body. The spleen has some influence upon the red cells; R. M. Pear c e

Transcription

1 THE SPLEEN AND THE RESISTANCE OF RED CELLS. By D. ORAHOVATS'. (From the Physiological Laboratory, Cambridge.) BARCROFT and others(l) showed that the spleen may be regarded as a reservoir for blood which is emptied into the general circulation in cases when the body requires more hemoglobin; this reservoir is filled again with blood when such a need ceases to exist. The only source from which the spleen may refill itself is the blood of the general circulation which is supplied to it. A part only of the passing blood is detained and this part may have been selected by the spleen out of the blood of the general circulation, or it may have been detained without any selective choice. If it is selected it must differ in some way from the blood of the body. The spleen has some influence upon the red cells; R. M. Pear c e with Krumbhaar and Frazier(2) have shown for instance that the resistance of the red cells of the blood from the general circulation to hypotonic salt solutions is greatly increased after splenectomy. The experiments described below were performed to investigate the resistance of the red cells of the spleen pulp itself compared to the resistance of the red cells of the body with the aim of obtaining data about the quality of the red cells detained by the spleen and to repeat the experiments of Pearce. The resistance of the red cells of the spleen pulp was compared with the resistance of the red cells of the body (1) to hypotonic salt solutions, (2) to saponin solutions. At the same time the resistance curves for both kinds of blood, as shown by Brinkman, were determined and compared. Eight experiments were performed in each case. Blood of the general circulation was taken by puncturing a given vessel. Blood from the spleen was obtained by making a cut of 4 to 5 mm. on the surface of the spleen a few millimetres deep. The blood obtained in this way was blood contained in the spleen pulp. It may be mixed to a certain extent with blood which, at the moment of taking, was passing through the spleen, but it was thought that, if a difference should be found between this blood and the blood of the body, the known error would not disturb the conclusions because it would mean that the differences between the possible pure blood of the spleen pulp and 1 University of Sofia. Travelling Fellow of the Rockefeller Foundation.

2 THE SPLEEN AND RESISTANCE OF RED CELLS. 437 the blood of the body would be still bigger. It was considered better not to change the normal circulation through the spleen by ligating the artery or the veins. Blood of the splenic vein was not used; many workers have investigated the properties of the blood from this vein and have found extremely variable results. The blood of the splenic vein is the concentrated expression of every change in the volume of the whole spleen. Knowing how easily the spleen takes up and squeezes out blood, it is obvious that differences will easily occur in the qualities of the blood of samples subsequently taken from the splenic vein. To test the resistance of a sample of blood to a given concentration of salt or saponin solution, the number of cells was stated which remained intact at the certain concentration of the haemolitic agent, but not the amount of hemoglobin which has left the cells and has gone into the diluting fluid, as it is usually done. A modification of the method of Lieb er - mann (8) was used. The blood sample was taken with the usual pipettes for counting red cells. The blood was diluted to the mark 101 with a salt solution of a given concentration instead of diluting it with a normal salt solution. As hypotonic salt solutions, sodium chloride solutions were used and the set of different concentrations was prepared by the drop method of Hamburger. With eight pipettes, eight samples of blood were taken simultaneously and every one of them was diluted with one of a set of salt solutions, these latter differing from each other in concentration by 0 04 p.c.; the solution of lowest concentration causes a complete haemolysis, the highest concentration is a normal solution which does not change the cells. In this way there are eight equal samples of blood each diluted with equal quantities of salt solutions varying in concentration; these concentrations are decreasing in an arithmetic progression. The pipettes were shaken well and left for 20 minutes, then shaken again and the cells in each were counted with the Buerker chamber by the usual method for red cells. The percentage of haemolysis was calculated, using as basis the normal number of cells. It was first found that the process of hamolysis is completed during this 20 minutes and that no further changes in the number of red cells occurred either when in the pipettes or when brought into the counting chambers. Many preliminary experiments demonstrated this. The number of red cells resistant after 20 minutes to a certain hypotonic concentration of salt does not change in the chamber for hours. Thi is not the case when saponin solutions are used. The saponin acts more slowly and to obtain a constant number of cells permanently resistant to a given concentration of saponin one has to wait not less than 1 hour. In all experiments

