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The erythrocyte sedimentation rate in plasma detergent mixtures
Brit. 07. Db. Chest (i959) 53, 68.
T H E E R Y T H R O C Y T E S E D I M E N T A T I O N RATE IN PLASMA D E T E R G E N T M I X T U R E S BY IAN DICKSON From The Chest Clinic, Townsville, N. Queensland, Australia
THE inter-relationship between the mass of the different protein fractions in the blood plasma, and the rate at which the erythrocytes settle in the plasma (the erythrocyte sedimentation rate), was demonstrated by Fahreus (1921). Fibrinogen was shown to have the greatest and albumin the smallest effect on the rate. The variation in the erythrocyte sedimentation rate (E.S.R.) is governed by the size of the aggregate formed by the clumping together of the rouleaux of red blood ceils, the aggregate size being constant for a particular plasma. Advances in the technique of plasma analysis, first by salt fractionation methods, and more recently by electrophoresis, have led to attempts to obtain a mathematical relationship between the mass of the various proteins present in the plasma and the E.S.R. (Meyer et al., 1953). These attempts have had little success, one possible reason being that analytical techniques are still not sufficiently accurate to ensure a critical separation of all the protein fractions present in the plasma. By means of synthetic plasmas Hardwicke and Squires (1952) were able to obtain a mathematical relationship between the maximum velocity of fall of the erythrocytes and the concentration of the macromolecules. They were, however, unable to apply this formula to natural plasma. Sykes (1948) drew attention to a finding of Schutt (1928) that the addition of very small amounts of ether to plasma caused an increase in the E.S.R., whereas addition of a larger amount resulted in a slower rate. Since that time, however, with the advent of the detergents, a more potent method of effecting changes in the chemical and physico-chemical properties of protein has been found. Addition of minute quantities of detergent to a protein solution will bring about profound changes in the protein (Anson and Edsall, 1948). The present paper is based on experiments performed on blood from a series of tuberculous patients with raised E.S.R.s to determine what effect the modification in the plasma proteins by addition of detergent would have on the E.S.R. For the purpose of these experiments a cationic detergent, cetyl trimethyl ammonium bromide (cetavlon), was used. The anionic detergents were found to be less effective. METHOD
Twelve millilitres of blood were aspirated through a wide bore needle into a silicone lubricated syringe with a minimum of venous stasis. Two millilitres of the blood were added to o. 5 millilitre of a 3.8 per cent. solution of sodium (Receivedfor publication November 6, I958.)
ERYTHROCYTE SEDIMENTATION RATE I N PLASMA DETERGENT MIXTURES
69
citrate in each of six tubes. Five of the tubes contained different amounts of cetavlon (see Table 1). The E.S.R.s for the six citrated bloods were then determined, using the method described by Rourke and Ernstene (193o), and observing precautions against the sources of error described by Ham and Curtis (1938) . The results are given in Table I. Addition of the detergent to the blood causes a reduction of the E.S.R. until, in the higher concentrations, the rate is similar to that of the individual erythrocyte. Microscopy of the blood at this concentration of detergent reveals that there is no aggregate formation, and that the rouleaux are composed of no more than five to ten erythrocytes. PLASMA
VISCOSITY
Various workers (T'ang and Wang, 1941 , Harkness etal.,1946 ) have shown that increased viscosity of the plasma is associated with an increased E.S.R., and Harkness and his co-workers consider the plasma viscosity a more reliable index of the disease process than the E.S.R. The viscosities of the various plasma-cetavlon solutions used in the above experiments were determined, using the simple capillary viscometer described by Steele (I948), and following the modifications described by Hardwicke and Squires (1952). The results are given in Table i. It will be seen that with increasing amounts of cetavlon there is progressive lowering of the E.S.R., but there is an increase in the plasma viscosity. This is in direct contrast to the normal finding of increased E.S.R. with increased viscosity. In order to determine if the detergent acts solely on the plasma, or on erythrocytes, or on both these constituents of the blood, a series of experiments was performed in which two samples of two miUilitres of blood were taken from each patient and added to 0. 