STUDIES ON X-AGENT XIII. EFFECT OF X-AGENT ON ERYTHROCYTE SEDIMENTATION RATE. Shonan Hygiene Institute, Kamakura, Japan. (Received March 27, 1961)

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1 Japan. J. Microb., Vol. 5, No. 2, 1961 UDC: : STUDIES ON X-AGENT XIII. EFFECT OF X-AGENT ON ERYTHROCYTE SEDIMENTATION RATE HIDEO MORIYAMA Shonan Hygiene Institute, Kamakura, Japan (Received March 27, 1961) Evidence has accumulated to show that there is an unidentified physical factor which affects the growth of bacteria(1)(2), the germination of plant seeds(3), the turbidity of protein solutions(4)(5)(6), the degree of hemolysis of blood cells(7)(8), and the cloudiness of kaolin suspensions(9)(10) This factor is called X-agent for the time being. It has been demonstrated that the X-agent is a kind of a high-energy radiation similar to cosmic rays(11)(12) It apparently consists of a variety of components varying in hardness(10)(12). If it falls upon a certain material substance, a secondary radiation is likely to be emitted from the latter. The agent is varying in a pronounced way with time and also with place. Occasionally, very large increases in its intensity are observed, as if there occurred a storm of the agent. Evidence has also been presented which suggests that the agent frequently tends to progress along a direction extending north and south. The action of the agent upon various substances cited above may consists in its ability to induce some changes in colloidal particles which are composed of either proteins or silicates. If free radicals are set free in the particles by the effect of the agent, the particles may combine with each other, while if the radicals are lost by the agent, the particles may be disintegrated into smaller particles, resulting in the alteration in the turbidity of a colloidal solution. Such a change induced in protein molecules, constituting an organism, may naturally have some influence on the organism. Thus, the X-agent seems to have extensive effects on all living organisms. On the other hand, since colloidal particles of silicates are present abundantly in the air, it is expected that the agent is more or less involved in meteorological phenomena. The appearance or disappearance of the free radicals occurring on the surface structure of the silicate-colloid particles floating in the air must have a great effect on the particles functioning as nuclei around which the vapor is condensed to form a cloud, whereas condensation of the vapor into water droplet, or dissipation of the latter into vapor, is accompanied by a change of temperature, followed by a barometric variation and rise of wind.

2 224 MORIYAMA Vol. 5, No. 2 That the weather is subject to the agent can thus be deduced even when the colloidal particles only are under consideration. It is highly probable, as will be reported in a forthcoming paper, that the agent can induce some changes not only in the silicates or proteins but also in various other substances. It has been found, as presented in this paper, that the X-agent is likewise involved in erythrocyte sedimentation rates. Since the chief component of blood cells is proteins, it is only natural that the rate should be influenced by the X-agent even when the proteins are the sole substance under the effect of the agent. The liberation or the disappearance of free radicals in the protein present on the cell surface should naturally have some effect on the sedimentation rate. The present paper is concerned with the phenomena observed in the sedimentation rate in which the X-agent seems to be involved. MATERIALS AND METHODS Citrated blood is used as a rule to study the sedimentation rates. However, the present work was conducted using red-cell suspensions which were prepared from the citrated blood in the following manner: The red cells were separated from the fluid portion by centrifugation, and after washing three times with saline, they were suspended in 0.9 per cent NaCl solution at about 30 per cent. The blood cells of different persons could be mixed after washing without accompanying any agglutination. Therefore, in the present investigation, in most cases, the blood cells from several persons were mixed together into a suspension and used. A red cell-suspension thus prepared was dispensed into ordinary sedimentation tubes (after Westergren) which were arranged in a row on a usual rack; the distance between every two tubes was 3 cm. As stated in the previous papers(5)(7)(9). a group of test tubes, arranged in a row, containing such substances as bacteria or proteins would develop different turbidities when left for a certain period of time. This is understood to be attributed to a dissimilar distribution of the X-agent among tubes, since each tube may be affected by the secondary radiation emitted from the neighboring tubes. The present work was initially undertaken to determine the nature of the phenomenon, if the phenomenon could be observed also in the sedimentation rates. As is generally known, the sedimentation rate is considerably increased when the tube is placed in a slanted position. Therefore, utmost care was taken so as not to set the tube out of its perpendicular position.

