COXGUIATIO?; O F BLOOD AND M I L K BY ELECTROLYTES A S D THE SISIILARITY B E T R E E S T H E CLOTTING OF BLOOD AYD T H E FORMATION O F JELLIES BY N. R. DHAR AND SATY-.4 PRAKASH
In publications' from these laboratories, we have proved that sols can be divided into two groups:ti) The first group of sols consists of ferric hydroxide, aluminium hydroxide, chromium hydroxide, hydrated manganese dioxide, etc. This group of sols does not appreciably adsorb ions carrying the same charge as the sols when coagulated by KC1, K2SOl,BaC12, etc, and this class of sols does not become stable on dilution, does not show the phenomenon of positive acclimatisation, and shows additive relationships when coagulated by mixtures of electrolytes of different valencies. These sols do not show appreciable decrease of viscosity on the addition of small quantities of electrolytes. (ii) The second class of sols consists of arsenious sulphide, antimony sulphide, mastic, Prussian blue, etc. These sols adsorb appreciably ions carrying the same charge as the sols. They become more stable on dilution, show the phenomenon of positive acclimatisation, and show marked ionic antagonism when coagulated by mixture of electrolytes of different valences; their viscosities decrease appreciably when small quantities of electrolytes are added to them. I n this communication, we are recording our experimental results obtained with blood and milk, showing that blood and milk belong to the second class of colloids which adsorb ions carrying the same charge as the sols. Ordinary sheep's blood was collected in a bottle of capacity zoo cc. containing I cc. of toluene. During the course of experimentation a part of the blood clotted. The part which remained unclotted was utilised for the experiments. 50 cc. of unclotted blood were diluted to 2j0 cc. The volume was kept I O cc. in every case and 2 cc. of this diluted blood were used for coagulation. In the case of experiments with concentrated blood, 2 cc. of original undiluted blood were taken. Time of observation was kept one hour. I n the case of blood, TABLE I Electrolytes
Sodium citrate Potassium oxalate Potassium fluoride Sodium tartrate Ammonium nitrate Hydrochloric acid
Concentration necessary for coagulation in mol9 Dilute blood Concentrated blood
o 704 I oj I 6j3 I 139 4 6jj o 436
lf 31 hl
-
o 835 hl
368 M 013 A I 4 482 11 0 387 hl I I
?*I A1
' J. Phys. Chem., 29, 435, 6 j 9 (1925);31, 649 (1927)
S . R. DH.%R A S D hITP.1 PRAKASH
4.60
appcwance of turbidity was taken as the coagulation point. For experiments n.ith milk boiled cow’s milk was utilised. 2 0 cc. of it xere diluted t o I O O cc. arid 2 cc. of this diluted milk were used in each case. z cc. of original undiluted milk were taken in experiments with concentrated milk. The cxperiiiients were carried on at 3oOC. and t,he results are given in Table I. ‘r.\BLE 11 Conientration necessary for coagulation in mols Dilute milk Concentrated milk
Electrolytes
Podium tartrate Potassium fluoride Potassium oxalate Sodium acetate Hydrochloric acid
I ,2IO
31
31 1.96031 1.121
2.4jo 31 1 . 1 5 5 31 3 . 3 1 1 11 0.228
1.050
11
2.838 11 o.rj9 31
31
The experimental results show that diluted blood and milk are more stable towards sodium tartrate, citrate, potassium oxdate, fluoride, hydrochloric acid and ammonium nitrate. I n some previous communications’ we have investigated the sensitising influence of gelatine in different sols and we have advanced a general explanation of the phenomenon of sensitisation. I n this paper we have investigated the influence of traces of gelatine and saponin in the coagulation of blood and milk and the experimental results given in Table 111 have been obtained.