5 millilitre of a 3"8 per cent. solution of sodium citrate. Sufficient cetavlon to cause inhibition of the E.S.R. had been added to one of the 0. 5 millilitre of sodium citrate solution. The amount of cetavlon required had been previously determined experimentally by the method already described. The sedimentation rate was determined for the blood plus citrate mixture (sample A), and for the blood plus citrate-cetavlon mixture (sample B). Plasma and cells of both samples were then separated by centrifuging at 3,o00 revs./min, for eo minutes. After repeated washing of the blood corpuscles with normal saline, the cells from sample A were added to the plasma from sample B, and the cells from sample B were added to the plasma from sample A. The reconstituted bloods were then each mixed and the E.S.R.s again determined. It was found that the E.S.R. in the mixture containing the normal plasma corresponded to the E.S.R. in the normal blood sample (sample A), whereas the sample containing the plasma from the blood-cetavlon mixture (sample B) showed the same inhibition that was found in the original sample B. It was
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ERYTHROCYTE SEDIMENTATION RATE IN PLASMA DETERGENT MIXTURES
7I
thus shown that the factor causing inhibition of the E.S.R. when cetavlon is added to blood is confined to the plasma, since both sets of cells behaved normally in the normal plasma, and has no apparent effect on the erythrocytes themselves. In the various experiments performed with the different samples of plasma, it was found that the amount of detergent required to obtain the maximum effect on the E.S.R. varied within narrow limits (o-175-0.255 mg. per 2 millilitres blood). Addition of further amounts of detergent beyond this amount caused ha~molysis of the erythrocytes. SURFACE TENSION Another manifestation of changes in the physical properties of the colloidal solution of plasma proteins is alteration in the surface tension. In most colloidal solutions the addition of detergent causes a sharp drop in the surface tension followed by a gradual rise towards its original value (the Du Nuoy phenomenon). The explanation is thought to be that the detergent first spreads over the solution-air interface, causing the sharp drop in the surface tension, and later, reacting with the colloidal particles, is absorbed on to the surface of the particles, with the result that the surface tension rises. The surface tension of the plasma cetavlon mixture was measured by the stalagmometer as described by Jirgensons and Straumanis (I 954)- The method is based on the determination of the work required for the formation of a fresh surface of the liquid in the form of drops. A fixed volume of the liquid is allowed to drop from the lower glass surface of the stalagmometer at the rate of one drop every two to three seconds. The number of drops formed by the fixed volume of fluid is counted, and compared with the number of drops formed by an identical volume of distilled water. The surface tension is then found by the formula: S.T.:73~×D Zw : n u m b e r of drops of water Z ~ n u m b e r of drops of liquid D ~density of liquid
For the untreated plasma the surface tension was found to be 68 dynes per cenfimetre. For plasma with added detergent the surface tension was 44 dynes per centimetre. No Du Nuoy phenomenon was observed in the plasmacetavlon mixture in the concentrations used. ANALYSIS OF THE PROTEIN-DETERGENT MIXTURE There was no formation of a fibrin clot when calcium chloride solution was added to the plasma-cetavlon mixture. This failure of the plasma-cetavlon mixture to clot may be due to alteration in the fibrinogen, or to modification of the prothrombin. This is now being investigated.
72
DICKSoN
ELECTROPHORESIS OF SERUM DETERGENT MIXTURE
Analysis of untreated serum and of serum to which detergent had been added was carried out by paper electrophoresis,using a barbiturate buffer solution of p H 8, and staining with azocarmine after the method described by Pluckthun and Gotting (I951). The dyed filterpaper strips were then scanned according to the method described by Crook et al. (I954). The resultant traces are shown in Fig. I. t
Fio. I.--Electrophoresis traces of serum and serum--detergentmixtures.
