ON THE MOVEMENT OF MATERIALS ACROSS LIVING MERiIBRANES AGAINST CONCENTRATION GRADIENTS' RAYMOND C. INGRAHAbl, HOWARD C. PETERS, MAURICE B. VISSCHER
AXD
Department of Physiology, Unzversity of Minnesota, Minneapolis, Minnesota Received October 9 , 193'7
The ability of living systems to move materials against concentration gradients is one of their most important characteristics. Life under changing circumstances would be impossible without this ability. This generalization is as valid for complex organisms as it is for the simplest forms. The living organism must maintain its internal environment relatively constant in spite of changes in its external environment in order to maintain its integrity. In order to keep its salt content constant an organism must be able to do osmotic work. One of the most important cases is in connection with sodium chloride. In order to maintain constancy of composition with respect to this important component of the body, higher organisms must be able to prevent its loss from the intestines and kidneys when the rate of its intake is low and bring about its excretion when its intake is high. The problem of how salt may be moved against its diffusion gradients in accomplishing these ends is the subject of this paper. The problem has been approached by a study of the circumstances surrounding the movement of salts against their concentration gradients from the intestine to the blood. It has long been known that all of the phenomena of intestinal absorption could not be accounted for on the assumption that the intestinal epithelium behaves like a dialyzing membrane. Reid (9) showed that fluid is moved across various epithelial tissues when there is no difference in osmotic pressure on the two sides. Heidenhain (4) found that water is absorbed from hypertonic solutions in the intestine. Goldschmidt and Dayton (2) found that in the case of solutions containing both sulfate and chloride, the latter was absorbed when its concentration was less than the concentration of chloride in the blood. Burns and Visscher (1) made an exhaustive study of the influence 3f various anions upon the movement of chloride into and out of the gut m the living animal. They found that in the presence of a sodium salt of Presented a t the Fourteenth Colloid Symposium, held a t Minneapolis, Minneiota, June 10-12, 1937. 141
142
R. C. IXGNAHAM, 13. C. PETERS, AND M , B. ’\’ISSCHER
sulfate, phosphate, citrate, or ferrocyanide, sodium chloride was very rapidly absorbed from the intestine against steep concentration gradients. Ingraham and Visscher (5, 6, 7, 8) have studied this problem further and have found, among other facts, that under favorable circumstances sodium chloride remoral may proceed until the concentration ratio for chloride bemeen blood and intestinal fluid may be 200 to 1. Details of methods will bc found in these papers. In figure 1 are the analytical results of a typical experiment showing sodium chloride removal. A solution isotonic with the blood plasma containing equi-osmotic fractions of sodium chloride and sodium sulfate was placed in a closed loop of lower small intestine. In one and one-half hours the concentration of chloride had fallen to 2.5 mg. per cent, or less than 1 per cent of the blood concentration. This occurred by movement into the blood. Obviously, osmotic work was performed by the system in moving chloride from a place of lower to one of higher concentration. Also shown in figure 1 are the respective concentrations of ammonia in the intestinal fluid and in the blood. The ammonia is a product of the nietabolism of the intestinal epithelium, and its relation to thc activity of the intestine is seen in connection with the action of certain toxic substances which abolish the ability of the intestine to perform osmotic work; this will be referred to later. The ability to do osmotic work is not specific for sodium chloride, for under suitable conditions it may be shown that sodium bromide moves at exactly the same rate against equally great concentration gradients ( 5 ) . Moreover, this univalent ion impoverishment in the presence of polyvalent ions of the same sign is not limited to the anions, for when a polyvalent cation is present the univalent cation is likewise moved against large concentration gradients (8). These phenomena are dependent upon certain specific properties of intestinal epithelium. This can be seen from results of the experiments in which specific poisons h a w been used (7). As little as 0.0001 M mercuric chloride stops the process of specific impoverishment, as does 0.0005 NaeHAsOa. Somewhat higher concentrations of sodium fluoride, hydrogen sulfide, and sodium cyanide produce a sirnilar poisoning. I n figure 2 the results are presented of a typical experiment on this question. Two adjacent loops of small intestine (lower ileum) of a dog were filled each with 100 cc. of an isotonic soIution containing initially equi-osmotic quantities of sodium sulfate and sodium chloride. To the fluid in one loop there was added 0.025 M sodium fluoride. The results of the two experiments are plotted together, the one marked llcoiitrol” and the other “NaF.” In the case of the control the chloride content of the intestinal fluid fell to 22 mg. per cent in the course of 90 min., whereas in the loop containing sodium fluoride the chloride concentration instead rose to