3 438 D. ORAHOVATS. with saponin the count was begun after 1 hour and 20 minutes. The saponin solutions were made as described by Rywosch(4). A saponin standard solution of 1: 100 of a normal salt solution was prepared and kept sterile. In every experiment from this 1:100 saponin solution eight further solutions of 0.01 c.c. to 1-3 c.c. to 10,000 of a normal salt solution were prepared with a difference of 0-2 between each of them. The diluted saponin solutions should be used the same day. There is no difficulty in the counting of the cells which have remained resistant to a certain solution. The cells in the chamber vary slightly in size; the bigger ones have taken up water and probably lost hoemoglobin; the smaller ones have not been changed but the contours of all cells are intact and thus the counting is not disturbed. The cells remain as such until bursting, after which they disappear in the optically heterogeneous mass of fluid which is not visible in the counting chamber. L. E. Bayliss(7), discussing the work of Brinkman on reversible haemolysis, has found that, with the increase of hypotonicity, the background visible under the microscope is composed mostly of indistinct cells and detritus. He has used slides made of ordinary glass, not chambers of uniform depth, and the red cells observed by him were taken from the paste of a centrifuged blood which was diluted with a hypotonic salt solution equal in amount to the lost serum. Thus in his preparation half of the mass visible under the microscope consisted of cells, whereas in the blood counting chamber the proportion of cells to the whole mass of fluid are in the ratio of *5 to 101; hence the difference in observations. Results with hypotonic solutions. The experiments were performed on cats: urethane was used as an anaesthetic in all cases and chloroform was given only at the start in a few experiments. The urethane was injected subcutaneously 1 hour before the experiment in doses of 2 c.c. of *25 p.c. solution per kilogram body weight. In preliminary experiments it was ascertained that the resistance of the red cells of the body does not change during the experiment under the influence of the aneesthetic or other possible factor. In some experiments the samples were taken first from the general circulation and then from the spleen and in other experiments the opposite order. Brinkman(3), investigating the resistance curves of red cells of different animals treated with balanced solutions containing NaCl, NaHCO3, HCI, CaCl2 and C02, obtained curves as shown in Fig. 1 a. He concluded that in the circulating blood there are clearly expressed three groups of red cells with different resistances against giyen hypotonic salt

4 THBE SPLEEN AND RESISTANCE OF RED CELLS. 439 solutions. First a group which is most resistant and which forms about 10 p.c. of all cells, then a group to which the greatest part of the red cells belong, about 80 p.c., with a middle resistance, and a third group of cells of very little resistance which are laked at a very low degree of hypotonicity. He supposes the first group to consist of young cells, the third of old, and the middle group of the average grown cells. As is shown in Fig. 1 b the resistance curve of the red cells from the general circulation obtained by us is very nearly the same as that of Brinkman. The angles of the curve are not so sharp, but the three groups of cells are well distinguished from each other. In speaking of groups it must be understood that the term is an artificial one used for convenience; actually there is a complete graduation in Fig. 1 from corpuscles which resist a salt solution of 0*64 (or 0.32) to those which resist 0-36 NaCl (or 0-18 balanced solution), but the vast majority are laked by salt solutions of In Fig. 1 c this graduation is shown better; there ~~~~~~~~~~~100' ~~~~~~~~~ ( ~ ~ C ~~~~~~~~~~~ xx Fig. l a and b. Fig. 1c. Fig. 1 a and b. Ordinates. Percentage of corpuscles haemolysed. AbWeiMgce. Concentration of solutions. a-, Brinkman. b x x x x, NaCl. Fig. 1 c. Percentage concentrations of hoemolysis added at each subsequent degree of hypotonicity. the curve represents the amount of haemolysis which is added at each subsequent degree of hypotonicity. In all experiments, when the red cells of the blood withdrawn from the general circulation are treated with salt solutions near the isotonic concentration, a certain number of cells are laked with each small decrease in concentration; with greater dilutions, however, there is a considerable range within which decrease

