OSMOMETRIC STUDY OF GUM ACACIA SOLUTIONS FOR INTRAVENOUS INJECTION’ HUGH R. BUTT
AND
USED
ANCEL KEYS
Division of Biochemistry, The Mayo Foundation, Rochester, Minnesota Received August 1, 1957
Intravenous injections of gum acacia solutions are widely used, clinically, to increase the colloid osmotic pressure (C. 0. P.) of the blood plasma. In addition to the lack of available data on the effects of such injections on the total C. 0. P. of the blood plasma, there is a discrepancy in the reported C. 0. P. values, or mean particle size, in simple gum acacia solution itself. This is probably the result of several factors, among which importance may be given to variations in individual samples of gum acacia, especially with regard to particle size and sodium and calcium content. The basic structure of gum acacia is a galactoso-glucuronic acid (3, 8). Primarily it is a mixture of polymerized anhydrides of galactose and arabinose in varying proportions. In the crude state the calcium, potassium, and magnesium salts of arabic acid predominate. It has been repeatedly demonstrated that the sodium salt of gum acacia gives higher C. 0. P. values than the calcium salt (2, 7). Perhaps these two factors alone account for part of the discrepancy in the reported values (table 1). However, a variability in the C. 0. P. from 8 to 275 mm. of mercury in a single concentration of gum acacia seems large, even if we assume different particle size and salt concentrations. All of these authors, as far as we are aware, assumed that a protein-tight membrane was also impermeable to gum acacia particles. In no instance is there mention that the membranes used were “acacia-tight” or that the outer medium was tested for the presence of gum acacia. In this investigation we first attempted to measure the C. 0. P. of 6 per cent gum acacia, using tested protein-tight collodion sacs suspended in a suitable medium a t 0°C. Regardless of the outer medium (phosphate buffer, physiologic saline solution, or distilled water) the C. 0. P. value continued to fall during a period of at least twelve days. Qualitative tests showed that gum acacia entered the outer medium in each instance. When phosphate buffer (pH = 7.4) was the outer medium it was noticed Presented a t the Fourteenth Colloid Symposium, held a t Minneapolis and Rochester, Minnesota, June 10-12, 1937. 21
22
HUGH R. BUTT AND Ah7CEL KEYS
that small, grayish-white crystals collected on the outside of the collodion sacs and a few crystals settled out in the bottom of the tube containing the buffer. These were assumed to be, without direct evidence, crystals of calcium phosphate. The membranes employed possessed a permeability to distilled water varying between 0.005 to 0.014 cu. mm. per minute per square centimeter per 1 cm. of water pressure. The properties of gum acacia sol are greatly changed by the presence of proteins. When 6 per cent gum acacia is added to human blood serum, a stable equilibrium is quickly reached in collodion sac osmometers and therc is no evidence of leakage of the acacia through the membrane. There is a rise in the C. 0. P. of the serum approximately in proportion to the amount of acacia added (table 2). In the presence of relatively TABLE 1 Measurements o j the colloid osmotic pressure of 6 per cent solutions of acacia COLLOID OSMOTIC
S A L T C O N T E N T OF M E D I U M
AUTEOR
PREBSURI
mm. Hg.
0. . . . . . . . . . . . . . . . . . . . . . . . . 0. . . . . . . . . . . . . . . . . . . . . . . . . Na salt . . . . . . . . . . . . . . . . . . . Con-free
Na s a l t . . . . . . . . . . . . . . . . . . . . Cos-saturated 0.9 per cent NaCI.. . . . . . . . 0.65 per cent NaCl.. . . . . . . Locke, slightly modified.. . 0 .. . . . . . . . . . . . . . . . . . . . . . . . . 0.9 per cent NaCl., . . . . . . 0.9 per cent NaCl.. . . . . . . 0.9 per cent NaCl.. . . . . . . .