TABLE I11 olution of Kahlbaum ‘‘Golddruck’! gelatine v a s used z cc. of dilute blood used, volume kept I O cc. Time of observation--r hour. Concentration necessary for coagulation in mols Dilute Blood with gelatine
Electrolytes
Sodium citrate Sodium tartrate Potassium oxalate Potassium fluoride Hydrochloric acid
o.jo4 31 1 . 1 3 9 11
1 . 0 5 11 1 . 6 5 3 31 0.452 31
0 . 2 C.C.
0.5 C.C.
0.660 31 1 . 1 2 1 31 o . y i j 11
0 . 6 2 7 ;\I
__
0.452
AI
068 31 11 1.58611
1.0 C . C .
I
1 . 0 3 2 31
0.945
0.990
__
AI
1.528 31 0.44j 31
TABLE 11One percent solution of saponin was uatd. Electrolytes
Concentration n w e s a r y for coagulation in mola Dilute Blood with saponin 0.2 c.c
Sodium citrate Potassium oxalate Potassium fluoride Hydrochloric acid -
o.jo4 31 J . 0 j 31 I , I O 31 0..+52
31
0.693 11 0 . 9 9 0 31 I ,653 0.3j2
11 11
’ Jiolloid-Z., 38, 14 (1926);39, 346 (1926:;41,229 (1927).
0 .j C . C .
__ 1.00; I . 606
31
0 . A l j
31
11
I 0 C.C.
__
1.0,lj
-_ -_
31
COAGULATIOS O F BLOOD A S D MILK B Y E L E C T R O L Y T E S
TABLE T' IC;; solution of gelatine and 1% of saponin were used. 2 C.C.of dilute milk made up to Time of observation-1 hour.
I O C.C.
Concentration necessary for coagulation in mols Electrolytes
Dilute milk
with gelatine 0.2c.C.
O.jC.C.
with saponin I.0C.C.
O.ZC.C.
Podium tartrate ~.zro?rI I . I ~ ; A I 1 . 1 2 1 1 1 1.08511 Hydrochloric acid 0.2281\1 0.22811 0 . ~ 2 8 1 1 o.zz8M 0 . 2 1 6 M
0.jC.C.
0.20511
1.Oc.C.
-
0.18~11
The above experimental results show that saponin and g e k i n e render blood and milk unstable ton-ards salts. With acids gelatine does not sensitise blood and milk. It seems likely, therefore, that the seneitising influence of gelatine is really due to t'he hydrogen ions present in it. The sensitising influence of saponin is difficult to explain. It will be interesting to note that saponin sensitises the coagulation of blood and riiilk by salts as well as by acids. It appears, therefore, that the hemolytic action of saponin on blood is due to its rendering blood unstable towards electrolytes. I n presence of saponin the blood particles are rendered unstable and hence coagulat'ion and hemolysis become easier. I n previous papers, we have shown that the scnsitising influence of gelatine towards different sols is UP to its containing acidP. From our experimental results, it will be seeii iat hydrochloric acid coagulates blood ancl milk more readily than salts the saiiie concentration. I n other ~vortli;. both blood and milk are w r y xmitive to the coagulat,ing influence of H' ioni. I n this respect, blood and milk resemble mastic, gamboge and other readily hydrolysable sols. The view that the sensitising influence of gelatine i; due to the presence of H' ions present in gelatine is supported by the fact that there is no sensitisation by the presence of gelatine when blood, and milk are coagulated by hydrochloric acid. Consequent,ly, the sensitising influence of gelatine on blood and milk is due to the presence of acid in gelatine. have carried on experiments on the coagulation of a so1 of henioglobin by different electrolytes and we have found that, the sol of hemoglobin is rendered iinstable towirds electrolytes by thc presence of saponin. The stnbilisation of blood and milk hy the addition of sinal1 quantities of oxalate, citrate fluoride, tartrate and sulphate ions is certainly duc to the adsorption of the negative ions from various electrolyte., and the conwlucnt increase of the negative charge on blood and milk. Ti-- cupric =iilphate in prciencc: of :.niall quant,ities of sodiuni citrate. sodiuni ncrtate, potassinn1 fluoride, sodium tartrate ant1 potassium hyrlroside.