3 April, 1961 STUDIES ON X-AGENT. XIII. 225 RESULTS Observations made by the side of a window facing either south or north. Each of sedimentation tubes in a group, arranged in a line, showed varying sedimentation rate, as indicated in Table 1 and Fig. 1. This may indicate that the X-agent was acting upon the line of sedimentation tubes just like upon a row of tubes containing bacteria or proteins. Each of the tubes showed different sedimentation rate, but not independently of other tubes placed nearby, so that the varying rates tended to give an undulating pattern. The patterns presented in this figure were all obtained with tubes arranged Table 1. Variation in blood sedimentation rates among tubes arranged in a line in north-south direction (see Fig. 1) Distance between every two tubes was 3 cm. Test was made at laboratory temp. Fig. 1. Patterns of erythrocyte-sedimentation rate observed in a line of tubes arranged in north-south direction. Observations were made in southern room (see Table 1). Day of observation; I: Oct. 5, II: Oct. 6, III: Oct. 13, IV: Oct. 21, In cases of III and IV, the tubes were covered with black paper sack.

4 226 MORIYAMA Vol. 5, No. 2 Fig. 2. Patterns of erythrocyte-sedimentation rate observed in a line of tubes arranged in north-south direction. Observations were made in northern room. Day of observarion; I: Oct. 19, II: Oct. 21, III: Oct. 22, IV: Oct. 28, The rates were read three times: 1, 11/2, and 2 hours after sranding. Table 2. Tube No. and the sedimentation rate A. Data obtained from the experimental results presented in Fig. 1

5 April, 1961 STUDIES ON X-AGENṬ XIII. 227 in the north-south direction, and the observations were conducted beside a window, on a table located in a southern room. In Fig. 2 are shown the results of similar observations made in a northern room. It is evident that similar patterns could be attained regardless of the room in which the rates were measured. The sedimentation rate, however, when the observation was performed in the southern room, had a tendency to become higher as the tube was placed closer to the window, as if something capable of increasing the rate was coming from the window. This is not attributable to an ordinary light coming from the window, because similar tendency was seen when the row of tubes were covered with a sack of thick black paper; curves III and IV in Fig. 1 were obtained in this way. Moreover, as will be described later, no change in the rates was found even when the tubes were irradiated with an electric heater (600 w.) or an electric lamp (100 w.) placed 2 meters away. The above mentioned variability in the rate was never ascribed to dissimilarities in any individual characters of the sedimentation tubes, if existed any. The volume of each tube was carefully examined, and those which proved not correctly calibrated were discarded. In Table 2, it is shown that the sedimentation rate was not concerned with any individual characters of tubes. The data were obtained from the experimental results shown in Fig. 1. Observations made in tubes arranged in various directions. As described previously(5), a row of test tubes containing bacterial cultures or protein solutions revealed patterns which had a tendency to vary with the direction in which the tubes were arranged. The study was made to establish whether or not this was the case with the sedimentation rates. The investigations were carried out as follows: Eight racks, each holding Fig. 3. Eight stands, each holding five tubes, were arranged in this manner.

6 228 MORIYAMA Vol. 5, No. 2 Fig. 4. Patterns of erythrocyte-sedimentation rate in various directions. Tubes were arranged in manner illustrated in Fig. 3. The rates were read 1, 11/2, and 2 hours after standing. five sedimentation tubes arranged in a row (the distance between every two tubes was 3 cm as before) were placd in a manner shown in Fig. 3. The results obtained on 4 successive days are summarized in Fig. 4. In this figure, no correlation is found between the direction and the shape of the pattern in contrast to the case of test tubes containing bacteria or proteins. This may be ascribed to the difference existing between the usual test tubes and the sedimentation tubes. The test tubes used by the author were about 1.5 cm in diameter and they generally contained 5 ml of a culture medium or a protein solution, whereas the sedimentation tubes were very thin (about 0.25 cm in inside diameter) and could hold only 1 ml of the cell-suspension wh en the suspension was filled to the mark graduated at 20 cm high. In order to emit the secondary radiation sufficient to exert an effect on the adjacent tubes, each tube must contain a considerable amount of a solution. It may be reasonable to consider that the very small quantity of the cell suspension admitted in the thin tube could not affect the adjoining tubes. Therefore, the patterns obtained in a row of sedimentation tubes may be different in nature from those previously obtained using test tubes containing bacteria or protein solutions. Observatoins made in a group of tubes arranged separately. Since it is conceivable that the X-agent can produce a secondary radiation from the substance upon which it falls, any location may have a variety of

