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LYSOLECITHIN AND HEMOLYTIC . THE SIGNIFICANCE OF LYSOLECITHIN PRODUCTION IN THE DIFFERENTIATION OF CIRCULATING AND STAGNANT

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 . THE SIGNIFICANCE OF LYSOLECITHIN PRODUCTION IN THE DIFFER- ENTIATION OF CIRCULATING AND STAGNANT BLOOD1 By KARL SINGER (From the Laboratory, Boston Dispensary and the Joseph H. Pratt Diagnostic Hospital, Boston) (Received for publication October 4, 1940) Considerations regarding the etiology of hemo- interpreted as a precursor of lytic are inextricably bound up with the and the morphological expression of an presence of spherocytosis and the associated in- alteration in the structure of erythrocytes due to crease in hypotonic fragility, and the effect the action of different types of hemolytic sub- of in the amelioration of this dis- stances. Dameshek and Schwartz (3) demon- order. Although many different opinions are ad- strated the presence of 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 or an increase in 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 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 on the bone marrow (1). tion of this lysin might be the cause of congenital Careful studies of the 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 output) and also that spherocytes from cases of acquired hemolytic increased erythrocyte production (as evaluated by anemia reacted like normal cells. These findings the 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 . 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 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 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 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. Ten Lysolecithin content (expresed as Hemolysing hemolyzing value or titer) value of i cc. of blood serum are precipitated with 10 times its quantity of 95 per cent alcohol, filtered, and the filtrate evaporated in a vacuum apparatus at room temperature E EICind of__xDe XD00~~~~~ DS to complete dryness. The residue is again dissolved in 5 cc. alcohol, filtered, mixed with 6 times its quantity of ether, and placed in the refrigerator overnight. The 6 Uincubated precipitate contains the lysolecithin. After centrifuga- hemolysis, + + + - 1: tion the ether is removed by means of a suction pump, perhd:ew 100 100 100 73 0O and after the precipitate is dried the content of lysoleci- 6 Unincubated thin is determined. For this determination its hemolytic hemolysis, + + + + + . _ _ 1: 39 power is used. The dried precipitate is now dissolved in 0.8 cc. of physiological salt solution. From this basic per cen 100 100 100 73 016 : solution a geometric series of dilutions is prepared in the usual manner by further dilution with saline. An approximate 2 per cent saline suspension of washed hemolysis, + + + + + + - - 1:32 per cent 100 100 100 100 100 100 70 erythrocytes (containing about 100,000 cells per cmm.) Iumeaoved: is taken up in leukocyte pipettes to the 1 mark and di- 8 Unmnuateed luted with the various single dilutions of lysolecithin to hemolysis, + + + + + -ol1:32b hetorltsic, 100b100-minte 98a93e1 61m70 the 11 mark. While hemolysis is taking place, the pi- percenti UnMoved pettes are set aside for one hour; with very weak concen- phercenhemolysis, -52ermet--0 ofr h saeeto trations of the lysin present, the reaction may be facili- percent 58 0 tated by placing the pipettes in the incubator for the same period. The solutions are then examined in a Time of incubation was 20 to 24 hours. hematocytometer. By determining the number of undis- t Shaking of the serum was performed by means of a motor twice a minute. solved erythrocytes, an exact measure of the degree of hemolysis is obtained. Hemolysis above 90 per cent is regarded as complete. At this degree of hemolysis the Thesehlo emocanexperimentss,expect+confirm yslcthncotnthe statement1:128 of opaque fluid in the pipettes becomes clear and transparent. Bergenhem and FTahraeus (5). In determining The lysolecithin content of a serum is expressed in the the relationship between the lysolecithin content