Continuous line=serum. Interrupted line=serum+detergent. These traces show a modification in the gamma globulin as demonstrated by a disappearance of the gamma globulin peak in the serum-detergent trace. At the same time there may be some change in the alpha 2 and beta globulins as shown by the change in size and position of these peaks in the serumdetergent trace. Discussion
With the addition of small amounts of detergent to protein solutions Putnam and Neurath (I945) were able to show electrophoretically that excess protein migrated as a distinct entity from the protein-detergent complex, suggesting that the whole protein molecule becomes involved in the complex, or remains entirely dissociated from the detergent. The complex appears, therefore, to be formed as the result of a primary chemical reaction. Viscosity measurements and X-ray diffraction studies as a means of investigating molecular shapes have shown that the detergents bring about an unfolding of the protein molecule, which loses its rigid configuration and assumes a more random orientation. The viscosity of the solution of the resultant protein-detergent complex may be greater than that of solutions of either constituent alone. Proteins of rod-like morphology (and both fibrinogen and g a m m a globulins conform to this description) have a greater viscosity than the globular proteins,
BRYTHROCYTE
SEDIMENTATION
RATE
IN P L A S M A
DETERGENT
MIXTURES
73
such as albumin. Houston and Lawrence (1955) have shown that both fibrinogen and g a m m a globulin are the protein fractions most likely to be increased in the plasma of tuberculous patients. The raised E.S.R. and the increased plasma viscosity are probably, therefore, both manifestations of the same factor, namely, the increased amounts present of these two protein fractions. The unfolding of the plasma protein molecules following the addition of detergent can be presumed from the increased viscosity,but this unfolding is paradoxically associated with a lowering of the E.S.R. The nature of the change brought about by the detergent in the colloidal solution of the plasma proteins is not known. The progressive diminution of the E.S.R. with increase in the concentration of t h e detergent, the absence of the Du Nuoy phenomenon and the results of the electrophoresis of the serumdetergent mixture suggest that there is an immediate and complete reaction between the two components, of a simple chemical nature. It is significant that, without any alteration to the protein mass, but with change in the chemical and physico-chemical character of certain of the protein fractions, and of the colloidal solution as a whole, modification or even inhibition of the formation of the erythrocyte aggregate can be achieved. Mass, therefore, is probably not the only factor to be considered in the evolution of a formula relating protein fractions to the E.S.R. One other factor to be considered, and one which Fahreus mentioned in his early work, is the influence of the electrical forces acting in the colloidal solution. Florey (194I) has shown that there is a marshalling of the protein molecules into a set pattern in colloidal solutions, and there is a hypothesis that this pattern derives from the electrical forces acting in the colloidal solution. The formation of aggregates from the negatively charged erythrocytes may be merely a reflection of this pattern of forces acting in the plasma. The cationic detergent, by reacting with the protein molecule, m a y cause a disintegration of this pattern, with consequent inhibition of aggregate formation.
Summary Experiments are described which show that addition of small amounts of a cationic detergent to blood plasma causes a diminution in the erythrocyte sedimentation rate and an increase in the plasma viscosity. In the concentration used the detergent did not have any effect on the erythrocytes. Electrophoresis of a similar serum-detergent mixture showed modification of the gamma-globulin and, to a lesser degree, in the alpha-2 and beta globulin peaks. Addition of calcium chloride solution to a plasma-detergent mixture failed to produce a fibrin clot. There was a fall in the surface tension of the plasma-detergent mixture, but no Du Nuoy phenomenon was observed. The significance of these findings is discussed, and the theory advanced that electrical forces acting in the colloidal solution of the plasma proteins have an effect on aggregate formation and, therefore, on sedimentation rate. I wish to acknowledge my indebtedness to Dr. A. C. Penman and to Dr. J. S. Miller, CheshireJoint Sanatorium, England, for helpful criticism and advice.
74
DICK$OI~
REFERENCES ANSON, M. L., and EDSALL,J. "17, (I948) : "Advances in Protein Chemistry." New York. CRoorm, E. M., HARMS, H., HASSAN, F., and WARP.EN, F. (1954) : Bioehem. 07., 56, 434FAHm~US, R. (I92 x) : " Suspension Stability of the Blood." Stockholm. FLORIn,, J. (194i):07. Amer. chem. S0c., 63, 3083 . HAM, T. H., and CtmTIS, F. C. (I938): Medicine, x7, 447. HARDWmrm, J., and SQUIRES,J. R. (1952):o7. din. Sci., 6x, 333. HARKN~SS, W., HOUSTON,J., and WHrrTINOTON, W. (1946) : Brit. reed. 3 - i~ 968. HOUSTON,J., and LAWm~NCE,J. S. (1955): Brit. 07. Tuberc. Dis. Chest, 49, 119. JmOENSONS, W., and STRAtrUANIS,W. (I954): " Colloidal Chemistry." London. MExr~R, A. J., Tm~VORROW,V., WASHBURN,A. H., and MUORAOE, E. R. (I953): Blood, 8¢ 893~ PLUCKTI~UN,H., and GOTTING,H. (1951) : Klin. Wochenschrifl, I5, 6. PUTNAM, F. W., and NF.URATn,J. (1945):O7. biol. Chem., 159~ 195. ROURKE, M. D., and ERNSTENE, A. C. (193 o) : 07. din. Invest., 8, 545. SCHUTT, E. D. (1928) : Svenshia Lff, ~'5, lOO9. ST~'~L~, F. (1948): Bull. Inst. reed. Lab. Tech., x4~ 78. SYrups, W. (1948) : Brit. reed. 07our., ii, 393. T'ANG and WANO (1941) : 07. trop. Meal. Hyg., 44, 5, 28.
