510 J. Physiol. (94I) 99, 50-514 6I2.0I4.44:612.III EFFECT OF LIGHT ON RED BLOOD CELLS. THE LIGHT SENSITIVITY OF BLOOD FROM DIFFERENT VERTEBRATE SPECIES BY W. MEYERSTEIN (From the Department of Physiology, University of Birmingham) (Received 22 January 1941) INVESTIGATIONS into the life span of erythrocytes have established the fact that the red cells have an obvious sensitivity to normal daylight [Meyerstein, 1928]. Suspensions of red cells in saline exposed to daylight haemolysed much sooner than similar suspensions stored in the dark. Earle [1928] at the same time reported that electric light haemolysed rabbits' corpuscles. An extension of this research [Meyerstein, 1928, 1929, 1932] indicated that the haemolytic effect of daylight or artificial light differed quantitatively on blood drawn from different animal species. This observation has now been extended and the technique refined, so that the experiments may be done with one drop of blood. METHOD The apparatus used is shown in Fig. 1. The light source is a projection lamp of 500 W, the filament of which is fixed at the focus of the parabolic concave mirror A. The light thus collected is reflected to another parabolic mirror B, which in turn brings the light to a focus, at which point the small flask containing the blood suspension is fixed. The flask is immersed in a glass vessel cooled by running tap water. 0 05 c.c. blood is taken without anaesthesia from capillaries or veins (it is immaterial which is punctured), and diluted with isotonic saline up to 5 c.c. No anticoagulant is used, the dilution being sufficient to prevent coagulation. 2 c.c. of this 1 % blood suspension are placed in a small flask with a long neck, the bulb of which is of 2 c.c. capacity and corresponds in size with the filament of the electric lamp. The bulb has two flat parallel sides. The flask is brought into the focus of the
_, Z, ~~~~~~~~~~~~~~~~~~~~~~~~... _~~ ~ ~ l*. _ EFFECT OF LIGHT ON RED BLOOD CELLS 511 parabolic mirror B, and is exposed to the light for 15 min. During this time the temperature of the blood suspension is taken every 5 min., and has never been found higher than 25-30 C. After exposure to light, the result is not at once manifest; some time must elapse before the haemolysis produced by the light can be detected. Hence, after exposure to light, the flask is kept for exactly 24 hr. in darkness at room temperature (18-19 C.), and the degree of haemolysis is then estimated. For this purpose, the flask is centrifuged, and the Fig. 1. supernatant fluid compared colorimetrically with the remainder of the blood suspension, completely haemolysed by freezing or by the addition of a small amount of powdered saponin. The erythrocytes of the following species have been investigated: man, guinea-pig, rabbit, rat, dog, cat, ox, pig, horse, monkey, hen, frog. All the animals were kept under normal conditions in daylight. In some cases many individuals were investigated; in the others at least two animals of every species were tested, and of every animal two blood samples. Table I shows the results obtained. Those for the dog and horse were obtained using round bulbs containing 10 c.c. and exposing to light for 1 hr. Experiments on the blood of other animals show that the results obtained by this variation in teclnique are comparable; therefore it is
512 W. MEYERSTEIN considered justifiable to include these results in the table. The results are averages, and with hens the fluctuations are larger than usual, the limits being noted in the table. The blood of the guinea-pig shows a remarkable constancy; therefore this blood was regularly used as control test. TABLE I Degree of haemolysis produced by light under the same conditions Frog 5 Hen 15-35 Monkey (Rhesus) 0 Cat 7 Human being 12 Pig 45 Rat (white) 80 Rabbit 80 Ox 85 Horse 90 Dog 95 Guinea-pig 98 The table shows that the nucleated erythrocytes of the hen and frog have a low sensitivity to light. Among the mammals we find extreme differences. There is a large group, guinea-pig, rabbit, -rat, dog, ox, and horse, which are highly