hemolyzing value of the substance derived from 10 cc. of serum. Only the solutions which give complete he- of 2 equal amounts of unincubated and incubated molysis are considered for the evaluation. blood of the same blood sample, we should be able to differentiate between stagnant (i.e. unmoved) As already mentioned, Bergenhem and Fahraeus and circulating (i.e. moved) blood. In- stagnant (5) found that the lysolecithin content of a serum blood we can expect that the lysolecithin content 3The erythrocyte suspension is usually made of the of the unincubated blood sample must be much patient's own red cells, but it is also possible to use other higher compared with the corresponding value in normal erythrocytes as there is practically no difference circulating blood, because this blood reacts like in the lysolecithin fragility of the erythrocytes of normal the blood sample in the incubator. The relation- persons. These erythrocytes are obtained from .oxalated blood and are then washed three times with normal saline ship of the lysolecithin content in equal amounts before suspension is prepared. of unincubated and incubated unmoved blood of 156 KARL SINGER the same sample is called the lysolecithin quotient beings only venous blood was used for the determi- (LLQ): nations. In dogs no difference was demonstrated venous blood of serum in the LLQ of arterial as against Lysolecithin content of unincubated animal, but rather considerable indi- Lysolecithin content of incubated serum the same -LLQ. vidual differences were present. The LLQ in the 8 dogs was always ¼ to Y. In stagnant blood we should find a LLQ of 1 or approaching 1, in contradistinction to circulat- TABLE II ing blood where the LLQ must be smaller due to LLQ in normal human blood the fact that the amount of lysolecithin can be in- creased by incubation. In stagnant or stored Lysolecithin content of serum blood this incubation has already taken place in Number LLQ the body. In the following experiments the LLQ Unincubated Incubated was always determined from 10 cc. of serum. 1 1:2 1:8 2: 8= 2 1:4 1:8 4: 8= To use smaller quantities of serum, the method 3 1:4 1:16 4:16= has been further modified: the test is made in 4 1:8 1:16 8:16 = M 5 1:2 1:16 2:16= y special pipettes that take just half of the content 6 1:4 1:16 4:16= 4 of normal leukocyte pipettes. The amount of 7 1:4 1:32 4:32 =Y 8 1:4 1:8 4: 8= serum used for each determination can thus be 9 1:8 1:16 8:16 = M reduced to 5 cc.; the dried lysolecithin for each 10 1:2 1:16 2:16=Y evaluation is dissolved in 0.4 cc. instead of 0.8 cc. of normal salt solution. B. The LLQ in splenic vein blood (stored blood)

RESULTS Determinations of the LLQs in blood obtained from the splenic artery and vein were performed A. The LLQ of peripheral blood in in 8 dogs in order to show possible differences normal conditions between the " reservoir blood " of the spleen and The LLQ was determined in 8 dogs and 30 that of the circulating peripheral blood. Due to normal human beings. The normal group com- the great difficulties in obtaining sufficient amounts prised healthy physicians, students and patients of reservoir blood directly from the spleen, deter- who showed no evidence of , spleen, or blood minations in the splenic vein blood were made be- disease or of any condition in which an abnormally cause it was thought that the splenic vein might lowered velocity of blood flow was suspected. not only contain blood passing directly through Table II contains the values obtained in 10 human this organ, but also a more or less large quantity beings and the first column of Table III shows of stored blood. The results are tabulated in the results in dogs. For simplification, only the column 2 of Table III. end results of the determinations are given. The In all experiments the LLQ in the splenic vein lysolecithin content of dog serum was tested blood was greater than that in the splenic artery against dog erythrocytes, but for the determina- (i.e., following incubation of splenic vein blood tion of lysolecithin in human serum, human ery- there was usually no increase in lysolecithin titer). throcytes