5 440 D. ORAHOVATS. in the concentration does not markedly affect the number of cells which are laked, until a certain concentration is reached at which the majority of cells disappear sharply and there remain a number of cells which resist for a long time further dilution in salt solution. The curve in Fig. 1 b is an average curve of eight experiments. When this curve is compared with the curve obtained from red cells of the Fig. 2 a. Concentration NaCl-in p.c. -, Cells of general circulation. x x x x, Spleen pulp cells Fig. 3. Fig. 4. Fig. 3. Concentration NaCl in p.c. --, Cells of general circulation. x x x x, Spleen pulp cells. Fig. 4. Concentration NaCi in p.c., Cells of general circulation. x x x x, Spleen pulp cells.

6 THE SPLEEN AND RESISTANCE OF RED CELLS. 441 spleen pulp taken at the same time and treated with the same salt solutions, a difference in the behaviour of both kinds of cells is obvious. Fig. 2 a represents the two average curves of all experiments; Figs. 3 and 4 show two single experiments with the greatest and the smallest differences. There are two points in which the curves differ from each other: (1) the red cells of the spleen pulp are less resistant to hypotonic salt solutions than the red cells of the general circulation, and (2) the curve of the spleen blood shows a different shape. Not all three clearly expressed parts in the resistance curve of the cells from the general circulation exist in the spleen blood curve. The most resistant group of cells is the same in both curves, only less resistant in the spleen blood curve, but the two other groups of cells in the spleen blood curve cannot be distinguished from each other. The curve rises in a straight line and the difference in the resistance between the spleen blood and the blood of the body is the greatest in this part of the curve. The least resistant group in the curve of the general circulation either does not exist in the curve from the blood of the spleen pulp or it is laked at once. In Fig. 2 b the difference in the resistance between the spleen blood _ _ X 10, Fig. 2 b. Concentration NaCl in p.c. - -, Spleen pulp blood. -, Blood from the general circulation. and the blood of the body is shown in the same way as in Fig. 1 c; the curve shows the amount of haemolysis added at each subsequent degree of hypotonicity; it may be seen that a greater number of cells of the spleen blood pulp are laked at lower degrees of hypotonicity than the number of cells of the body and that the hemolysis begins at a higher salt concentration. The differences in the concentrations of the salt

7 442 D. ORAHOVATS. solutions at corresponding percentages of haemolysis are given in Table I. TABLE I. Percentage of haemolysis lo0 Differencesin salt concentrations *06 * necessary to cause haemolysis of same degree for both kinds of blood The conclusion arrived at by comparison of both curves would be that the spleen blood has not only a diminished resistance, but that the spleen pulp blood is of a different mixture of cells. To avoid possible errors the resistance curves were determined in control experiments for the blood of the spleen pulp, the blood from a brachial artery and the brachial vein. The curves obtained were the same as before and there was no difference between the curve for the blood of the artery and of the vein either in regard to the shape of the curve, or the degree of resistance. In other controls blood was taken from muscles by making cuts in the latter, and compared with the blood from arteries and veins of the body; no difference in the resistance was found. Only in the first experiment was there a non-typical result in so far as the spleen blood curve crossed the curve of the blood of the body, nevertheless both curves had the typical shape. Another unexplained result was obtained with the blood from the vena hepatica, an ear vessel and the spleen pulp. The curve of the liver blood had _ the same shape as the blood from 100 the ear but was less resistant than 90 the latter, lying between the curve 80 / of the blood from the ear and that 70 from the spleen pulp. 60 Results with saponin. The re- 1 sistance of the red cells of the spleen 50 pulp and the red cells of the general 40 circulation was then tested against 30 saponin solutions. The experiments 20 were performed in the way already 10 described. The results obtained 0 may be seen in the average curve l.l 1.3 of all experiments shown in Fig. 5. Fig. 5. Concentrations of saponin solutions. The differences in the concentra-, Cells of general circulation. x x x x, Spleen pulp cells. tions of the saponin solutions at corresponding percentages of haemolysis for both kinds of blood are