T H E E R Y T H R O C Y T E S E D I M E N T A T I O N RATE IN PLASMA D E T E R G E N T M I X T U R E S BY IAN DICKSON From The Chest Clinic, Townsville, N. Queensland, Australia
THE inter-relationship between the mass of the different protein fractions in the blood plasma, and the rate at which the erythrocytes settle in the plasma (the erythrocyte sedimentation rate), was demonstrated by Fahreus (1921). Fibrinogen was shown to have the greatest and albumin the smallest effect on the rate. The variation in the erythrocyte sedimentation rate (E.S.R.) is governed by the size of the aggregate formed by the clumping together of the rouleaux of red blood ceils, the aggregate size being constant for a particular plasma. Advances in the technique of plasma analysis, first by salt fractionation methods, and more recently by electrophoresis, have led to attempts to obtain a mathematical relationship between the mass of the various proteins present in the plasma and the E.S.R. (Meyer et al., 1953). These attempts have had little success, one possible reason being that analytical techniques are still not sufficiently accurate to ensure a critical separation of all the protein fractions present in the plasma. By means of synthetic plasmas Hardwicke and Squires (1952) were able to obtain a mathematical relationship between the maximum velocity of fall of the erythrocytes and the concentration of the macromolecules. They were, however, unable to apply this formula to natural plasma. Sykes (1948) drew attention to a finding of Schutt (1928) that the addition of very small amounts of ether to plasma caused an increase in the E.S.R., whereas addition of a larger amount resulted in a slower rate. Since that time, however, with the advent of the detergents, a more potent method of effecting changes in the chemical and physico-chemical properties of protein has been found. Addition of minute quantities of detergent to a protein solution will bring about profound changes in the protein (Anson and Edsall, 1948). The present paper is based on experiments performed on blood from a series of tuberculous patients with raised E.S.R.s to determine what effect the modification in the plasma proteins by addition of detergent would have on the E.S.R. For the purpose of these experiments a cationic detergent, cetyl trimethyl ammonium bromide (cetavlon), was used. The anionic detergents were found to be less effective. METHOD
Twelve millilitres of blood were aspirated through a wide bore needle into a silicone lubricated syringe with a minimum of venous stasis. Two millilitres of the blood were added to o. 5 millilitre of a 3.8 per cent. solution of sodium (Receivedfor publication November 6, I958.)
ERYTHROCYTE SEDIMENTATION RATE I N PLASMA DETERGENT MIXTURES
69
citrate in each of six tubes. Five of the tubes contained different amounts of cetavlon (see Table 1). The E.S.R.s for the six citrated bloods were then determined, using the method described by Rourke and Ernstene (193o), and observing precautions against the sources of error described by Ham and Curtis (1938) . The results are given in Table I. Addition of the detergent to the blood causes a reduction of the E.S.R. until, in the higher concentrations, the rate is similar to that of the individual erythrocyte. Microscopy of the blood at this concentration of detergent reveals that there is no aggregate formation, and that the rouleaux are composed of no more than five to ten erythrocytes. PLASMA
VISCOSITY
Various workers (T'ang and Wang, 1941 , Harkness etal.,1946 ) have shown that increased viscosity of the plasma is associated with an increased E.S.R., and Harkness and his co-workers consider the plasma viscosity a more reliable index of the disease process than the E.S.R. The viscosities of the various plasma-cetavlon solutions used in the above experiments were determined, using the simple capillary viscometer described by Steele (I948), and following the modifications described by Hardwicke and Squires (1952). The results are given in Table i. It will be seen that with increasing amounts of cetavlon there is progressive lowering of the E.S.R., but there is an increase in the plasma viscosity. This is in direct contrast to the normal finding of increased E.S.R. with increased viscosity. In order to determine if the detergent acts solely on the plasma, or on erythrocytes, or on both these constituents of the blood, a series of experiments was performed in which two samples of two miUilitres of blood were taken from each patient and added to 0. 