were used. The hemolytic titer of dog In 6 of the 8 experimental animals the unincubated serum is apparently greater than that of human blood of the splenic vein contained as much ly- serum, although this may simply be due to the solecithin as the incubated; the LLQ was 1. In greater fragility of the red cells of the dog to 2 animals the lysolecithin content of the unincu- lysolecithin. Normally the LLQ is always a frac- bated sample was not an optimal one and could tion oi 1, the lysolecithin content (expressed in still be increased by. incubation. But compared hemolytic titer) of the incubated serum being with the corresponding arterial blood, a higher 2 to 8 times greater than that of the unincubated lysolecithin content was observed in these 2 dogs serum. Relatively great differences were en- as well. In order to decide whether the ery- countered in different individuals. In human throcytes coming from the stored blood of the LYSOLECITHIN AND HEMOLYTIC ANEMIA 157

TABLE III C. The LLQ in hepatic vein blood LLQ in dog serum To decide whether blood coming from other blood reservoirs than the spleen might show the Lysolecithin content in serum from same changes, the LLQ in hepatic vein blood ob- Dog Peripheral blood* Splenic vein blood Hepatic vein bloodt tained after ligature of the inferior vena cava was num- ber determined in 3 dogs (Table II, column 3). *Un- Incu- Un- Incu- Un- Incu-L Some investigators (16, 17) believe that not only nu-bated LLQ incu- bated LLQ incu- bateLL the spleen but also the liver may be regarded as 1 1: 8 1: 64 8 1:64 1: 64 1 1:8 1:64 Yg an organ containing more or less large blood 2 1: 8 1: 32 Y4 1:32 1: 32 1 not determined 3 1:16 1: 64 Y4 1:64 1: 64 1 notdetermined depots. In contradistinction to the splenic vein 4 1:16 1: 64 Y4 1:64 1: 64 1 notdetermined blood, hepatic venous blood showed the same LLQ 5 1: 8 1: 64 8 1:64 1: 64 1 not determined 6 1: 8 1:64 M/ 1:64 1: 64 1 1: 8 1 1: 641 Ys as found in the peripheral circulation. 7 1:32 1:128 4 1:64 1:128 4 not determined 8 1:32 1:128 4 1:64 1:128 M 1:321 1:1281 X D. The LLQ in the blood of varicose veins * Peripheral blood was drawn from jugular vein or As already pointed out, a LLQ of 1 may, under carotid artery. t Hepatic vein blood was obtained after preceding liga- certain conditions, demonstrate the presence of ture of inferior vena cava. stagnant blood, or at least of a very sluggish cir- culation. To confirm this interpretation, the LLQ spleen might have already adsorbed lysolecithin was determined in other conditions in which the with a resultant tendency to spherocytosis and in- presence of a sluggish circulation of blood might creased fragility, the lysolecithin fragility of red be assumed. Following a suggestion by Dr. cells from the splenic vein and the splenic artery was compared. Bergenhem and Fahraeus (5) TABLE V noted such a tendency, and Heilmeyer (15) found The LLQ in varicose vein blood by direct measurement of the thickness of the red cells from the splenic artery and the splenic vein Lysolecithin content in serum from in patients with hemolytic anemia that the splenic Case Cubital vein blood Varicose vein blood vein erythrocytes were considerably thicker. number Such a tendency should lead to increased fragility. Unincu- Incu- Unincu- Incu- Table IV (as an example of numerous experi- bated bated LLQ bated bated LLQ ments) shows, however, that no difference in the 1 1:8 1:16 3 1:16 1:16 1 2 1:8 1:16 M 1:16 1:16 1 lysolecithin fragility of the erythrocytes in both 3 1:8 1:16 M 1:16 1:16 1 splenic vessels could be demonstrated. In order 4 1:8 1:16 M 1:16 1:16 1 5 1:4 1:16 X 1:16 1:16 1 to test the lysolecithin fragility, 2 exactly equal 6 1:2 1:8 X 1:8 1:8 1 suspensions of erythrocytes from the splenic ar- 7 1:4 1: 8 M 1:4 1: 8 Y2 tery and the splenic vein (100,000 cells per cmm.) were tested against 2 exactly equal concentrations Robert Brandt of Augusta, Georgia, determina- of a lysolecithin extract. tions of the LLQ in blood from varicose veins were performed. The investigations of Magnus TABLE IV (18) have shown that the blood in large varicose Lysolecithin fragility of splenic