MOVEMENT OF CHLORIDE ACROSS LIVING MEMBRANES
143
approach the blood plasma level of 340 mg. per cent. The sodium fluoride destroyed the impermeability of the membrane to sulfate (not shown in the graph) and abolished the qecific absorption. It also practically abolished the ammonia formation. These effects are produced by all of the poisons mentioned (7). The conditions for the occurrence of specific absorption from the intestine are extremely exact. If the tissues are injured in any way, chemically, mechanically, or thermally, the epithelium is no longer able to perform osmotic work. The amount of work performed in a given time is not identical for different individual experiments, although from the same animal adjacent loops of intestine give almost identical results, and suc-
FIG.1 FIG.2 FIG.1. Results of a typical experiment showing sodium chloride removal. Also shown are respective concentrations of NHs in intestinal fluid and in the blood. FIG.2. Effect of sodium fluoride on intestinal membrane
cessive trials are in agreement. Occasionally the intestine of an animal shows spontaneously poor ability to do osmotic work. This might seem to be a disadvantage to the study of the phenomenon, and it is adisadvantage to the extent that a larger number of experiments have had to be performed than would have been necessary if the material were more uniform. However, one advantage has accrued from the instances of spontaneous failure of ability to do osmotic work, namely, that they have given opportunity for the. study of means of bringing about more rapid performance of osmotic work. The most striking effect produced by any substance studied is that resulting from the addition of relatively small quantities of Al(0H)a. As a routine procedure in a number of experiments two adjacent loops of small intestine were studied, being filled with the
144
R. C . IXGRAHAM, H. C. PETERS, AND M. B. VISSCHER
usual isotonic mixture of sodium chloride and sodium sulfate. In the case of one of the loops there was an addition of 1.5 cc. of alumina cream per 100 cc. of fluid. It was regularly found that the rate of chloride removal was greater from the loop containing the Al(OH)3 than from the control. This effect is shown in figure 3. In this instance there was very little C1 impoverishment in the control loop, whereas in that containing the alumina cream the chloride concentration fell to a low level. The same effect of aluminum was observed when small quantities of Alz(SiO& are employed. There is a suggestion that this effect depends upon the sign of the aluminum ion, since the negative colloidal dyes, trypan red and brilliant vital red, produce a reverse effect, whereas in a number of instances methylene blue has accelerated chloride impoverishment. The influence of methylene blue need not, of course, be due t o the simple effect of charge, and the njechanism of the A1(OH)3effect cannot be considered to be settled.
L
I
i
Yl i Time. Hours
I IYl
FIG.3. Effect of Al(OH)a on chloride removal
As has been pointed out in a previous publication (6) it has been found to be impossible to account for univalent ion impoverishment on the basis of a membrane equilibrium of the Donnan type, or as resulting from processes going on in approaching such equilibrium. After exhaustively considering and subsequently rejecting every known type of process based on ion exchange or membrane equilibrium, a kinetic picture has been developed, which is based upon highly probable assumptions and which, as will be apparent from the data presented, will satisfactorily account in a quantitative way for the phenomena as observed. The type of uni-univalent salt impoverishment which is observed in the small intestine can be accounted for by assuming that there is a flow of water into the blood carrying out the uni-univalent salt a t the concentration a t which it exists in the gut, and a simultaneous flow of water free from that salt into the intestine at another point. It is necessary to postulate a membrane which is in essence a mosaic, one portion of which is strictly semipermeable and through which water enters the gut, and
MOVEMENT O F CHLORIDE ACROSS LIVING MEMBRANES
145
another portion of which is permeable to uni-univalent salts through which the solution of such salts passes into the blood. This scheme is analogous to the physical model diagrammed in figure 4. If the vessel contains originally V oliters of a solution of sodium chloride a t a concentration Co millimoles per liter, and pure water flows into the vessel a t some rate Ri and sodium chloride solution a t a concentration C (the concentration at any time t ) leaves the vessel a t some rate Ro, the concentration, C, of sodium chloride in the vessel will decrease. If the rates of flow, Ra and Ri, are constant or vary in a uniform manner with time, the Concentration, C, will likewise decrease in a uniform way with time. In the following derivation only the case in which Ri and ROare constant is considered.