8 THE SPLEEN AND RESISTANCE OF RED CELLS. 443 given in Table II. As is seen from these curves the red cells from the spleen pulp compared with the red cells from the general circulation behave towards a given saponin solution in a way opposite TABLE II. Percentage of hlemolysis Differences in saponin concen- 09 *11 *12 *12 *11 10 *08 *06 *08 *10 *16 trations necessary to cause haemolysis of same degree for both kinds of blood to that towards a hypotonic solution. They are more resistant to saponin solutions of given concentration than are the red cells from the general circulation. The shape of the resistance curve of the body blood shows the same three groups of cells as the resistance curve to hypotonic salt solutions. It is to be noted that the least resistant group appears smaller, and that the whole curve is steeper. On the other hand, the curve of the spleen pulp cells against saponin, instead of having three well-marked portions, has only two: that which corresponds to the corpuscles of lowest resistance, and which tends in the blood curve to become tangential, is absentfromthe spleen curve. The end points of both curves 100 ;ex. are nearer together and the differ- 90.., ences in the concentration neces sary to cause the same percentage of hwmolysis are smaller. In general 60 both curves are less characteristic, 50 but the main result demonstrates 5 the fact that to saponin the red 40. cells of the spleen pulp are more 30 - resistant than the cells from the 20 * general circulation. In four ex- 10. / $ periments a number of samples of a - both kinds of blood were tested, and other samples taken at the Fig. 6. same time were tested against,cells of general circulation against hypotonic salt solutions. The aver- hypotonicity. age of the curves obtained are -, Cells of general circulation against shown in Fig. ~~, shown in Fig , Spleen saponin. pulp cells against hypo- R yw o s c h testing the resistance tonicity. of the red cells of different animals x x x, Spleen pulp cells against saponin. to different htemolytic agents observed the fact that there exists a certain antagonism between the resistance to saponin and the resistance to

9 444 D. ORAHOVATS. hypotonic salt solutions. If different animals are graded as regards the resistance of their red cells to saponin, the following order would be observed: lamb, goat, ox, cat, grey mouse, pig, grey rat, dog, white rat, rabbit and guinea-pig. The resistance of their red cells against hypotonic salt solutions arranged them in the following order: guineapig, white rat, dog, grey rat, rabbit, pig, grey mouse, cat, ox, goat and lamb. In other words just the reverse of the former grouping. Port() pointed out that the order of animals as regards the phosphoric acid content of their cells, as given by Abderhalden(6), is similar to the order of resistance to saponin and the reverse of the order of resistance to hypotonic salt solution. In other words, the higher the content of a cell of phosphoric acid, the more resistant it is to hypotonic salt solution, and the less resistant it is to saponin. Applied to the results of the present experiments, this conception would mean that the difference in the degree of resistance of the cells from the spleen pulp and from the general circulation to hypotonic salt solutions on the one hand, and the reverse relationship of their resistance to saponin solutions on the other hand, could be explained by differences in the salt content of the same kind of cells, especially by differences in their content of phosphoric acid. The red cells of the spleen pulp would contain less phosphoric acid than the red cells from the general circulation. And the three groups of cells, seen in the curve of the resistance of the cells of the general circulation, would mean differences in the content of phosphoric acid among the cells of the general circulation. Brinkman calls them young, old, and the middle group. Judging from the present results the "old" cells, the least resistant, are the cells which are most changed by the spleen, because the part of the curve of the blood from the general circulation formed by them disappears in the curve of the spleen pulp cells; but there is a further consideration: being the least resistant to hypotonic salt solutions, they would seem to contain the smallest amount of phosphates of any of the cells of the blood of the general circulation, for they are the most resistant to saponin, and in the saponin curve for the cells of the general circulation they occupy the same position as that of the young cells in the curve against hypotonic salt solutions. But just this group seems to be the least changed in the spleen pulp red cells' curve; so that according to the results of the present work there are but meagre data upon which to base more than the statement that the red cells which are detained by the spleen are different from the red cells in the general circulation in regard to their content of salts. The question arose: What is the cause of this difference? Is the