N. R. DHAR A S D SATYA PRAKASH
TABLE VI z C.C. of blood made up to I O cc. Time = I hour. Amount of electrolyte added in mols Concentration of ammonium nitrate necessary for coagulation
&I
KO salt added Sodium citrate Potassium fluoride Sodium acetate Potassium hydroxide
2
0 022
4 482 &I 5 2 5 8 11 4 741 11
n1
o 114 11 0 095 31 o 002 31
4 913 11 4 999
TABLE T’II c c. of dilute blood made up to Time = I hour
Amount of electrolyte added in mols
IO
?*I
c.c
Concentration of copper sulphate necessary for coagulittion
No salt added
AI 51 o 0009jo AI 0 00002j
Sodium acetate Sodium tartrate Sodium citrate Potassium hydroxide
0966 XI 0178 AI 0022
0020
0 000175
;\I M
0 000200
o
000700
11 AI
The following results were obtained in the coagulation of dilute milk by cupric sulphate in presence of sodium acetate, sodium tartrate, sodium citrate and potassium hydroxide. z
C.C.
TABLE VI11 of dilute milk made up t o Time = I hour
Electrolytes added in mols
Concentration of copper sulphate necessary for coagulation
KO salt added Sodium acetate Sodium tartrate Sodium citrate Potassium hydroxide
I O C.C.
0.0011
0.00946 bI 0 . 0 0 3 5 6 hl
0.00Ij 0.0015
31 hI 11
nr
0.0022
11
0.0027
0,002
31
0.0017 AI
The experimental results given in Tables VI-VIII, prove conclusively that blood and milk show ionic antagonism markedly when coagulated by ammonium nitrate or copper sulphate in presence of small quantities of sodium citrate, potassium fluoride, sodium acetate and potassium hydroxide. In presence of the above substances the particles of blood and milk are stabilised by the adsorption of OH’ and other negative ions from the above electrolytes. This stabilisation of blood and milk is due to the increase in the amount of negative charge on the particles and is exactly identical with the behaviour of sols of arsenious sulphide, antimony sulphide, Prussian blue, etc., already investigated.
COAGULATION O F BLOOD AND MILK BY ELECTROLYTE%
463
Our experimental results show that the explanation of the stability of blood and milk due to the presence of small amounts of citrate, tartrate, oxalate, fluoride, etc., based on the view of the removal of the precipitating calcium ions by the above negative ions is incorrect. The real explanation of the stabilisation of both blood and milk by the presence of sodium or potassium salts of the the above acids, is the increase in the charge on the particles of blood and milk due to the adsorption of the negative ions and of OH' ions derived from the hydrolysis of the sodium or the potassium salt,% of the weak acids already mentioned. Recently medical men are using solutions of sodium citrate with great success for stopping the outflow of blood with sputum in cases of tuberculosis. Concentrated solutions of sodium citrate are intraveneously injected and the out'flow of blood is stopped in a short time. From the experimental results recorded in this paper, it will be evident that the stopping of outflow of blood is due t o its coagulation by concentrated solutions of sodium citrate. These results are interesting in view of the well-known fact that dilute solutions of sodium citrate hinder the coagulation of blood. Our results explain satisfactorily the peculiar behaviour of blood, that it is stabilised and clotting is hindered in presence of dilute solutions of sodium citrate, oxalate, fluoride, etc., and that the outflow of blood is stopped by concentrated solutions of sodium citrate. I n previous communications1 from these laboratories, we have investigated the conditions of the formation of jellies of different substances. We have proved that jellies can be divided into three groups. The first group consists of gelatine, starch, agar, silicic acid, soaps, manganese arsenate, zinc arsenate, vanadium pentoxide, ceric hydroxide, etc. In this group, the particles forming the jellies, consist of some kind of network. These substances produce most stable jellies and are formed very readily. The second group of jellies is obtained by the slow coagulation of the sol throughout its whole mass. Possibly, hydroxides of iron, aluminum, and chromium, form typical members of this group and the particles of this group need not consist of a network. The stability of this group is less-than t h a t of the first group. Both these groups have marked affinity for water. The third group forms the von \J7eimarn jellies wbich are the least stable ones. These jellies consist of finely divided substances which are precipitated very suddenly. We are of the opinion t h a t clotted blood is nothing but a jelly which is similar to that obtained in the first group of substances consisting of particles forming a net-work. I n previous papers, we have proved that the stable jellies of ceric hydroxide, vanadium pentoxide, and silicic acid undergo syneresis in course of time due to the decrease of the hydration tendency of the particles forming the jelly. It seems likely that the blood as a whole is an unstable hydrophile colloid, which sets t'o a jelly very readily. I n many respects, blood is something like ceric hydroxide sol. On keeping blood, it forms a gel very readily; in 'Z. anorg. allgem. Chem., 152, 399 (1926); 164, 63 (1927).