Soidwon lrnvmp thru fiypofktm2 fdkr "F"wkuk LS rmprmeadCe fo polyvaL-wt ions- a? mtc E.
FIG.4. Model for explaining the type of uni-univalent salt impoverishment which is observed in the small intestine.
In the case of the intestine the solution contains both sodium citrate and sodium chloride. The gut is freely permeable to sodium chloride but almost completely impermeable to sodium citrate. Then if a hypothetical 6lter, F, is assumed to be present a t the outlet which is impermeable to jodium citrate, the above scheme will represent a physical model of the chloride-impoverishing mechanism of the small intestine on the basis of this theory. If the rates ROand Ri are constant it is possible to derive the mathematical relationship between concentration and time in terms of the original volume and concentration of the fluid in the intestine and the rates of fluid movement. By comparing the concentration of sodium chloride experimentally determined a t various intervals of time in the course of an actual experiment with those values of C calculated from the theoretical formulae at the same time, one can determine whether chloride impoverish-
146
R . C. INGRAHAM, H . C. PETERS, AND hf. B . VISSCHER
ment as it occurs in the small intestine can be quantitatively accounted for by the type of fluid movement postulated. The formula relating time and concentration is derived as follows: Let V O= the original volume of the solution in liters, Co = the original concentration of sodium chloride in the solution in millimoles per liter, V , C = the volume and concentration at any time t in the same units, t = time in hours, Ro -- rate of flow of sodium chloride solution, concentration C, from the intestine to thp blood in liters per hour, R, = the rate of flow of pure water into the intestine from the blood in liters per hour, D = (no - R,) or the rate of volume decrease, since in all experiments the volume in the intestine decreases with time, and I -:the amount of salt present a t any time. In such a system it i s evident that:
c = f (1, V )
(1)
I and V are functions of the third variable t . The total derivative of equation 1 in respect to t is then:
dC - aC d I
ac dV dt
__I-
dt
a I dt -I-
At any time
C = vI
(3)
Therefore,
The change in salt content in respect to time
(dt)
is equal to the rate of
exit of salt solution multiplied by the concentration of salt in the moving fluid, or,
dP - -CRO
z-
The change of volume with time Wuid leaving and fluid entering:
is
equal to the difference in the rate of
CIV == ( - R o +. R,) dt
3
-k,
('9)
MOVEMENT O F CHLORIDE ACROSS LIVING MEMBRANES
147
Substituting equations 4, 5, 6, and 7 for the proper terms in equation 2:
From equations 3 and 7 : dC =
[T -
-(Ri C - Ro)]dt V
(9)
Simplifying: dC = ( u->CRi dt
(10)
Since V = V O- Dt dC
C
- -Ridt Vo - Dt
On integration this yields:
R.
log c = -4 log (VO - Dt)
D
+ log A
(14)
Or
At zero time
Therefore
Simplifying and from equation 12 Ri -
c = co(;)D Or, in logarithmic form, log,, By plotting the log
C Ri V co = -D- log,, vo
C Ti against log - one should obtain a straight line,
CO
VO
148
R . C. INGRAHAM, H . C. PETERS, A S D M. B. VISSCHER
R
the slope of which represents 2, if the formulation fits the real phenom-
D
enon. The results of three of the most complete experiments we have performed are plotted in this way in figure 5. The experimental data and some of the calculations are given for these experiments in table 1. For the period of the first hour in each case there is substantially a straight-
c
V
CO
VO
line relation between log - and log -, as is predicted from this reasoning. For the point at one and one-half hours, there was in two instances a very considerable deviation from the predicted straight line. This does not appear to be a serious discrepancy, however, because the assumption that
M 06
05
.P2
.ais
" 1 1 ' 5 b I b.910
FIG.
" 1 1 1 1 1 .A
.5 6 ?.b.YI.O
5 . Plot of log
"
I
.2 2 5 ,?