artery and splenic vein erythrocytes veins possesses a very sluggish circulation, par- ticularly when the patient is in a horizontal posi- Lysolecithin dilutions tion. Nobel (19) and Wolf and Remenovsky Kind of erythrocytes - (20) demonstrated this tendency to stagnation 1:2 1:4 1:8 1:16 1:32 1:64 by x-ray studies of the injected veins. In 6 pa- From artery: hemolysis, + + + + - tients with very large varicose veins the LLQ in per cent 100 100 100 94 38 0 blood from the cubital vein and in blood from the From vein: hemolysis, + + + + - per cent 100 100 100 92 35 0 varicose vein was compared. Blood was taken in the morning before the patient got up. Table V 158 KARL SINGER shows that blood obtained from the varicose veins the patient's own erythrocytes. As demonstrated always presented a LLQ of 1 in contradistinction in another paper (6a), the spherocytes of con- to blood from the cubital veins. Case 7 shows genital hemolytic jaundice have a decreased re- identical values for blood of the upper and lower sistence towards lysolecithin and therefore give extremities; no varices were present and this case higher hemolyzing values. All these cases show was taken as a control. no great difference from the normal cases as far as the lysolecithin content of the serum is con- E. The LLQ in cases of congenital cerned. Only Case 1 had a very low value in the hemolytic anemia unincubated blood. In no case was there any According to the hypothesis of Bergenhem and evidence indicating an increased production of Fahraeus (5), lysolecithin may be considered to lysolecithin. be the lysin responsible for the spherocytosis and increased blood destruction in this disorder. It DISCUSSION seemed, therefore, of great interest to determine The results thus far obtained have confirmed the LLQ in such cases. Table VI shows the re- the findings of Bergenhem and Fahraeus (5) re- sults obtained in 6 cases of congenital hemolytic garding lysolecithin. The demonstration has fur- jaundice. thermore been made that the concentration of this physiological lysin is increased by keeping the TABLE VI serum quietly in the incubator for several hours; The LLQ in cases of hemolytic jaundice no such increase occurs in serum which has been Lysolecithin content of shaken. This increase in the amount of lysole- serum* from Cas cithin in incubated serum may be expressed by num- State of blood ber Unincu- Incu- the lysolecithin quotient, in which the relationship bated bated LLQ of the lysolecithin content of unincubated to that blood blood of incubated (unmoved) serum of the same blood Hemoglobin 50% 1 Erythrocytes 2,500,000 1:1 1:16 h6 sample is determined. 9% In circulating blood this quotient is always a Hemoglobin 54c%; fraction of 1. In stagnant blood the LLQ is 1 2 Erythrocytes 3,000,000 1:8 1:16 Y2 or approximates 1, probably because the enzymatic Reticulocytes 7.8% process causing an increase in lysolecithin in the Hemoglobin 90% incubated test tube has already been active within 3 Erythrocytes 4,200,000 1:2 1: 4 Y2 Reticulocytes 8.4% the body. In splenic vein blood of dogs the LLQ was found to be 1 in most animals, probably due to Hemoglobin 64% 4 Erythrocytes 3,600,000 1:4 1: 8 M storage of blood in the spleen. At least from the Reticulocytes 8.2% standpoint of lysolecithin production, the spleen Hemoglobin 70% may be considered to be the " incubator " of the 5 Erythrocytes 3,600,000 1:4 1:16 X That a 1 Reticulocytes 18% body. LLQ of is the serological mani- festation of stagnant blood was also indicated in Hemoglobin X 106% investigations of blood obtained from varicose 6 Erythrocytes 4,900,000 1:8 1:16 Y2 Reticulocytes 7.2% veins. The extremely sluggish circulation in these veins, particularly in patients placed in a hori- * Blood was obtained from the vein of the antecubital zontal position for several hours, has been demon- fossa. strated by Magnus (18), Nobel (19) and others In all cases the saline fragility of the erythro- (20). The finding of a LLQ of 1 in blood from cytes was increased and the lysolecithin fragility these veins in contradistinction to a LLQ of %8 test was likewise pathological. The