5 millilitre of a 3"8 per cent. solution of sodium citrate. Sufficient cetavlon to cause inhibition of the E.S.R. had been added to one of the 0. 5 millilitre of sodium citrate solution. The amount of cetavlon required had been previously determined experimentally by the method already described. The sedimentation rate was determined for the blood plus citrate mixture (sample A), and for the blood plus citrate-cetavlon mixture (sample B). Plasma and cells of both samples were then separated by centrifuging at 3,o00 revs./min, for eo minutes. After repeated washing of the blood corpuscles with normal saline, the cells from sample A were added to the plasma from sample B, and the cells from sample B were added to the plasma from sample A. The reconstituted bloods were then each mixed and the E.S.R.s again determined. It was found that the E.S.R. in the mixture containing the normal plasma corresponded to the E.S.R. in the normal blood sample (sample A), whereas the sample containing the plasma from the blood-cetavlon mixture (sample B) showed the same inhibition that was found in the original sample B. It was
70
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ERYTHROCYTE SEDIMENTATION RATE IN PLASMA DETERGENT MIXTURES
7I
thus shown that the factor causing inhibition of the E.S.R. when cetavlon is added to blood is confined to the plasma, since both sets of cells behaved normally in the normal plasma, and has no apparent effect on the erythrocytes themselves. In the various experiments performed with the different samples of plasma, it was found that the amount of detergent required to obtain the maximum effect on the E.S.R. varied within narrow limits (o-175-0.255 mg. per 2 millilitres blood). Addition of further amounts of detergent beyond this amount caused ha~molysis of the erythrocytes. SURFACE TENSION Another manifestation of changes in the physical properties of the colloidal solution of plasma proteins is alteration in the surface tension. In most colloidal solutions the addition of detergent causes a sharp drop in the surface tension followed by a gradual rise towards its original value (the Du Nuoy phenomenon). The explanation is thought to be that the detergent first spreads over the solution-air interface, causing the sharp drop in the surface tension, and later, reacting with the colloidal particles, is absorbed on to the surface of the particles, with the result that the surface tension rises. The surface tension of the plasma cetavlon mixture was measured by the stalagmometer as described by Jirgensons and Straumanis (I 954)- The method is based on the determination of the work required for the formation of a fresh surface of the liquid in the form of drops. A fixed volume of the liquid is allowed to drop from the lower glass surface of the stalagmometer at the rate of one drop every two to three seconds. The number of drops formed by the fixed volume of fluid is counted, and compared with the number of drops formed by an identical volume of distilled water. The surface tension is then found by the formula: S.T.:73~×D Zw : n u m b e r of drops of water Z ~ n u m b e r of drops of liquid D ~density of liquid
For the untreated plasma the surface tension was found to be 68 dynes per cenfimetre. For plasma with added detergent the surface tension was 44 dynes per centimetre. No Du Nuoy phenomenon was observed in the plasmacetavlon mixture in the concentrations used. ANALYSIS OF THE PROTEIN-DETERGENT MIXTURE There was no formation of a fibrin clot when calcium chloride solution was added to the plasma-cetavlon mixture. This failure of the plasma-cetavlon mixture to clot may be due to alteration in the fibrinogen, or to modification of the prothrombin. This is now being investigated.
72
DICKSoN
ELECTROPHORESIS OF SERUM DETERGENT MIXTURE
Analysis of untreated serum and of serum to which detergent had been added was carried out by paper electrophoresis,using a barbiturate buffer solution of p H 8, and staining with azocarmine after the method described by Pluckthun and Gotting (I951). The dyed filterpaper strips were then scanned according to the method described by Crook et al. (I954). The resultant traces are shown in Fig. I. t
Fio. I.--Electrophoresis traces of serum and serum--detergentmixtures.
