LYSOLECITHIN AND HEMOLYTIC ANEMIA. THE SIGNIFICANCE OF LYSOLECITHIN PRODUCTION IN THE DIFFERENTIATION OF CIRCULATING AND STAGNANT BLOOD Karl Singer J Clin Invest. 1941;20(2):153-160. https://doi.org/10.1172/JCI101207. Research Article Find the latest version: https://jci.me/101207/pdf LYSOLECITHIN AND HEMOLYTIC ANEMIA. THE SIGNIFICANCE OF LYSOLECITHIN PRODUCTION IN THE DIFFER- ENTIATION OF CIRCULATING AND STAGNANT BLOOD1 By KARL SINGER (From the Hematology Laboratory, Boston Dispensary and the Joseph H. Pratt Diagnostic Hospital, Boston) (Received for publication October 4, 1940) Considerations regarding the etiology of hemo- interpreted spherocytosis as a precursor of lytic jaundice are inextricably bound up with the hemolysis and the morphological expression of an presence of spherocytosis and the associated in- alteration in the structure of erythrocytes due to crease in hypotonic saline fragility, and the effect the action of different types of hemolytic sub- of splenectomy in the amelioration of this dis- stances. Dameshek and Schwartz (3) demon- order. Although many different opinions are ad- strated the presence of hemolysins of the immune vanced regarding the etiology of the disease, the body type in cases of acquired hemolytic anemia outstanding ones are those postulating a faulty and showed the disappearance of these antibodies bone marrow or an increase in hemolysin produc- and of spherocytosis after splenectomy. In con- tion. The theory of faulty bone marrow produc- genital hemolytic anemia, however, the demonstra- tion, as introduced by Naegeli (1), claims that tion of hemolysins has not been consistently made. spherocytes are produced by the bone marrow and Recently, Bergenhem and Fahraeus (5) showed that splenectomy does not change the production the presence in normal blood serum of a hitherto of abnormal red cells. The dramatic effect of this unknown hemolytic substance called lysolecithin. operation is explained by removal of the inhibi- They postulated that an increase in the produc- tory effect of the spleen on the bone marrow (1). tion of this lysin might be the cause of congenital Careful studies of the hemoglobin metabolism in hemolytic anemia. Singer (6) demonstrated the this disorder, however, have revealed that, after increased fragility of spherocytes present in the splenectomy, increased blood destruction (as congenital condition towards this lysin but found measured by the urobilinogen output) and also that spherocytes from cases of acquired hemolytic increased erythrocyte production (as evaluated by anemia reacted like normal cells. These findings the reticulocyte counts) return to normal values have recently been confirmed by Gripwall (2). despite the continued presence of spherocytes. The present paper deals with the problem of lyso- Some observers (2) believe that the spleen is the lecithin production in the body and the possible most important organ in this disease but do not relationship of this physiological lysin to the de- deny production of spherocytes by the bone velopment of congenital hemolytic jaundice. marrow. The hemolysin theory, on the other hand, states THE RESEARCHES OF BERGENHEM AND FAHRAEUS that the shape of the red cells is altered outside led to the the bone marrow through the action of hemoly- Bergenhem and Fahraeus (5) were sins. Dameshek and Schwartz (3) produced discovery of their lysolecithin by observing char- spherocytosis in experimental animals by the in- acteristic changes in blood which was allowed to jection of specific hemolytic sera and Haden (4) stand quietly for several hours at body tempera- was able to induce the same abnormality in vitro ture. This incubated blood showed considerable by the addition of hypotonic saline solutions to retardation of the sedimentation rate of the ery- normal erythrocytes. The former authors have throcytes, a change in the shape of red cells from the biconcave to the spherical form and a notice- 1 This work has been aided by grants from the Milton able reduction of aggregation (rouleaux forma- fund of Harvard University and the 'Dazian Foundation tion). These authors demonstrated that these for Medical Research. changes were due to the action of a substance 153 154 KARL SINGER formed by an enzyme which was capable of genhem and Fahraeus (5) demonstrated that ly- splitting this substance from the serum lipoids. solecithin is also destroyed or rendered inactive Adsorption of the latter substance by the red by shaking or moving the serum. The most in- blood cells resulted in the above alterations. If teresting phenomenon was the demonstration that stronger concentrations of this substance devel- the lysolecithin content present in a freshly drawn oped, actual hemolysis of the erythrocytes was blood sample can be increased if the unmoved noted. The enzymatic process in the serum serum is incubated for several hours; if, however, reached its maximum at a temperature of 420 C. the serum is kept in motion during incubation, and a pH of 7.2 and was inactivated at 560 C. this increase does not