The Dynamics of Living Matter

Hober § has recently called attention to another possibility. Ham- burger had found that acids, e.g. CO2, increased the permeability of red blood corpuscles for certain anions. Hober has shown that such an increased permeability for anions must lead to a difference of potential between the inner and outer surface of the semipermeable elements, the inner surface assuming a positive charge. It is possible that such differences of potential, in case they lead to an electric current, may

bring about a cataphoresis of the water or the particles dissolved in it, into or out of the cell. But in view of the observations of MacCallum I am more inclined to believe that contractile phenomena inside the cell furnish at least part of the energy of secretion and absorption in those cases where the osmotic forces alone cannot explain these phenomena. To illustrate what possible form these forces may assume, I may point out the rhythmical squeezing out of the liquid contents of the vacuole in Infusorians. Here the work of secretion is obviously done by protoplasmic contraction, and not by osmotic pressure. It is quite possible that, mutatis mutandis, something similar may occur in all cells, although this is only a surmise.

6. FURTHER LIMITATIONS OF TRAUBE'S THEORY OF SEMIPERMEABILITY Traube's idea that all living cells are surrounded by a membrane which is absolutely permeable for water, does not seem correct for a number of marine animals. Fundulus heteroclitus, a marine fish, lives and develops exclusively in sea-water, i.e. in a solution whose osmotic pressure is, roughly estimated, like that of a half-grammolecular ( -- } solution of NaCl. I have found that this fish as well as its eggs

can be put permanently into distilled water without the least injury. No swelling of the eggs or the tissues occurs under these conditions.* It may also be put into sea water whose osmotic pressure has been increased by the addition of a certain percentage of NaCl without perceptible shrinkage. This shows that water does not diffuse rapidly through the skin of the animal or the membrane of the egg. It cannot be stated, however, that it does not diffuse at all, since it is possible that a slight diffusion of water into the cells may be compensated by an increased secretion of water from the cells. In addition, the egg and animal must be but slighly permeable for salts, as otherwise the salts would diffuse from the blood and the tissues of the animal into the distilled water, and this would cause the death of the animal. The skin and the egg cannot be said to be absolutely impermeable, since gases like O and CO2 diffuse into the eggs, and since the latter rapidly dry out and die when taken out of the water and exposed to dry air. More- over, K and other toxic salts are able to diffuse slowly into the egg, as can be shown by the fact that if potassium salts are added to sea water, the heart of the embryo soon stops beating.

The behavior of Fundulus to distilled water is not the rule for marine animals, as most of them when subjected to it die rapidly. I have recently made a series of experiments with a marine Crustacean Gam- marus, of the Bay of San Francisco.* The osmotic pressure of the sea-water of the bay varies at different times of the year between that of about an -- and a f m NaCl solution. When Gammarus is brought suddenly from bay water into distilled water, its respiratory motions

stop, as a rule, in about half an hour. This standstill becomes per- manent, unless they are put back into sea water within a short time (about ten minutes). If, however, Gammarus be put into a cane m sugar, dextrose, or lactose solution of any concentration from — to f m upward, they die just as rapidly, if not more so, than in distilled water. The same is true when the animals are put into a pure NaCl solution isosmotic with the sea water. They die still more rapidly when put into distilled water, to which all the other salts found in the sea water are added, with the exception of NaCl, and in the concentra- tion in which those salts occur in sea water. If they are put, however, into a solution of NaCl, KC1, and CaCl2, in that proportion in which these salts occur in the sea water, the animals may live as long as forty- eight hours; and if some MgCl2 is added to this solution, the animals may live as long as in sea water. If we prepare solutions composed of only two of the salts contained in the sea water; namely, NaCl + KC1, or NaCl + CaCl2, or NaCl + MgCl2, the Gammarus lives only a few hours. These experiments prove that the medium surrounding the Gammarus must not only have a definite osmotic pressure, but that this pressure must be supplied by specific salts. Perhaps the follow- ing data may explain, in part at least, why this lack of specific salts leads to the death of the animal.

When the eggs of Fundulus are put immediately after fertilization into a pure solution of NaCl, whose concentration roughly equals that in which this salt is contained in the ocean, — , or f m, no egg is able to form an embryo. The eggs begin to segment and may go as far as the 64-cell stage, but after this they die. But if to the NaCl solution a small but definite amount of a bivalent metal (with the exception of the most poisonous ones, like Hg), is added, just as

many eggs form embryos as in normal sea water.* It is a striking fact that not only the salts of such bivalent metals which occur in the sea water or the body, e.g. Ca and Mg, render the pure NaCl solution harmless ; but also such salts as do not occur in the body, or are posi- tively poisonous, e.g. Sr, Ba, Co, Zn, Pb, and others. An example will illustrate these antagonistic effects between the salts of the univalent and bivalent metals. It is remarkable how small a quantity of calcium suffices to render the NaCl solution harmless. The anion has nothing to do with this effect of the calcium salt, as the result remained the same when any other soluble calcium salt was used, e.g. Ca(NO3)2 or CaCl2. The results also remained the same when in the place of the Ca salts, Sr, Ba, Co, Zn, or Pb salts were used, and even the quantities of the salt required to make the NaCl solution harmless were about the same for all the salts. I think it is one of the most striking facts known in toxicology that a pure solution of NaCl of that concentration in which this animal lives is poisonous, while this solution can be rendered less harmful or harmless by adding so poisonous a substance as Ba, Co, Zn, Pb, etc.