sensitive to light, with degrees of haemolysis from 80 to 98 %, and another group, man, rat and monkey, which are highly resistant to the haemolytic action of light, with a degree of haemolysis from 0 to 12 %. The strongest resistance is found in the monkey with no haemolysis. But this resistance is not absolute, for if the time of exposure to light is prolonged haemolysis can be obtained. An intermediate position between the two groups is taken by the blood of pigs with a degree of haemolysis of 45 %. The reasons for the differences in behaviour of erythrocytes of different species when exposed to light are difficult to explain. Plasma plays no obvious part in haemolysis produced by light. The corpuscles were removed from the plasma by repeated washing in isotonic saline, and the light resistance of the washed corpuscles estimated. The following table compares the effect of light on the unwashed cells with that after washing. TABLE II Before washing After washing Rabbit 80 90 Pig 45 60 Man 15 25
EFFECT OF LIGHT ON RED BLOOD CELLS These results indicate either that the diluted plasma gives certain protection, or what is more probable, that the repeated washing of the red cells has produced trauma. Snapper [1912] stated that the osmotic resistance of red cells is diminished by washing; this supports the latter hypothesis. Lepeschkin [1931], also, reported that if erythrocytes were slightly damaged by exposure to hypotonic saline, the haemolysis produced by light increased. The effect of light on corpuscles of different species bears no relation to their osmotic resistance, as is shown in Table III. The figures for osmotic resistance have been obtained from Isaacs in the Handbook of Haematology [1938]. TABLE III Haemolysis by light Osmotic resistance % Man 042-0-48 12 Guinea-pig 0-42 98 Monkey 0-46 0 Dog 0-46 95 513 Experiments have been done to investigate the nature of the rays which actively produce haemolysis. Ultra-violet light can be excluded (1) because the light is filtered through several layers of glass before impinging on the cells, and (2) because previous experiments [Meyerstein, 1929, 1932] showed that the use of uviol glass instead of ordinary glass in the apparatus, did not affect the results. Infra-red rays are absorbed in part by the cooling water. To test this, however, the flask containing the suspension of guinea-pig blood was immersed in a solution of iodine in carbon bisulphide. By this method almost all rays except infra-red rays are excluded. The haemolysis produced in this experiment was 8 % compared with the control test of 95 %. Therefore it can be stated that the major effect of light on red cells is produced by rays of the visible spectrum. Further experiments are in progress to determine the factors concerned in this action of light on erythrocytes. SUMMARY 1. The red blood cells have an obvious light sensitivity, since haemolysis is produced by light. This sensitivity is small with the liucleated erythrocytes of hens and frogs. Among the mammals, two groups are distinguishable, the first with high sensitivity, guinea-pig, rabbit, rat, dog, ox, horse, and the second with big resistance, man, cat, monkey. An intermediate position between these two groups is taken by the blood of pigs.
514 W. MEYERSTEIN 2. The light haemolysis is caused by alteration of the red cells, not of the plasma. 3. The different behaviour to the action of light has no connection with the osmotic resistance of the erythrocytes. 4. The light which is responsible for the action described is almost exclusively in the visible region. REFERENCES Earle, W. R. [1928]. J. exp. Med. 48, 667. Isaacs, R. [1938]. Handbook of Haematology, ed. by Hal Downey, 1, 108. Lepeschkin [1931]. Protoplasma, 17, 11. Ref. Kongre8ecentralblatt ge8. inn. Med., 1932, 65, 618. Meyerstein, W. [1928]. Klin. Wochen8chrift, 7, 2244. Meyerstein, W. [1929]. Wiener Arch. inn. Med. 18, 359. Meyerstein, W. [1932]. MHinchner med. Wochenschrift, 32, 312. Snapper, I. [1912]. Biochem. Z. 43, 266. CAMBRIDGE: PRINTED BY WALTER LEWIS, M.A. AT THE UIBIVRSITY PRESS