7.3 4.5 4.2 4.292
246-275 134 274 83 16 18.6 57.5 56-58 8.8 11 18
Pfeffer, I877 hfoore and Roaf, 1907 Gasser, Erlanger, and Meek, 1919 Gasser, Erlanger, and Meek, 1919 Krogh and Nakazawa, 1927 Drinker, 1927 Went, 1929 Dodds and Haines, 1934 Dodds and Haines, 1934 Onozaki and Sanada, 1935 Butt and Keys, 1937
large amounts of protein, the change in C. 0. P. attributable to the gum acacia is such as to indicate a mean particle size of the order of 100,000. Experimentally, Drinker (6) has observed that when a sufficient amount of horse serum is added to an acacia solution, the capillary membrane in contact with the mixture retains its normal semipermeability but does not display the free leakage which appears when acacia alone is used. Obviously the true C. 0. P. of simple gum acacia solutions cannot be obtained by the use of ordinary protein-tight membranes. Membranes of less permeability were prepared by incorporating a Rater-soluble, nonvolatile swelling agent (glycol) in anhydrous ether-alcohol collodion as described by Pierce (14). These membranes had a permeability to distilled water varying from 0.003 to 0.004 cu. mm. per minute per square centimeter per 1 cm. of water pressure. Some of these membranes were
23
OSMOMETRIC STUDY OF GUM ACACIA SOLUTIONS
"acacia-tight" and with them equilibration was attained usually, within forty-eight hours. The values obtained indicate that a 6 per cent gum acacia solution has a C. 0. P. that approximates that of the normal human blood plasma (table 3). The second phase of the investigation was concerned with the results of intravenous injection of gum acacia solution in humans. Determinations were made of the C. 0. P., the serum proteins (albumin and globulin), TABLE 2 The effect of acacia on the colloid osmotic pressure of normal serum i n vitro
C;&P...;d:; ;;Tv;vrl "",;~~;;I~;g;~
DILUTIONS OF HUMAN SERUM WITH BUFFER AND ACACIA
5
5
NITROQEN PER 100 CC. OF SERUM
p
~ ~ OF WATER PRESSURE
cc. of serum cc. of buffer
0.005
0.003
5 cc. of buffer 0 . 5 cc. of 6 per cent acacia
1
5
I
4 1
cc. of serum cc. of buffer cc. of 6 per cent acacia
255
0.002
281
0.011
,
TABLE 3 Colloid osmotic pressure determinations of gum acacia 80LUTION
12 per cent arabic acid... . . . . . . . . . . . . . . . . . . . . . . . . . 6 per cent arabic acid., . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 per cent gum acacia (Na salt). . . . . . . . . . . . . . . . . . . 4 per cent gum acacia (Na salt). . . . . . . . . . . . . . . . . . .
1
p~
7.227 7.227 4.292 4.292
1
C.0.P.I N M M . OF MERCURY
+
33 13 18 5.2
and the fate of the injected acacia in three cases of cirrhosis of the liver and in one of subacute toxic degeneration of the liver. The gum acacia employed in each case was a 6 per cent (with 0.9N sodium chloride) solution sterilized in the autoclave. Mention is made that these solutions were autoclaved, because this process supposedly increases the C. 0. P. of the solution (5). Following the intravenous injection of a solution of 6 per cent gum acacia the C. 0. P. of the blood
~
~
24
HUGH R. BUTT AND ANCEL KEYS
I 1
j, Norma? levelf
1100 1050-
!
850
750 800
-
mm. HZO 200 pressure -
p e r qm.
- - = -
150-
protein N.
-
pw !OO C.C.
Normal levelf I
I
I
FIG.1. The effect of the intravenous injection of gum acacia solution in an individual with cirrhosis of the liver. Ill00
,
I
900 850
mm. H20 250 F
~
3
~
protein N. per 100 C.C. serum
Normal level> 2 0 0
~
1
1
,
I
I
I
25
OSMOMETRIC STUDY OF GUM ACACIA SOLUTIONS
serum is assumed usually to be elevated only for a period of forty-eight hours (1). However, it will be seen in figure 1 that the C. 0. P. of the blood serum remained above the patient's basal level, following the third 500-cc. injection of gum acacia, for a period of four days. In the case summarized in figure 2 there was only one 500-cc. injection of gum acacia. The C. 0. P. of the blood serum in this instance was maintained above or a t the basal level for only twent,y-four hours. It is frequently stated in the literature that the serum proteins, particularly the albumin fraction, fall to a low concentration in man and animals following the intravenous injection of gum acacia solution (4). Only (llO0
loso-
"C
1000 -
2
900-
L
1 1
-
8'
R
Normal
A
~QVQ~-'
t .t' 950-
k
830-
@ < 800-
mm. KO 2 5 0 1
pressure
PEP
qm.
A
2,"-
Prota1n N,150 - Normal I e v e P per 100 C.C.
