as 100 per cent hemolysis. This solution after dilution to approximately the same hemoglobin concentration as the supernatant saline was used as a standard for the colorimetric determination of hemolysis. Some of the original control tubes were centrifuged to pack the red cells. The supernatant fluid was carefully removed and then more dilute saline was added up to the original volume. After the cells were thoroughly mixed with the saline, a specimen was removed for a hematocrit determination. The contents of the test tube were then centrifuged again and the supernatant fluid analyzed for chloride and degree of hemolysis. Results of preliminary work with isotonic solutions are recorded in table 1. Since the marked hemolysis made these results unsatisfactory, the work was then carried out in hypertonic solutions. These results are recorded in table 2.
174
D. H. DRUMMOND, J. P . TOLLMAN, AND F. L. RICHARDS
TABLE 2 Results with hypertonic solutions
A. Fresh cells NO.
~
EEMATOCRIT
1
HEMOLYSIS
1
CBLOBIDE CONCENTRATION
R
~
~
Control equilibrated with 0.336 m NaCl after washing with 0.142 m NaCl 1. . . . . . . . . . . . 2. . . . . . . . . . . .
I
0.5
~
0.263 0.273 0.268 (mean) 1 ~
~
28.4 (mean)
I
1.00 0.99
Reequilibrated with 0.154 m NaCl
1. . . . . . . . . . . . 2. . . . . . . . . . . . ~
36.0 36.0
0.190
0.178
j
1.26 1.26
Cells equilibrated with 0.336 m NaCl and then treated with x-ray, 2 hr. after irradiation
1 2
0.270
'
0 272
I
1.13 1.14
~~
Same after standing 15 hr. after irradiation 1. . . . . . . . . . . .
2. . . . . . . . . . . . B. Cells after being kept in ice-box for 5 days NO.
1
I
1. . . . . . . . . . . . 2. . . . . . . . . . . . .
EEMATOCRLT
98.5 28.5
1 i 1 j
HEMOLYSIB
~
CHLOBIDE CONCENTRATION
0.267 0.267
0.5 0.5
I i
i
R~~~~~~
1.00 l.OO
Reequilibrated with 0.142 m NaCl
1 . . . . . . . . . . . . I. 2 . . . . . . . . . . 1.
34.0 35.0
~
I
0.5 0.5
i
1
0.173 0.174
1.19
Cells equilibrated with 0.314 m NaCl; 15 hr. after irradiation
1 . . . . . . . . . . . .1 2 . .. . . . . . . . . . ! 3. . . . . . . . . . . . 4. . . . . . . . . . . . ~
32.5
I
1.5
32.0 32.0
1
1.5
0.258 0.260 0.262 0.260
1
1
1.16 1.13 1.13 1.13
k
~
EFFECT OF ROENTGEN IRRADIATION
175
O N ERYTHROCYTES
TABLE 2-Concluded C. Cells after being kept in ice-box for 10 days
Control equilibrated with 0.308 m NaCl ~-
I 1 2
I
I
I
p a cent
30.5 30.5
0.243 0.245
1
1.07 1.07
Reequilibrated with 0.135 m NaCl 1
36 3 36 3
2
1
'
0 8 0 8
1 1
0.167 0.167
~
1 28 128
Cells equilibrated with 0.308 m NaC1; 4 hr. after irradiation 1 . . . . . . . . . .. . ' 2. . . . . . . . . . . 3 . . . . . . . . . . .1 4. . . . . . . . . . ~
32.5 32.5 32.5 32.5
~
1.0 1.0 1.0 1.0
1
1
I ~
0.223 0.223 0.222 0.222
I I
1.18 1.18 1.18 1.18
DISCUSSION
It is seen from table 2 that after irradiation the cells showed uniform swelling and some degree of hemolysis. Also the chloride concentration decreased, showing that some chloride had diffused into the cells. If there were no injury to the membrane then water would be withdrawn from the saline with the swelling, causing a rise in the concentration of the saline. It is evident that both water and chloride diffused into the irradiated cells. The question now arises as to whether or not the entire swelling is due to membrane injury. If the cell membrane were injured so that it would freely transmit ion4 from the outside, sodium chloride would diffuse into the cell. The osmotic concentrations inside and outside would then be equalized and the cell \ \ o d d assume a volume equal to that which it nould assume if it were placed in a n isotonic solution. Consequently i t is necessary to know volume changes in the normal cell for different concentrations of the saline. Since the cells had been repeatedly washed with saline, the ion* which could be replaced by chloride within tlic cells have been entirely replaced, owing to repeated equilibration according to the Gibbs-Donnan law. Consequently the concentration of chloride outside the cells is a mcasure of the hypertonicity of the solution, since no chloride shift can occur. In figure 1 there is plotted relative volume against chloride-ion concentration in millimolrs pcr litrr. Thr re5iilts ohtaincd in this work arc indicated by
176
D . H . DRUMMOND, J. P. TOLLMAN, AND F. L. RICHARDS