evaluation of ¼' etc. in blood obtained from the cubital vein in the lysolecithin content was performed with nor- the same patient supports the interpretation sug- mal erythrocytes in order to avoid errors which gested in this paper. might be caused by the increased susceptibility of The concept of Rein (16) and associates, Ep- LYSOLECITHIN AND HEMOLYTIC ANEMIA 159 pinger (17), and others, that the liver may also ripheral blood in that disorder was found to be be regarded as an organ containing blood depots, normal.4 In order to explain the presence of could not be corroborated by determining the spherocytosis and greatly increased blood destruc- LLQ. The finding of a normal LLQ in the tion regularly seen in congenital hemolytic jaun- hepatic veins may be explained either by the as- dice, Bergenhem and Fahraeus (5) assumed an sumption that no " storage " blood was obtained in increased production of lysolecithin. The find- our experiments, or that the mechanism of storage ings of both normal content and quotient appear of the blood in the liver may be quite different to rule out the lysolecithin as of any significance from that in the spleen. The recent investigations in the pathogenesis of this disease. We believe, of Knisely (21) on the histology of the spleen therefore, that lysolecithin, although it has hemo- indicate that the red cells are separated from the lytic qualities, is not the hemolysin responsible for fluid of the blood in the venous sinuses of the the production of hemolytic jaundice. spleen. As a LLQ of 1 was obtained from serum of the splenic vein blood in 6 of 8 examined ani- SUM MARY mals, we may conclude that not only the erythro- 1. Bergenhem and Fahraeus demonstrated that cytes but also the plasma must be stored in the normal blood contains a hemolyzing substance spleen for the reason that only unmoved serum called lysolecithin. They believed that this lysin gives a LLQ of 1. Due to this storage the " res- was developed in high concentration in the stored ervoir blood" of the spleen probably has an in- blood of the spleen and was therefore involved in creased content of lysolecithin. the physiological mechanism of blood destruction. Although these experiments confirm the hy- Congenital hemolytic jaundice was assumed to be pothesis of Bergenhem and F'ahraeus (5) that due to an increased production of this substance. splenic reservoir blood contains more lysolecithin 2. A quantitative method is described for deter- than circulating blood, there is at present no proof mination of the lysolecithin content in relatively for the assumption that, since this substance has small amounts of blood. The content of lysole- hemolytic power when extracted from the serum, cithin in peripheral blood of dogs and normal it is therefore actively engaged in normal blood human beings was determined. It was demon- destruction. It is also very doubtful whether the strated that this content could be increased 2 to concentration of lysolecithin obtainable from 8 timVs by incubating unmoved blood for several serum may be considered sufficient for the range hours, but that moving the blood during incuba- of normal blood destruction in the body. The tion did not produce this increase, probably due recently published experiments of Ham and Castle to the fact that moving destroyed or inactivated (22), who believe that splenic hemolysis is due to the lysin. Circulating (i.e. moved) blood con- metabolic changes of the erythrocytes produced tains less lysolecithin than stagnant (i.e. un- by intravascular stasis, likewise rule out any ac- moved) blood. To relate circulation of blood to tion of lysolecithin in this mechanism. Further- its lysolecithin production the lysolecithin quotient more, lysolecithin can by no means be regarded LLQ _ as the lysin primarily responsible for the hemato- Lysolecithin content of unincubated serum logical changes in congenital hemolytic jaundice. Lysolecithin content of incubated serum According to the hemolysin theory, spherocytosis was determined. is produced outside of the bone marrow by action 3. The LLQ of normal peripheral blood is al- of hemolysins upon