1
h I
I
>
Loq of time in m i n u t e s Gum acacia injacted at a r r o w s FIG.3. A case of cirrhosis of the liver showing the occasional reduction of concentration of albumin following the injection of gum acacia solution.
in the case summarized in figure 3 was the albumin concentration decreased appreciably following this procedure, and a t no time was this reduction of the magnitude reported in the literature. DISCUSSION
It seems that the failure to consider the variability of individual gum samples in regard to particle size and salt content, together with the employment of protein-tight rather than acacia-tight membranes, probably accounts for most of the errors in the reported C. 0. P. determinations of gum acacia. It is significant that the divergent values reported in the literature are not supported by any acceptable criteria of equilibrium and are in sharp disagreement with the results of cryoscopic measurements.
26
HUGH R. BUTT AND ANCEL KEYS
It appears that the disperse phase in simple gum acacia sols is composed of particles of variable size, with molecular weights ranging perhaps from less than 2000 up to several hundred thousand or more; the observed colloid osmotic pressure of such sols will depend partly on the method of preparation, but more particularly on the permeability of the membrane used. It is difficult to explain why gum acacia when added to human serum will not leak through a collodion sac from which it previously would escape. Drinker observed increased permeability, dilatation, and edema when a frog’s leg was perfused with a buffered 3 per cent gum-saline solution, but these increases disappeared upon the addition of horse serum in amounts greater than 15 per cent to the perfusion medium. His conclusion that serum is necessary for the maintenance of normal capillary tone does not help the understanding of the mechanics of the phenomenon. In dogs with intact circulations, Stanbury, Warweg, and Amberson (15) have replaced the plasma proteins with gum acacia in saline solution SO that the plasma protein level was maintained below 0.2 per cent. These animals were maintained in normal health for months without any symptoms of edema or shock. It appears that some of the functions of the plasma colloids can be replaced, in the experimental animal, by gum acacia solutions sufficiently purified and properly buffered, and in which at least 20 per cent of erythrocytes are suspended. This, however, does not explain why, a t the isolated capillary membrane, gum acacia freely leaks through when not mixed with serum. It has been suggested (10) that the value of gum acacia in maintaining blood volume may be the result of its adsorption on the capillary walls, whereby the exit of fluid from the vessel is impeded. However, Amberson and his coworkers have demonstrated that gum acacia alone is not effective in maintaining blood volume if there is an oxygen lack. SUMMARY
In the concentration usually employed for clinical use (6 per cent), gum acacia behaves as a typical lyophilic colloid that is highly aggregated. It exerts a C. 0. P. (18 mm. of mercury) that is nearly equivalent to that of the normal colloids of the plasma. Gum acacia particles readily leak through ordinary protein-tight membranes but are restrained by membranes of less permeability. The effect of human serum on the permeability of membranes to gum acacia has been discussed. In four human cases with liver injury the injection of gum acacia was followed by a rise in the C. 0. P. of the blood serum with no significant change in the concentration of the albumin or globulin fractions. The C. 0. P. of the serum remains markedly elevated for some hours after injections of gum acacia, but this effect disappears in a few days.
OSMOMETRIC STUDY OF GUM ACACIA SOLUTIONS
27
REFEREKCES (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16)
AMBERSON,W. R.: Biol. Rev. 12, 48-86 (1937). BAYLISS,W. M.: Proc. Roy. Soc. Med. B89, 380-93 (1916). BUTLER,C. L., AND CRETCHER, L. H.: J. Am. Chem. Soc. 61, 1519-25 (1929). DICK,M. W., WARWEG, EDNA,AND ANDERSCH, MARIE:J. Am. Med. Assoc. 106, 654-5 (1935). DODDS,E. C., AND HAINES,R. T . M.: Biochem. J. 28, 499-503 (1934). DRINKER,C. K . : J. Physiol. 63, 249-69 (1927). GASSER,H. S., ERLANGER, JOSEPH, AND MEEK,W. J.: Am. J. Physiol. 40,31-53 (1919). HEIDELBEROER, M., AND KENDALL, F. E.: J. Biol. Chem. 84, 639-53 (1929). KROGH,A., AND NAXAZAWA, F.: Biochem. 2.188,241-58 (1927). KRUSE,THEO.:Am. J. Physiol. 41, 195-6 (1920). MOORE,BENJAMIN, AND ROAF,H. E.: Biochem. J. 2, 34-73 (1907). ONOZAKI, NOBOSUKE, AND SANADA, YUKIKAZU: TohBku J. Exptl. Med. 28, 120-30 (1935). PFEFFER, W.: Quoted by W. R. Amberson. PIERCE,H. F . : J. Biochem. 75, 795-815 (1927). STANBURY, S.: Quoted by W. R. Amberson. WENT,S.: Quoted by W. R. Amberson.