occur. Since circulating Bergenhem and F'ahraeus (5) concluded that this blood is under conditions of constant motion, it is hemolytic substance was either identical with the to be expected that the amount of lysolecithin so-called lysolecithin or very closely related to it. present in the circulating blood must be smaller Lysolecithin is a definite chemical substance2 than in blood which is stagnant. which has previously received attention in the Bergenhem and Fahraeus (5) believe that ly- study of the biological effect of snake venom. solecithin may be of great importance in the From the investigations of Flexner and Noguchi physiological mechanism of erythrocyte destruc- (8), Kyes and Sachs (9), Luedecke (10), Man- tion. They point out that the spleen must be con- waring (11), Delezenne and Ledebt (12), it is sidered as an organ containing a more or less known that cobra toxin hemolyzes red blood cells constant reservoir of blood, as shown by the well- only in the presence of serum and that the blood known researches of Barcroft (14) and his asso- lipoids are necessary for this hemolytic activity. ciates. Blood stored in the spleen is temporarily Snake venom also contains a lecithinase which excluded from the circulation, and in this splenic changes lecithin to lysolecithin, the latter having reservoir an increased formation of lysolecithin hemolytic qualities. Small amounts of lysolecithin may occur. By adsorption of this substance by obtained by the action of cobra toxin on lecithin, the erythrocytes, direct hemolysis of the red blood when added to normal blood, produce the same corpuscles in the spleen, or preparation of these changes as the substance obtained from incubated erythrocytes for further destruction, presumably serum. If normal serum is inactivated, and cobra takes place. In hemolytic jaundice, where the en- toxin then added, the hemolytic power of the larged spleen contains large quantities of blood, serum extract can be reestablished. Formation an increased production of lysolecithin is said to of lysolecithin by the lecithinases present in cobra occur and is held to be responsible for the in- toxin and in normal serum can be prevented by creased erythrocyte destruction. For lack of a adding quinine which is a typical inhibitor of the biological micromethod for determination of ly- action of lipases. solecithin, Bergenhem and Fahraeus (5) were Recent investigations by Bergenhem (7) have unable to carry out direct measurements of this revealed certain other qualities of lysolecithin. substance in blood. They therefore worked with This author pointed out that lysolecithin is in large quantities of horse serum, measuring some manner related to the complement function changes in the red cells by means of the sedimen- of the serum and that there are several parallelisms tation rate. As changes in the sedimentation rate in the behavior of this lysin and of complement are frequently slight and difficult to evaluate, a which are of particular interest. It has been simpler and more accurate method for determina- known for many years that such manipulations as tion of lysolecithin appeared desirable. In an- shaking of the serum, dilution with distilled water, other paper (6) a method for determination of etc. may destroy or inactivate the complement the lysolecithin fragility of various red cells (nor- function (cf. Sachs and Klopstock (13)). Ber- mal and spherocytic) was described. By adapta- tion of this method the problem of the significance of lysolecithin in the mechanism of hemolysis in 2Lysokephalin is supposed to have quite similar quali- and conditions was ties. Both lysolecithin and lysokephalin are sometimes physiological pathological called lysocithin (7). facilitated. LYSOLECITHIN AND HEMOLYTIC ANEMIA 155 METHODS can be increased if unmoved blood is incubated In the paper (6) on the lysolecithin fragility test, a for from 8 to 24 hours, but that this increase does method for extraction of this lysin from serum was de- not take place if blood is moved during the in- scribed. This method has been adapted for the quanti- cubation period. As an example, Table I shows tative determination of the lysolecithin content of serum. the results of determination in the lysolecithin Blood is drawn and put into the ice chest until it is com- pletely clotted and the serum is expressed. Then the content of 2 aliquot samples of unincubated and serum is removed from the clot by means of a capillary incubated serum in 2 different dogs and also the pipette. Usually, the obtained serum is centrifuged in results of movement during the whole incubation order to remove the last vestiges of erythrocytes. It was period. noted that the process of centrifuging the serum for several minutes apparently does not diminish the content TABLE I of lysolecithin. The serum, which is now completely freed from any erythrocytes, is then divided into two Lysolecithin content of unincubated and incubated serum equal parts. One part is precipitated immediately by means of alcohol, the other part after incubation.