7 April, 1961 STUDIES ON X-AGENT. XIII. 229 secondary radiations which are emitted from various nearby substances in addition to the primary radiation. Therefore, it is expected that each area should have its peculiar pattern of the X-agent. If it is true that the secondary radiation emitted from the cell-suspension contained in the tube cannot be involved in the formation of a pattern in a row of sedimentation tubes, the pattern must be that which belongs to that particular area. The patterns presented so far in this paper were all obtained from sedimentation tubes which were arranged in a row at intervals of 3 cm. If any mutual effect of the secondary radiation emitted from the tubes were involved in the patterns obtained in such a row of tubes, placing of tubes further apart would make the effect of the secondary radiation insignificant. In order to see whether or not any change which may be accounted for by the detached arrangement of the tubes can occur in the pattern, the investigation shown in Fig. 5 was performed. Sixteen sedimentation tubes, each containing the cell suspension, were arranged in a manner illustrated in the left part of the figure The distance between two tubes was 15 cm or more, that is, the distance was at least 5 times greater than before. The tubes were arranged in a certain area on the wooden Fig. 5. Sedimentation rates of a group of tubes arranged in manner shown above. The rates were read 4 hours after standing. Solid line: tubes placed outside; broken line: tubes placed inside. Date of experiment, I: Feb. 6, II: Feb. 8, III: Feb. 11, IV: Feb. 15, V: Feb. 17, 1960.

8 230 MORIYAMA Vol. 5, No. 2 floor of an empty room with the outside light source being excluded by black curtains. The results obtained in succession on 5 different days are presented in the right part of the figure. It is evident that the rates were different for each tube, indicating that the distribution of the X-agent was dissimilar. It is noteworthy that the pattern of the distribution varied with the day of observation, despite the fact that the observation was performed in one and the same place. This indicates that the X-agent was altering day after day during these observation periods. Since it was confirmed that the distribution of the X-agent is dissimilar even in such a small, limited space, it is reasonable to regard the above mentioned patterns obtained in a row of tubes as the distribution picture of the X-agent itself in an area where the tubes were arranged. Observation made on two groups of tubes each of which was exchanged in location in the midst of the investigation. Evidence has been presented to show that the X-agent is fluctuating incessantly(8) In this respect, it is natural that the distribution pattern of the agent should change day after day as above described. Moreover, it is expected that the agent is undergoing a continuous change even in a short period of a Table 3. Variation of the sedimentation rates with place and time (see Fig. 6). The figures in parentheses represent the sedimentation rates after th e change of position.

9 April, 1961 STUDIES ON X-AGENT. XIII. 231 Fig. 6. Variation in sedimentation rates with place and time (see Table 3) I: First observation was made in a southern room (solid curves); second observation was made after the tubes were transferred to northern room (broken curves).ii : First observation was made in northern room (solid curves); second observation was made after the tubes were transferred to a southern room (broken curves).iii : Sedimentation rates of I, after transferring to the northern room. IV: Sedimentation rates of II, after transferring to the southern room. Day of observation: Apr. 13, day. This is actually demonstrated as shown in Table 3 and Fig. 6. In the investigation shown in this table and figure, two groups of tubes were located in two separate rooms, that is, in a southern and a northern room. After 3 readings were made in succession, the location of the tubes was exchanged. If the pattern was determined solely by the location, the three solid curves in II in Fig. 6 should be identical to the three broken curves in III, since these curves were all obtained in the same place. However, the pattern is not identical, at least, in the northern room, showing that the distribution pattern of the X-agent was changing at least in the northern room during the time of investigation. Although identical patterns failed to be found in the northern room, in the southern room the three solid curves in I are similar to the three broken curves in IV. Furthermore, although it is evident that the solid curves in II are not identical to the broken curves in III, they share something in common. In view of these facts, it may be concluded that the picture of the X-agent was not constant at least in the northern room during the time of invesigation. Thus, it is clear that the sedimentation rates were mostly determined by the site in which the tubes were located, so that the change of the locaion resulted in the change in the rates. This indicates that the sedimentation rates were never determined by a change in the blood cells which was caused by the X-agent at the beginning of the investigation. In other words, a change in the blood