Continuous line=serum. Interrupted line=serum+detergent. These traces show a modification in the gamma globulin as demonstrated by a disappearance of the gamma globulin peak in the serum-detergent trace. At the same time there may be some change in the alpha 2 and beta globulins as shown by the change in size and position of these peaks in the serumdetergent trace. Discussion
With the addition of small amounts of detergent to protein solutions Putnam and Neurath (I945) were able to show electrophoretically that excess protein migrated as a distinct entity from the protein-detergent complex, suggesting that the whole protein molecule becomes involved in the complex, or remains entirely dissociated from the detergent. The complex appears, therefore, to be formed as the result of a primary chemical reaction. Viscosity measurements and X-ray diffraction studies as a means of investigating molecular shapes have shown that the detergents bring about an unfolding of the protein molecule, which loses its rigid configuration and assumes a more random orientation. The viscosity of the solution of the resultant protein-detergent complex may be greater than that of solutions of either constituent alone. Proteins of rod-like morphology (and both fibrinogen and g a m m a globulins conform to this description) have a greater viscosity than the globular proteins,
BRYTHROCYTE
SEDIMENTATION
RATE
IN P L A S M A
DETERGENT
MIXTURES
73
such as albumin. Houston and Lawrence (1955) have shown that both fibrinogen and g a m m a globulin are the protein fractions most likely to be increased in the plasma of tuberculous patients. The raised E.S.R. and the increased plasma viscosity are probably, therefore, both manifestations of the same factor, namely, the increased amounts present of these two protein fractions. The unfolding of the plasma protein molecules following the addition of detergent can be presumed from the increased viscosity,but this unfolding is paradoxically associated with a lowering of the E.S.R. The nature of the change brought about by the detergent in the colloidal solution of the plasma proteins is not known. The progressive diminution of the E.S.R. with increase in the concentration of t h e detergent, the absence of the Du Nuoy phenomenon and the results of the electrophoresis of the serumdetergent mixture suggest that there is an immediate and complete reaction between the two components, of a simple chemical nature. It is significant that, without any alteration to the protein mass, but with change in the chemical and physico-chemical character of certain of the protein fractions, and of the colloidal solution as a whole, modification or even inhibition of the formation of the erythrocyte aggregate can be achieved. Mass, therefore, is probably not the only factor to be considered in the evolution of a formula relating protein fractions to the E.S.R. One other factor to be considered, and one which Fahreus mentioned in his early work, is the influence of the electrical forces acting in the colloidal solution. Florey (194I) has shown that there is a marshalling of the protein molecules into a set pattern in colloidal solutions, and there is a hypothesis that this pattern derives from the electrical forces acting in the colloidal solution. The formation of aggregates from the negatively charged erythrocytes may be merely a reflection of this pattern of forces acting in the plasma. The cationic detergent, by reacting with the protein molecule, m a y cause a disintegration of this pattern, with consequent inhibition of aggregate formation.
Summary Experiments are described which show that addition of small amounts of a cationic detergent to blood plasma causes a diminution in the erythrocyte sedimentation rate and an increase in the plasma viscosity. In the concentration used the detergent did not have any effect on the erythrocytes. Electrophoresis of a similar serum-detergent mixture showed modification of the gamma-globulin and, to a lesser degree, in the alpha-2 and beta globulin peaks. Addition of calcium chloride solution to a plasma-detergent mixture failed to produce a fibrin clot. There was a fall in the surface tension of the plasma-detergent mixture, but no Du Nuoy phenomenon was observed. The significance of these findings is discussed, and the theory advanced that electrical forces acting in the colloidal solution of the plasma proteins have an effect on aggregate formation and, therefore, on sedimentation rate. I wish to acknowledge my indebtedness to Dr. A. C. Penman and to Dr. J. S. Miller, CheshireJoint Sanatorium, England, for helpful criticism and advice.
74
DICK$OI~
REFERENCES ANSON, M. L., and EDSALL,J. "17, (I948) : "Advances in Protein Chemistry." New York. CRoorm, E. M., HARMS, H., HASSAN, F., and WARP.EN, F. (1954) : Bioehem. 07., 56, 434FAHm~US, R. (I92 x) : " Suspension Stability of the Blood." Stockholm. FLORIn,, J. (194i):07. Amer. chem. S0c., 63, 3083 . HAM, T. H., and CtmTIS, F. C. (I938): Medicine, x7, 447. HARDWmrm, J., and SQUIRES,J. R. (1952):o7. din. Sci., 6x, 333. HARKN~SS, W., HOUSTON,J., and WHrrTINOTON, W. (1946) : Brit. reed. 3 - i~ 968. HOUSTON,J., and LAWm~NCE,J. S. (1955): Brit. 07. Tuberc. Dis. Chest, 49, 119. JmOENSONS, W., and STRAtrUANIS,W. (I954): " Colloidal Chemistry." London. MExr~R, A. J., Tm~VORROW,V., WASHBURN,A. H., and MUORAOE, E. R. (I953): Blood, 8¢ 893~ PLUCKTI~UN,H., and GOTTING,H. (1951) : Klin. Wochenschrifl, I5, 6. PUTNAM, F. W., and NF.URATn,J. (1945):O7. biol. Chem., 159~ 195. ROURKE, M. D., and ERNSTENE, A. C. (193 o) : 07. din. Invest., 8, 545. SCHUTT, E. D. (1928) : Svenshia Lff, ~'5, lOO9. ST~'~L~, F. (1948): Bull. Inst. reed. Lab. Tech., x4~ 78. SYrups, W. (1948) : Brit. reed. 07our., ii, 393. T'ANG and WANO (1941) : 07. trop. Meal. Hyg., 44, 5, 28.