' .A
I L " l 5
6 7.6910
C v - against log co vo
the membrane through which the water enters the gut is absolutely impermeable to chloride might be in error to the extent that perhaps 1 per cent of the chloride in the blood could pass. Such a minor correction as this could completely account for the deviation when the chloride content of the intestine has fallen to such low figures as 2 per cent of the blood chloride level. It should also be mentioned in this connection that one is here approaching the limits of analytical accuracy for chloride in biological fluids. Summarizing the mathematical results, it seems that one is entitled to say that until extremely low chloride concentrations are reached the theory as developed will completely account for the phenomena observed.
149
MOVEMENT O F CHLORIDE ACROSS LIVING MEMBRANES
R.
From the values of 2 the absolute value for R ; itself can be ealculated
D
because D is experimentally determined. In several experiments this value has ranged between 150 and 250 cc. per hour for a loop of intestine 25 cm. in length. It seems important to note that this is a distinctly reasonable result. Ri is in a mathematical sense an arbitrary constant as far as the data we have presented are concerned. It should be noted, TABLE 1 Observed and calculated data o n the rate of active absorplion TIME
hours
0 0.167 0.383 0.583 1.oo 1.50
0 0.167 0.333 0.5 1 .oo 1.5
~
CCl
V
(DXPERIYENTAL)
i'
I
millimoles per litm
cc.
I1
-
77.9 69.2 48.2 28.1 6.24 2.2
1 0.89 0.62 0.36 0.08 0.028
50 47.5 44.4 41.4 35.3 28.0
59.5 40.1 28.7 15.6 3.5 2.2
1.oo 0.67 0.48 0.26 0.059 0.037
75.0 70.7 66.3 62.0 49.0 36.0
1.oo 0.94 0.88 0.83 0.65 0.48
75 .O 68.3 62.0 55.3 35.7 16.0
1.oo 0.91 0.82 0.74 0.47 0.21
1 0.95 0.89 0.83 0.70 0.56
Experiment 3 0 0.17 0.33 0.50 1.00 1.50
59.7 43.5 23.8 12.05 1.56 1.2
1 0.73 0.40 0.20 0,026 0.020
however, that it is a factor amenable to measurement although not in a direct way, because fluid is both entering and leaving the intestine simultaneously. Up to the present time the best evidence that we have that water actually enters the intestine at the rate Ri is derived from the fact that values of this constant calculated from the movement of substances with widely different diffusion constants are in substantial agreement with one another. The mechanism for driving water in this system has not been considered
150
R . C. INGRAHAM, H. C. PETERS, AND M. B . VISSCHER
up to this point. Since in these experiments there is no difference in osmotic pressure between the two fluids, ordinary osmosis will not account for the flow. Abnormal osmosis can, however, occur. Practical difficulties have prevented us from making crucial studies on this question up to the present time, and it would be unprofitable to go into an extended discussion from a theoretical basis, since too many important points are unknown. It is nevertheless worth while to say that the theory of Sollnei (10) and of Grollman and Sollner (3) offers possibilities for the solution of this aspect of the problem. REFERENCES (1) BURNS,H. S., AND VISSCHER, M. B.: Am. J. Physiol. 110,490(1934). (2) GOLDSCHMIDT, S., AND DAYTON, A. B.: Am. J. Physiol. 48,459 (1919). (3) GROLLMAN, ARTHUR,AND SOLLNER,KARL:Trans. Electrochem. SOC. 61, 487
(1932). (4) HEIDENHAIN, R.:Arch. Physiol. 66,579 (1894). (5) INGRAHAM, R.C.:Proc. SOC.Exptl. Biol. Med. 33,453(1935). (6) INGRAHAM, R. C., AND VISSCHER, M. B.: Am. J. Physiol. 114,676 (1936). (7) INGRAHAM, R.C.,AND VISSCHER, M. B.: Am. J. Physiol. 114,681 (1936). (8) INGRAHAM, R. C., IND VISSCHER,M. B.: Proc. SOC. Exptl. Biol. bled. 36, 201 (1937). (9) REID,E . W.: J. Physiol. 21,408 (1897). (10) QOLLNER, KARL:Kolloid-Z. 62,31 (1933).