10 232 MORIYAMA Vol. 5, No. 2 cells caused by the agent, by which the sedimentation rates were influenced, was not a fixed one in nature. However, the change could vary with the picture of the X-agent, although evidence has been presented previously that the change has a tendency to be fixed(6) As suggested above, the change in the cells which influences the rates may be a physico-chemical change in some cell material, and this change may be variable and to reflect a constant variation in the pattern of the X-agent. Observations made of different temperatures. As is well known, temperatures have a considerable effect on the red-cell sedimentation rates. According to the investigation of the author, the variation of temperatures by a few degrees is revealed in the variation of the rate as shown in Table 4. In this table are presented the results obtained in groups of tubes arranged in a row with mutual distance of 3 cm. Although the mean values of the rates increased with a rise in temperature, the lack of uniformity in the rates was observed at all temperatures. There is no doubt that the temperature was not involved in the formation of pattern of dissimilar rates. That light itself is also unlikely to be concerned in pattern formation is likewise demonstrated in this table; similar patterns could be demonstrated in rows of tubes arranged in an incubator completely shut out from the external light source. Table 4. Effect of temperature on blood sedimentation rate. Observations made concerning thermal radiation. Sometimes thermal radiation seems to be involved in the effect of the X-agent as communicated previously(10) and will be described in forthcoming papers. The X-agent appears to consist of various components with varying hardness, and the softest component is believed to have a striking resemblance

11 April, 1961 STUDIES ON X-AGENT. XIII. 233 Fig. 7. Thermal radiation and sedimentation rates. Above: Observation was made in northern room on Oct. 14, Below: Observation was made in a southern room on Jan. 29, : irradiated; -: non-irradiated. The first reading was made 1 hour affer standing, followed by successive readings of intervals of 1/2 hour. to a thermal radiation or to be a kind of thermal radiation itself. It was impossible, however, to demonstrate any effect of thermal radiation on the erythrocyte-sedimentation rates. The investigation was carried out in the following way: A row of tubes, each containing the cell-suspension, was exposed to the thermal radiation emitted from an electric heater (600 w.) placed about 2 meters away. Some tubes were protected from the radiation by a pile of bricks placed close to the tubes so as to avoid the radiation. The results are presented in Fig. 7. No difference in the rates between the irradiated and non-irradiated tubes was found. Thus, an attempt to demonstrate the effect of thermal radiation failed, although it is considered that the demonstration may be possible if some adequate method is employed. The pattern presented in the lower part of Fig. 7 was obtained by using two racks, each holding 10 tubes, which were arranged closely in a row. It deserves attention that the rates have a tendency to become lower as the tubes are placed closer to the south window. Some factor acting to diminish the rate seemed to come from the window. In this connection, it should be noted that the patterns presented in Fig. 1, although they were obtained in the same place as those shown in Fig. 7, tend to ascend towards the south, as if something capable of increasing, not decreasing, the rate was entering from the window. Such a contrariness is a feature never uncommon in the phenomena in which the X-agent is concerned, a matter which will be discussed in detail in another paper.