this respect, it is certainly less stable than ceric hydroxide sol prepared in the cold. 1 1 h n blood set>, it unciergoes syner:>sis very readily. The syneresis in PI~ than in the case of other inorganic case of blood is a much C ~ U ~ C ! ~pixmss antl organic jcllic=. It wenis likely that the coagulation of blood or of Iiiilk by concentrated solution* of sodiiini tartrate, citrate? pot oxalate. ixtc., iq real coagulation and not clotting or gelation. wts in in the case of blood, the hydration tendency of the blood decreases and dehydrntioii begins with t,h(, styarntion t ~ ffibrin. 1i-e niust try t starch ,jelly anti gelatine jelly, when broken, d o not re-set readily. Consequently, in iiiariy respects blood and possilily rnillc rrsenible r? sol of vanadiuin pentoside or ceric hydroside or m a n y m c ~ rarsenate, though blood is far less stable than the oth(1r sol,. The iriiportant question which nmv arises and remains unanswered i,--vvhy do blood a r i d iiiilk not coagulate in the anima! tern'? In the case of blood, it secnIs likely that the clotting is prevented in the system by the rnot,ion of the fluid. I \ h n the motion is disturbed by sonic means or other, clots or thrombi are formed.
AIoreovcr, froin some recent espcrinwnts, it has b w n proved that the clotting tendency is most marked near the neutral point of blood. On the acid side and alkaline side, thc charge on the blood is increased and hence the hydration tendency is les? antl hence clotting happens with greater difficulty in alkaline or acid solutions of blood. If blood recrivcd in aveswl is not stirred, thi. clot fornis unifornily throughout the whole quantity of blood. converting it to a mlid, rather dry. firm mass. Such a. clot iq of uniform dark red colour. If it be squeezed, a dark red fluid
I'OAGULATIOS O F BLOOD A S D MILK BY ELECTROLYTES
465
is forced out vhich is identical with defibrinated blood. If the fresh blood be kept T-ery cold or if it be received in an oiled dish, it will not clot so quickly; and since the red corpuscles are heavier than the plamia, tlierc may be time for them to sink to the bottom in a very thick layer. The leucocytes are lighter and rest in a layer on top of the red corpudea. whilst above, t h r r r is some plasma almost free from cells. By t,his tiinc, clotting occurs throughout, the clot differing from the uniform red one fornied by rapid coagulation in the presence of a greyish yellow upper layer TThieh contains iiio;.t of tho leucocytes and platelets. This slow clotting showing the eftect of gravity is icen w r y commonly in the heart a t autopsy, for the'intnct endothelial lining of the h a r t keeps its contents a long time n.ithout clotting. Then the tieep rrd portion of the clot is in tlie dependent part, while the tough, clastic, translucent j-ello~~-ish .substance occupies the uppermost part' of the heart. Tlic foregoing results can be esplaincd from the view that blood eonsi mixture of colloids having different densities. If the clotting is x rapid as it usually happens \Then the blood is shed, network or riirilies fornied and t,he whole of the liquid is adsorbed in the netwo~,li,and n uniform jelly is obtained, there being no tinic: for the separation of t h r colloid-: aecording t o their difference in densities. \Then the clotting is rctnrdctl. thc3rc is time for different colloids to be wparatrd in Merent, layers, nxitl xvc gf't clotting in different layers. IVe are of the opinion, therefox, that, blood eonsi-ts of a niisture of colloids and forniq a very unstable systrrn. anti ha.: a great tendency to clot. This clotting tendency is w r y likcly e a u w l hy the smie forces \r.hich cause the gelation of 501s of silicic acid. vanatliurii pentoside, civic hydroxide, starch, gelatine, etc., nnd i? prohsbly t h e to the incri: the hydration tendency of the particles Tvhich forin :Lnctwork. of
