582.6 SOME OBSERVATIONS UPON SODIUM ALGINATE. By 0. M. SOLANDT. From the Physiological Laboratory, Cambridge. (Received for publication 13th December 1940.) ALGINIC acid was discovered by Stanford in 1883 in the course of experiments on the extraction of iodine from Scottish kelp [Stanford, 1883]. It is now prepared commercially in the British Isles from the sea-weed Laminaria hyperborea. It is coming into general use as an ingredient of foods and to replace agar in many of its uses. Alginic acid is probably a polymer of d-mannuronic acid with an empirical formula of C6H1007 and an equivalent weight of 194. The molecular weight varies with the degree of polymerisation from 40,000 to 250,000. The viscosity in solution is very high, and rises rapidly with increasing molecular weight. Alginic acid itself is unstable and only slightly soluble in water. The sodium salt is quite stable at room temperature and ph 5-5 to 8-5 and is soluble in water to the extent of 3 to 10 per cent. depending upon the molecular weight [Albright and Wilson, 1938; Rose, 1937; Nelson and Cretcher, 1929, 1930; Barry and Dillon, 1936; Dillon and McGuinness, 1931]. Sodium alginate is available under the trade name of Manucol in a variety of grades which differ in their average molecular weights. The range of molecular weights which is available includes the molecular weights of the plasma proteins. This suggested that sodium alginate might be a suitable substance for use in the treatment of shock when blood or plasma was not available for transfusion. Experiments soon showed that sodium alginate is toxic when injected intravenously and is quite unsuitable for this purpose. TEE TOXICITY OF SODIuM ALGINATE. Sodium alginate solutions were tested for toxicity by intravenous injection into mice and rabbits. The tests were done with a 1 or 2 p.c. solution of Grade III sodium alginate (average molecular weight 85,000) in 0-9 p.c. saline. The minimum lethal dose was not determined accurately but is about 100 mg./kg. in rabbits. Four rabbits were given doses exceeding 125 mg./kg. and all died. Three were given a dose of 100 mg./kg.; one survived and two died. One rabbit received 85 mg./kg. and two 75 mg./kg. and all survived. The time of death varied from 15 minutes to about 15 hours after the injection. 25
26 Solandt The earlier deaths were always accompanied by mild convulsions, marked cardiac slowing, and early cessation of respiration. In mice (9) doses of 200-500 mg./kg. were fatal in from 1 minute to 12 hours. In the rabbits, whether death was immediate or delayed, there was always marked dilatation of the right heart and often considerable pulmonary cedema. Sections of the lungs from three of these rabbits showed, in addition to the usual picture of pulmonary cedema, a very marked cedema of the adventitia of the arteries and arterioles. There was no similar lesion around the veins or bronchi. Sections of liver showed no abnormality. There was no gross or microscopic evidence of embolism due to precipitation of sodium alginate or to clumping of red blood cells. The effect of the intravenous injection of sodium alginate on a lowered blood-pressure was tested in five cats under chloralose anesthesia, the blood-pressure having been lowered by hawmorrhage. In all cases there was a transient rise in blood-pressure, followed by a fall which ended in the death of the animal if the dose of sodium alginate was large. The lethal dose varied greatly with the rate of injection. Doses between 120 and 450 mg./kg. were fatal within a few minutes. Marked slowing of the heart preceded death, but the heart continued to beat while the blood-pressure was falling and after respiration had ceased. At autopsy the right side of the heart was greatly distended and there was some pulmonary cedema. No attempt was made to estimate the toxicity of sodium alginate when given by mouth or subcutaneously. It is widely used in food products and is apparently harmless. THE EFFECT OF SODIuM ALGINATE ON THE SEDIMENTATION RATE OF RED BLOOD CELLS. Sodium alginate greatly accelerates the sedimentation of red blood cells. Sedimentation rates were measured in Westergren tubes. Most of the experiments were done with a suspension of twice-washed red blood cells in 0 9 p.c. saline. A 40 p.c. suspension of cells was used, and this was diluted with an equal volume of the sodium alginate solution to be tested. In some cases whole blood was used and similarly diluted. The sodium alginate solutions were made up in normal saline so that all the solutions were isotonic or slightly hypertonic. The results for normal, twice-washed, human red cells are shown in fig. 1. This is the average of four experiments. The extent of sedimentation in each of the various strengths of sodium alginate in 30 minutes is shown. In these experiments sodium alginate Grade III with an average molecular weight of 85,000 was used. Similar results were obtained with Grade I (average molecular weight 40,000). The
Some Observations upon Sodium Alginate sedimentation of unwashed cells was similar or slightly more rapid. The increase of sedimentation rate in whole defibrinated blood was much the same. Similar experiments were performed using whole heparinised blood from rabbits and rats. In both these animals the effect of sodium 27 w. 2. ~~~~Q o Q) FIG. 1.-The sedimentation of washed human red cells in various strengths of Grade III (average molecular weight 85,000) sodium alginate solution in 30 minutes. (Average of four experiments.) C =control. FIG. 2.-The sedimentation of washed human red cells in various strengths of Grade VIII (average molecular weight 185,000) sodium alginate solution in 30 minutes. C = control. alginate upon the sedimentation rate in diluted whole blood is similar to that seen with human blood. Washed cells from the rabbit and the rat show a somewhat lower sedimentation rate with sodium alginate.