12 234 MORIYAMA Vol. 5, No. 2 DISCUSSION The effect of the X-agent has been demonstrated in such materials as culture media inoculated with bacteria, or solutions of protein or of kaolin. Generally, the solutions, having a volume of 5 ml or so, were admitted in ordinary test tubes. The solutions having such a considerable volume are expected to be emitting a secondary radiation of X-agent which is induced by the primary one. When test tubes containing any of such solutions were arranged in a row with mutual distance of several cm, each tube appeared to be influenced by the secondary radiation coming from the tubes placed in close proximity, so that each tube would be thought to be the cause of the dissimilarity found in the change occurring in each tube. However, the effect of such secondary radiation emitted from the tubes seems to be insignificant in the erythrocyte-sedimentation rates studied using the ordinary sedimentation tubes, since the sedimentation tube is very thin and its volume is extremely small. Therefore, the dissimilarity observed in the rates can be regarded as the reflection of the complicated pattern of the X- agent at the site where the row of tubes are arranged. In other words, the pattern of the X-agent, belonging to a place, is considered to be revealed in the pattern of dissimilar sedimentation rates. Thus, the study on a peculiar pattern of the X-agent prevailing in an area, is possible when the sedimentation rates are under investigation. It should be noted that in a pattern observed in a line of tubes, extending such a short distance as 20 or 30 cm., several crests of peculiar characters are observed. however, the boundaries between such patches do not appear to be clear-cut, a patch being gradually shifted to other patches. As pointed out above, each tube seemed to be changed, generally, in its rate not independently of other tubes placed next to it, so that the pattern of varying rates showed a tendency to wave. This indicates that a patch is shifted gradually to the other. The pattern of the X-agent seems, thus, to be lacking in uniformity even in a very small space. It is natural that the unevenness could become evident as the area was increased. This is shown in Fig. 7; thus, the pattern presented in the lower part of this figure were obtained in a line of tubes extending a distance of about 60 cm, while the rates of tubes placed at both ends of the line were very different. As emphasized repeatedly, the characteristic feature of the X -agent consists in its variability with both place and time. The variability with place is thus clearly demonstrated in the sedimentation rate, while at the same time the variability with time is likewise revealed in it. Thus, the pattern of a location is changed day after day, no two identical patterns being obtained even in the same place.

13 April, 1961 STUDIES ON X-AGENT. XIII. 235 SUMMARY 1. The effect of the X-agent could be demonstrated in the sedimentation rate of blood cells. 2. No identical rate was observed in different tubes containing the same blood suspension. This could be attributed neither to any different characters of individual tubes, nor to any differences in temperature or light. 3. The dissimilarity of the rate is believed to be due to a dissimilar distribution of the X-agent in the place where the investigation was made. The X-agent apparently varied in character from spot to spot. It could be demonstrated that even in a very small space, extending only several cm, there seemed to be several different patches of pattern of the agent. 4. The pattern of the agent peculiar to a place appeared to vary with time; no two identical patterns could be found even in the same place. The characteristic feature of the X-agent, which is believed to consist in its variability with time and place, could thus again be demonstrated in the sedimentation rate of blood cells. REFERENCES (1) Moriyama, H. and H. Ueno,. Studies on X-agent. I. Change in the growth-rate of Proteus vulgaris due to inoculation time. Jap. J. Microb., 3: , (2) Moriyama, H. and H. Ueno,: Studies on X-agent. II. Effects of materials existing outside the culture media on the growth of Proteus vulgaris. Jap. J. Microb., 3: , (3) Moriyama, H.: Studies on X-agent. III. Effect on the growth of seeds. Jap. J. Microb., 4: 7-17, (4) Moriyama, H.: Studies on X-agent. IV. Effect of X-agent on protein solution. Jap. J. Microb., 4: 83-95, (5) Moriyama. H.: Studies on X-agent. V. Effect of X-agent on the turbidity of protein or on the growth of bacteria arranged in lines of various directions. Jap. J. Microb., 4: , (6) Moriyama, H.: Studies on X-agent. VIII. Fixation of turbidity change in protein solutions induced by X-agent. Jap. J. Exp. Med., 31: 83-98, (7) Moriyama, H.: Studies on X-agent. VI. Effect of X-agent on red blood cells. Tohoku J. Exper. Med., 73: , (8) Moriyama, H.: Studies on X-agent. VII. Time-fluctuation of X-agent. Tohoku J. Exper. Med., 73: , (9) Moriyama, H.. Studies on X-agent. XI. Effect of X-agent on kaolin suspension. Jap. J. Microb., 4: , (10) Moriyama, H.. Studies on X-agent. XII. Striped patterns produced in kaolin suspension. Jap. J. Microb., 5: , (11) Moriyama, H., and H. Ueno,: Studies on X-agent. IX. Effect of lead shield upon X-agent with special reference to its action on the growth of bacteria and on the turbidity of protein solutions Jap. J. Exper. Med., 31: , (12) Moriyama, H.: Studies on X-agent. X. Effect of lead shield on X-agent with special reference to its action upon red cells. Jap. J. Exper. Med., 31: , 1961.