10 THE SPLEEN AND RESISTANCE OF RED CELLS. 445 resistance of the red cells to hypotonic solutions reduced during their residence in the spleen, or alternatively, does the spleen sift from the blood the least resistant element? In the latter case the resistance of the red cells in the spleen pulp would not alter with the length of time they were there. In the former case one might expect that the longer a cell remained in the pulp, the less resistant it would become. The next experiments were therefore as follows: The resistance of (1) the red cells of the general circulation, and (2) the red cells which had remained some time in the spleen pulp were compared with that of the red cells taken from the spleen pulp immediately after it had refilled itself with blood. In order to get blood which had just entered the spleen, the splenic nerves were uncovered as far as possible from the spleen, prepared and stimulated by an ordinary electrode. With a little experience the nerves may be found and prepared in a few moments. The stimulation causes a large and obvious contraction of the spleen; the spleen loses nearly half of its volume; the previously smooth surface becomes rough, granulated and hard, and the colour changes from bright to dark red. The contraction follows about half a minute after the stimulation. Three to five minutes after the stimulation has ceased the organ slowly refills itself, and the surface regains its smoothness and its former colour. Red cells from the refilled spleen were taken 5 minutes after the stimulation ceased. To avoid damaging the spleen, and so interfering with its power of contraction, only three samples of each kind of blood were taken. These were tested against the same hypotonic solutions and the results are shown in Table III. They show that the red cells which have TABLE III. Percentage haemolysis in blood from NaCl concentration General Spleen before Spleen pulp p.c. circulation contraction after contraction * * just been detained by the spleen have the same resistance as the red cells which have been contained in the spleen pulp before the contraction and are less resistant (to the same degree as the latter) than the red cells from the general circulation. The above experiments do not exclude the possibility that the resistance of the red cells is altered within a few seconds of their arrival in the pulp and undergoes no further change. This, however, seems to be a less likely contingency than that the less resistant cells are those which

11 446 D. ORAHOVATS. are most easily caught either because they are less plastic or for some other reason. In conclusion, it may be worth recording that we have repeated and confirmed the observation of R. M. Pearce, Krumbhaar and Frazier which originally drew our attention to this subject. The resistance of the red cells of the general circulation of four cats was determined. The next day the cats were splenectomised and the resistance of the red cells of the same vesselsear-determined at different intervals. The changes in the resistance are given in Fig. 7. The resistance increases rapidly for a certain time and then comes back to the previous level. The time of this recovery differs considerably in different animals. The cats stood the operation well, the wounds healing by primary intention. SUMMARY. 1. The red cells contained in the spleen pulp are less resistant to hypotonic salt solutions than the red cells '68 *64 '60 '56 '52 '48 *44 '40.36 '321 0 Days After Splenectory II -l 0 0~~~~ -x K x A x X x Fig. 7. Ordinates. Concentration of NaCl in p.c. 0-Beginning of hsemolysis. x -Complete hemolysis. from the general circulation, and the former are more resistant to saponin solutions than the latter. 2. The first statement means that it is probable that the red cells detained by the spleen have a different content of phosphoric acid than the red cells of the general circulation. 3. The red cells contained in the spleen pulp immediately after the spleen has been emptied and refilled have also a different resistance to hypotonic salt solutions from the red cells of the general circulation. 4. The statement of R. M. Pearce that after splenectomy the resistance of the red cells of the general circulation is increased for a certain time has been confirmed. I am much indebted to Prof. B arcro ft for the subject of this work, and his aid throughout the research. Qh x x x

12 THE SPLEEN AND RESISTANCE OF RED CELLS. 447 REFERENCES. 1. Barcroft, Harris, Orahovats and Weiss. This Journ. 60. p Pearce, R. M., Krumbhaar and Frazier. The spleen and anaemia Brinkman. Arch. Neerland. de Phys Rywosch. Pfluiger's Archiv, 116. p Port. Hober's Physikalische Chemie, Abderhalden. Zeitschr. f. physiol. Chem. 25. p Bayliss, L. E. This Journ. 59. p Liebermann, L. v. Deutsche Med. Woch