28 In defibrinated sedimentation rate, IHH Solandt Di horse blood, which normally has a very high I p.c. sodium alginate markedly diminished the HHHHhlMI.Oo 0 A 9 0 C 0z %~ r'j -: 0 FIG. 3. A. The sedimentation of washed human red cells in various strengths of Grade III sodium alginate solution. C =control. B. The sedimentation of washed human red cells in the same solution after it had been heated for 1 hour at 1000 C. The heating reduced the viscosity of the alginate solution approximately ten times and increased the sedimentation rate in the higher concentrations as shown. C =control. sedimentation rate. Lower concentrations of sodium alginate increased the sedimentation rate just as in blood from other species. Direct observation showed that the increased sedimentation rate in the presence of sodium alginate is due to the clumping together of the B c
Some Observations upon Sodium Alginate red cells into large aggregates. This clumping resembles true agglutination both macroscopically and microscopically but is readily distinguished by the ease with which the clumps can be shaken apart. It seems reasonable to suppose that the slow sedimentation seen in the higher concentrations of sodium alginate is due to the high viscosity of these solutions. The clumping of the corpuscles is clearly visible even though they do not settle. Fig. 2 shows the sedimentation rate of washed human cells in Grade VIII sodium alginate (average molecular weight 185,000). The sedimentation rate is much lower than in Grade III, and the most rapid sedimentation occurs in a more dilute solution. The effect of sodium alginate in producing an increased sedimentation rate cannot be due to its viscosity alone. If sodium alginate solution is heated to 1000 C. it loses its viscosity before it loses its ability to produce an increase in sedimentation rate. This is shown in fig. 3. It is apparent that heating for 1 hour at 1000 C., which causes an approximately tenfold reduction in viscosity, increases the sedimentation rate in 0 5 p.c. concentration and has very little effect on the sedimentation rate in 0-25 p.c. concentration. Heating for 4 hours produces a further decrease in viscosity and a marked diminution in the effect on the sedimentation rate. Direct observation of the mesenteric vessels in the cat during fatal poisoning with sodium alginate sometimes showed clumping of corpuscles visible to the naked eye and sometimes did not. In three of the rabbits blood was drawn from the heart at death. The sedimentation rate of this blood (heparinised) was 3 mm. in 30 minutes. This is more than is normal in rabbits but is very slow compared to the rates seen in vitro. The amount of sodium alginate injected into these animals was sufficient to give an immediate concentration of 0-14 to 0-2 p.c. in the blood, assuming a blood volume of 70 c.c. per kilo. CALCIUM AND THE TOXICITY OF SODIUM ALGINATE. Maas [1938] has recently shown that the immediate toxicity of some samples of gum acacia is due to the absorption of calcium from the blood by the sodium arabinate of which the gum is mainly composed. This is shown by the fact that the intravenous injection of calcium chloride prevents the lethal effect of such samples of gum and that calcium arabinate is non-toxic. It seemed probable that a similar binding of the blood calcium might account for some of the toxicity of sodium alginate since the two substances have many properties in common. There is, however, the important difference that calcium arabinate is soluble, whereas calcium alginate is very insoluble. In two rabbits the total serum calcium fell from 13.0 and 13*5 mg. per cent. to 11 2 and 11 8 mg. per cent. 1 hour after the injection of 29
30 Some Observations upon Sodium Alginate 100 mg./kg. of sodium alginate. It is probable that the lowering of calcium would be better seen in an ultra-filtrate of serum, but the investigation was interrupted before this could be done. In two rabbits a dose of 150 mg./kg. of sodium alginate was given and was followed within 10 minutes by the intravenous injection of 6 mg./kg. of calcium given as calcium chloride. Death was immediate in each case. Another rabbit was given 75 mg./kg. of sodium alginate intravenously, followed in 10 minutes by 5 mg./kg. calcium. There was an immediate convulsion, and death ensued in less than 5 minutes. Control experiments showed that doses of calcium up to 10 mg./kg. produced no obvious effect and that a dose of 75 mg./kg. of sodium alginate was not lethal. DISCUSSION. The toxicity of sodium alginate when injected intravenously is much greater than would be expected from its chemical composition and physical properties. Its action in accelerating the sedimentation of red blood cells is very marked but does not appear to account for this toxicity. Other substances such as gum acacia have a similar effect on blood sedimentation but are much less toxic [Kruse, 1919, 1920; Lucia and Brown, 1934; Hanzlik and Karsner, 1919]. Sodium alginate may, like some samples of acacia, combine with calcium in the blood and lower the ionised calcium significantly. However, the toxicity of sodium alginate cannot be directly due to the lowering of blood calcium because the subsequent injection of calcium increases, rather than decreases, the toxicity of an injection of sodium alginate. It seems probable that the toxicity of sodium alginate is related to its precipitation as the insoluble calcium salt. There is, however, no evidence of such a precipitation in the microscopic sections of liver and lung from the injected animals. REFERENCES. ALBRIGHT AND WILSON, LTD. (1938). Booklet on Manucol (Sodium Alginate). BARRY, V. C., and DILLON, T. (1936). Sci. Proc. Royal Dublin Soc. 21, 285. DILLON, T., and McGUINNESS, 0. (1931). Ibid. 20, 129. HANZLIK, P. J., and KARSNER, H. T. (1919). J. Pharmacol. 14, 379. KRUSE, T. K. (1919). Amer. J. Physiol. 49, 137. KRUSE, T. K. (1920). Ibid. 51, 195. LuCIA, S. P., and BROWN, J. W. (1934). Proc. Soc. exp. Biol. N. Y. 32, 189. MAAS, J. A. (1938). Quart. J. exp. Physiol. 28, 315. NELSON, W. L., and CRETCHER, L. H. (1929). J. Amer. Chem. Soc. 51, 1914. NELSON, W. L., and CRETCHER, L. H. (1930). Ibid. 52, 2130. RoSE, R. C. (1937). Ph.D. Thesis, University of London.