circles. In the higher concentrations there is quite good agreement. The scattering in the lower concentrations may be due to some difference in time of standing before the hematocrit determination, since Ponder and Saslow (3) have shown that there is some change in cell volume with time after change in the concentration of the suspending medium. These authors (4)have shown that the red cell does not act as a perfect osmom-
C
V FIG. 1. Plot of relative volume of the cells against chloride-ion concentration in millimoles per liter
eter, although Davson (1) has reported that the red cell is more nearly an ideal osmometer than Saslow and Ponder have found and that there is no diffusion of cations into the cell in hygertonic solutions. Warburg and Winge (5) have studied this problem, and their results are represented in the form of a graph showing the relative volume of erythrocytes on the abscissa and the relative salt concentration on the ordinate. It is seen that in the higher concentrations their curve approached a straight line, which is the nearest graphic representation of our data. By re-
EFFECT OF ROESTGEX IRRADIATION ON EHTTHI~OCTTES
lii
computing their results in the samc terms as our graph it was possible to incorporate their results. Their figures, taken from arbitrary points on their curve, are represented by the double circles in figure 1. It is seen that a t higher concentrations their curve straightens out into that represented by our results. The curve drawn in figure 1 is chosen to give the best representation of the combined points of our results and the Warburg and Winge curve. The curve on the graph is defined in the equation:
H
= -0.00287C
+ 1.76 + (12.7 X 104)/C3
(1)
The solid points represent the recomputed ideal results given by Ponder and Saslow (4) for cells acting as perfect osmometers without diffusion of ions. I t is not the purpose of this paper to discuss in detail the question of ionic permeability of red cells in hypertonic solution. I t is nevertheless
b
. . . . . .' .. .*. . . . . ...... .. '&*
s,
Cl,
..... ,., Ai': '.
*
. . : . e
FIG.2. (a) A tube before irradiation with an arbitrary separation of cells; tube after irradiation
evident that there is some marked impermeability, since cells shrink in hypertonic solutions. Furthermore there is a relationship between cell volume and saline concentration represented by figure 1. In subsequent developments this graph will be used for reading off relative cell volume for any given saline concentration. The problem now presents itself to determine from the data how much of the swelling is due to injury to the membrane and how much is due to swelling of the cytoplasm due to irradiation. In figure 2 the upper drawing represents a tube before irradiation with an arbitrary separation of cells. B1represents cells which will change volume without membrane injury after irradiation. bl represents cells which will swell as a result of injury to the membrane with diffusion inward of sodium chloride, together with the component h, which will hemolyze. After irradiation B1changes to Bz. Also the unhemolyzed portion of bl, namely,
178
D. H. DBUMMOND, J. P . TOLLMAN, AND F. L. HICHAHDS
(bl - h ) , has changed to ba. The actual ratio of volumes irrespective of hemolysis and membrane changes is Ba/B1 = k
(2)
where k is an arbitrary swelling due to irradiation, which is to be computed. If Hl and Hz are the initial and final hematocrit readings, then
H I = bl
+ BI
and HZ = bz
+ BZ
(3)
Since the total chloride present has not changed, although the concentration has done so, owing to diffusion into bl, it is evident that
Sic11 = (Sa
+ bi)Cla
(4)
and hence, bi
=
SIC11 - sscle Cls
where S and Cl represent the volume and chloride-ion concentration of the saline, respectively. To find BZtwo factors must be taken into account. One of these is the swelling due to irradiation and is measured by the factor k in equation 2. If k were unity there would still be a change in Ba because of a change in chloride concentration from Cll to Clz. This volume change can be read off from the graph; it is the factor read on the abscissa labeled V and denoted here by f c . Therefore
Bz
=
BI.k.f,
(5)
Similarly the volume bz is equal to the unhemolyzed portion of bl multiplied by the volume factor f i , which is that for an isotonic solution and is seen from the graph to be 1.38. b2
= (bl
- h) X
1.38
(6)