normal red cells. It is the ways a fraction of 1; in stagnant or stored blood morphological expression of an alteration in the the LLQ was found to be 1. By means of the physical-chemical structure of erythrocytes. Al- LLQ, the differentiation between circulating and though in a previous communication (6a) demon- stagnant blood appears possible. stration was made that the lysolecithin fragility of spherocytes in congenital hemolytic jaundice is 4 Unfortunately, no data concerning the lysolecithin production in splenic artery and splenic vein blood in remarkably increased compared with that of cases of hemolytic jaundice are yet available, although normal cells, the lysolecithin production in the pe- such investigations would be highly desirable. 160 KARL SINGER 4. Blood from the splenic vein in dogs has a ruecksichtigung der Physiologie der Milz. Ztschr. LLQ of 1, indicating the presence of stored blood f. d. ges. exper. Med., 1936, 97, 555. 6a. Singer, K., The lysolecithin fragility test. Am. J. coming from the spleen. The LLQ in blood from M. Sc., 1940, 199, 466. the splenic artery, however, was always a fraction b. Singer, K., Das Problem der normalen und patho- of 1. These findings demonstrate an increased logischen Milzhaemolyse. (Lecture November 4, lysolecithin production in the " splenic reservoir 1937, Gesellschaft fuer Innere Medizin, Vienna.) Wien. Arch. f. Inn. Med., 1937, 37, 161. (Gesell- blood." schaftsberichte.) 5. The LLQ obtained from blood from varicose 7. Bergenhem, B., Experimentelle Untersuchungen ueber veins, presenting an area of very sluggish circula- die spontanen Veranderungen des Blutes in vitro tion, was likewise 1. hinsichtlich Suspensionsstabilitat Komplementak- 6. The LLQ in blood from the hepatic vein was tivitat und Antithrombinfunktion. Acta Path. et the same as in peripheral blood. This finding Microbiol. Scandinav., 1939, Supp. 39, 1. 8. Flexner, S., and Noguchi, H., Snake venom in rela- might indicate that the mechanism of " storage of tion to hemolysis, bacteriolysis and toxicity. J. blood " in the liver might be different from that in Exper. Med., 1902, 6, 277. the spleen, where, presumably, separation of 9. Kyes, P., and Sachs, H., Zur Kenntnis der Cobra- plasma and erythrocytes inside the organ occurs gifte aktivierenden Substanzen. Klin. Wchnschr., (21). 1903, 57, 82. 10. Luedecke, K., Quoted by Bergenhem and Fahraeus. 7. No difference in the lysolecithin content and 11. Manwaring, W. H., Ueber die Lecithinase des Kobra- the LLQ in the blood of patients with congenital giftes. Ztschrft. Immun. Forschg., 1910, 6, 513. hemolytic anemia, as compared with that of 12. Delezenne, C., and Ledebt, S., Action du venin de normal persons, was found. cobra sur les serums de cheval. Compt. Rendus 8. The significance of lysolecithin as the physi- Acad de Sci. Paris, 1911, 152, 790. 13. Sachs, H., and Klopstock, A., Methoden der Hemoly- ological hemolysin involved in the mechanism of seforschung. Urban and Schwarzenberg, Berlin, normal blood destruction by increased production 1928. in splenic reservoir blood is not conclusive. It is 14. Barcroft, J., Stellung der Milz in Kreislaufsystem. also very unlikely that lysolecithin can be regarded Erg. Physiol., 1926, 25, 818. as responsible for the increased blood destruction 15a. Heilmeyer, L., and Albus, L., Die hemolytische Hy- persplenie. Deutsches Arch. f. klin. Med., 1936, in congenital hemolytic jaundice, since no increase 178, 89. in the production of this lysin was demonstrable b. Heilmeyer, L., Die Spharocytose als Ausdruck einer in the disease. It is therefore believed that lyso- pathologischen Funktion der Milz. Deutsches lecithin, although having hemolytic qualities, is Arch. f. klin. Med., 1936, 179, 292. not the hemolysin causing hemolytic anemia. 16. Rein, H., Die Blutreservoire des Menschen. Klin. Wchnschr., 1933, 12, 1. BIBLIOGRAPHY 17. Eppinger, H., Die Leberkrankheiten. Springer, Vienna, 1937. 1. Naegeli, O., Blutkrankheiten und Blutdiagnostik. 18. Magnus, G., Zirkulationsverhaltnisse in Varicen. D. Springer, Berlin, 1931. Ztschrft. f. 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