:i
It is wll-known that the action of copper on nitric acid is an autocatalytic p r o c r s ~ ,because the product nitrous acid niaiketlly accelerates the reaction. I t P e r m s likely that the clotting of blootl i q n k o autocatalytic in its nature. because the liquid given out inniicdiately after clotting aect~ler:itc* tlic clotting process. Hence we are of the opinion that thc clotting of blood is :i p ~ ' o c + r which takes place by itself clue t o the cxtreiiiely unstable nature of blood and is not initiated by thrombin or any other substance. It aeenla likely, however, that thrombin and other products derived after clotting niarkedly accelerate the clotting process. The syneresis that is observed is really an ageing phenomenon which is observed with most ot,her gels and is due t o the decrease in the free surface and the activity and hydration tendency of the particles forming the jelly. IT-e have repeatedly observed that sols of silicic acid, vanadium pentoside, ceric hydroxide, etc., form transparent jellies by merely keeping in a stoppered bottle a t the ordinary temperature. I n course of time these stable jellies undergo synere actly similar behaviour is observable with blood, the only difference being that the blood is far less stable than the inorganic sols; and the clotting and syneresis are quicker processes in blood than the gelatin and syneresis observed with the inorganic sols.
S . R . D I M R AND SATYA PRAKASH
Summary Dilute blood and milk are more stable than concentrated blood and milk towards their coagulation by sodium citrate, sodium tartrate, sodium acetate, pot'assiurii oxalate, potassium fluoride, ammonium nitrate, and hydrochloric acid. 2. I n presence of small quantities of sodium citrate, potassium fluoride, sodium acetatc, sodium t a r t r a k , or caustic potash, blood and milk are stabilised towards their coagulation by ammonium nitrate, or copper sulphate. This stabilisation is due to the increase in the amount of negative charge on the particles of blood and milk by the adsorption of OH' and other negative ions from the salt solutions. 3 . Blood and milk behave like sols of arsenious sulphide, antimony sulphide, Prussian blue, etc., which are known to adsorb ions carrying the same charge as the sols. 4. Saponin and gelatine render blood and milk unstable towards salts, with acids, gelatine does not sensitise blood and milk. It seems that the sensitking influence of gelatine is due to the H' ions present in it. 5 . It appears that the liemolj-tie action of saponin on blood is due to its sensitking influence and its rendering blood unstable towards electrolytes. 6. The esplanation of the stability of blood and inilk in presence of small amounts of nit,rate, oxalate, fluoride, etc., is based on the fact that the electric charge of the particles is increased by the adsorption of negative ions and not on the removal of precipitating calcium ions by the above negative ions as has been hitherto believed. ;. The clotting of blood seems to be guided by the same laws as the formation of jellies of vanadium pentoxide, ceric hydroxide, silicic acid, etc. The only difference seems to be that blood is far less stable than the inorganic sols; and the clotting and syneresis are quicker processes in blood than the gelation and syneresis observed in organic and inorganic sols. I.
Cheniicnl Laboratoru, Allahahad L'?ii!IersiQ, Allahabad, India. October 24, 1027.