This relationship holds, since if b1 is completely permeable to sodium chloride and water, then the osmotic pressure inside the cell will be equal to that outside; consequently it will assume a volume equal to that which it would abbume in an isotonic solution. The quantity h is the per cent actually hemolyzed as seen in figure 2. The per cent of hemolysis given in table 2 is based on 100 per cent hemolysis of the initial cell volume. Therefore h = per cent hemolysis (6) times HI. Finally, writing H2 - b2 for R2 and H I - bl for B1 (equation 3) and substituting from rquation 6:
Hz
-
(bl
- +Hi)
X 1.38 = (HI - b1)fc.k
(7)
E F F E C T OF ROENTGEN IHHADIATION ON LHYTHROCYTES
179
Solving for k : k
=
Hz
-
(bi
- +Hi) X 1.38 ____-
(Hi -
(8)
bi).fc
Using equation 8 the values for Iz were calculated and recorded in table 3. It is seen that the fresh blood shows the highest values for k . After standing for some time there is a decrease and finally k becomes less than unity, representing a shrinking corresponding to the coagulation described by Miss Woodard. As for the nature of this process, it, is felt that neither these results nor those of Miss Woodard throw any light upon this. Certainly there is some injury to the membrane of the cell. However, it is
.~
TABLE 3 Calculated values of k
A. Fresh cells 2 hr. after irradiation.
.,... . . .
1.16 1.16 1.21 1.26 1.20 (mean)
1 1
Fresh cells 15 hr. after irradiation.
1.06 1.06 H. Cells 5 days old; 15 hr. after irradiation. , . .
1.OS
1.06 1.07 (mean) 2
i
0.85 0.85 0.84
1
'
0.84
C. Cells 10 days old; 1 hr. after
0.85 (mean)
impossible to say whether this is primarily due to irradiation, or is due to injury from swelling. I t might be that as the result of the swelling from irradiation some membranes are injured enough to produce hemolysis, while those more hardy or those subjected to less distension allowed free access to ions without releasing proteins.. Further, the idea of Miss Woodard that this process is due to increase in the number of molecules in the protein is very questionable. Her idea would suggest more of a physical change of the molecular form of the protein, which one would c:xpc.ct to bc independent of changes in its chcmistry produced by standing. It would seem equally plausible that there arc some colloidal changes, Imssibly related to changes in isoelectric point, which produced some swell-
180
GEOFFREY BROUGHTON
ing on a chemical basis resulting in imbibition of water. This mechanism would be essentially different from inflow of water to equalize osmotic relationships. It would seem that the only answer to this would be direct molecular weight determinations on irradiated hemoglobin rather than dependence upon such complex relationships as those studied. SUMMARY
1. It was found that irradiation by Roentgen rays caused swelling of erythrocytes. 2. Associated membrane damage was found dependent either upon irradiation or upon minimal injury due to swelling of the cell. 3. Diminution of volume after irradiation was found in those cells stored in the ice-box for some time. 4. The findings of Miss Woodard are in general confirmed, although it is felt that no adequate explanation can be given for the phenomenon. The authors wish to express their appreciation to Dr. H. B. Hunt of the Radiology Department for the use of the therapy equipment and to Miss Gladys Anderson for carrying out the irradiations. REFERENCES (1) DAVSON, H.: Biochem. J. SO, 391 (1936). (2) FAILLA,G . : Address given at the meeting of the American Association for the Advancement of Science, December, 1936. (3) PONDER, E., AND SASLOW, G.: J . Physiol. 70, 169 (1930). E., AND SASLOW, G.: J . Physiol. 73, 267 (1931). (4) PONDER, (5) WARBURG, E., AND WINQE,K . : Acta Med. Scand., Supplement xvl, p. 500 (1928). (6) WHITEHORN, J. C.: J. Biol. Chem. 46, 449 (1921). H . Q.: J. Phys. Chem. 49, 47 (1938). (7) WOODARD,
CATALYSIS BY METALLIZED BENTONITES GEOFFREY BROUGHTON Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts Received March 4, 1989
Clays have long been known to possess catalytic activity, some varieties finding considerable industrial use as catalysts. Metallic ions present in the clays undoubtedly influence their activity; in some cases acid clays, Le., clays which have undergone electrodialysis or treatment with acid to remove metallic ions, are found to be more active than the original clays. Nevertheless, no attempt appears to have been made to investigate the