If the eggs of Fundulus are raised in a solution of a salt with another univalent cation than Na, e.g. K, Li, or NH4, we find that beginning with a certain concentration a solution of each of these salts becomes a poison for the eggs of Fundulus, that is to say, does not allow any egg to form an embryo. If, at that concentration of one of these salts, a small but definite amount of a salt with a bivalent cation is added, the eggs form embryos and they are able to develop. Trivalent cations, like Al and Cr, were also able to render the toxic concentrations of salts with a univalent metal less harmful. The antitoxic effect of a tetravalent cation, Th, however, was found to be only slight.

While a small quantity of a salt with a bivalent metal thus suffices to render a solution of a salt with a univalent cation harmless, it was found that it was not possible to produce similar antitoxic effects through the addition of a salt with an anion of higher valency. If sodium sulphate, sodium citrate, etc., was added to a — NaCl solution, the latter continued It is remarkable that not only the solutions of a salt with a univalent cation, like NaCl, can be rendered harmless by a salt with a bivalent cation, e.g. ZnSO4, but that also the reverse is true; namely, that a toxic solution of ZnSO4 can be rendered harmless by a solution of NaCl, provided the concentration of the ZnSO4 is not too high. In 100 c.c. of a f m NaCl solution no Fundulus egg forms an embryo.

When from 2 to 8 c.c. — solution of ZnSCX are added to this NaCl solution, just as many eggs form embryos as in sea water or distilled water. If, however, from 4 to 8 c.c. of a — ZnSO4 solution are added to 100 c.c. distilled water, not a single egg is able to form an embryo, although in pure distilled water these eggs live and develop as well as in sea water. The Zn-ions are therefore not only able to prevent the toxic effects of a pure NaCl solution, but the NaCl of this solution also prevents, in this case, the toxic effects of the Zn-ions.

The quantitative relations are of some interest. About 4 c.c. — solution of a salt with a bivalent metal are required to render 100 c.c. of a f m NaCl solution harmless. We may therefore say that for this concentration of NaCl one ion of the bivalent metal suffices to render 1000 molecules of the salt with the univalent metal harmless. When a NaCl solution of a lower concentration, namely, f m or - is used, less salt with a bivalent metal is required for the antitoxic effect than in the case of a f m solution. If we use a NaCl solution with a concen-

tration of or below — , it is no longer harmful for the eggs of Fundulus. If we use stronger solutions than f w, we soon reach a limit where the addition of a salt with a bivalent metal no longer renders the solution harmless. It is possible that, at this limit, the loss of water on the part of the egg acts harmfully, and this effect, of course, cannot be antagonized by the addition of another salt. If we try to determine how much NaCl is needed in order to render a solution of ZnSO4 harm- less, we find that a comparatively large amount of NaCl is required for

this purpose. In order to prevent the poisonous effect of a - - ZnSO4 solution, so much NaCl had to be added that the concentration of the NaCl in the solution was about -^-. About 50 molecules of NaCl were therefore required to render one molecule of ZnSO4 harmless. The fact that the antitoxic effects of the salts with bivalent cations are so much greater than those of the salts with univalent cations is possibly respon- sible for the fact that I did not succeed in rendering a f m NaCl solution harmless through the addition of a salt with a univalent cation. The concentration of the solution would become so high that this might be sufficient to kill the eggs. The salts of certain metals are especially toxic, it being impossible to use those like Cu or Hg lor antitoxic effects, as they cause coagulation of the contents of the egg in smaller concen- trations than are required for the antitoxic effects of such a solution. The development of the egg of Fundulus requires at summer tem- perature from about twelve to twenty-four days. If we use a Ca salt

to render a -- or f m NaCl solution harmless, an embryo can be formed, and it may hatch, but will then die; if, however, a Zn or Ba salt be used for this purpose, an embryo is formed, and it may develop for a number of days quite normally ; but it dies before its development is complete. If we allow the egg to complete its development in distilled water or sea water, and put the larva, after it has hatched, into a mixture of 100 c.c. - - NaCl, and a small amount of a Ba, or Co, or Zn salt, the

embryo dies even more quickly than if put into the pure NaCl solution. These facts indicate that for this fish the ZnSO4 remains toxic even in the presence of the NaCl, and that these two salts are only antago- nistic as long as the fish is surrounded by the egg membrane. This sug- gests the idea that the antagonism between these two salts is due only to the fact that they retard each other's rapidity of diffusion into the egg.* If the egg is put immediately after fertilization into a - - NaCl solution,

very soon so much NaCl diffuses into the egg that it poisons the fish. The same is true if a small amount of ZnSO4 is put into distilled water. But if both salts are put together into the distilled water, neither the NaCl nor the ZnSO4 can diffuse as rapidly into the egg, and the germ lives long enough to form an embryo. In a few days, however, death occurs, showing that the diffusion of the ZnSO4 was not prevented, but only retarded. Another fact corroborates the idea that it is only

the rate of diffusion of salts through the membrane which is retarded in this case. A pure NaCl of the concentration - - or f m only prevents the formation of an embryo when the egg is put into the solution imme- diately after fertilization. If, however, the egg is put for the first twenty- four hours after fertilization into normal sea water and then into the pure NaCl solution of the above-mentioned concentration, the — or f m NaCl solution is not so toxic. In all probability the membrane of the egg or the cells becomes more hardened or less permeable during the first twenty-four hours.

The antagonistic effects between two salts with a bivalent cation are not so general, yet I found that \ c.c. of a -f$ m SrCl2 solution diminished somewhat the toxicity of a T5g m solution of MgCl2. While in the case of the membrane of the newly laid Fundulus egg, the addition of a trace of a salt with a bivalent cation sufficed to anni- hilate or diminish the toxic effect of a pure NaCl solution, we never find such simple relations for the Fundulus after it is hatched or for any living cell that is exposed directly to the solution without the inter- ference of a dead membrane, like the one which surrounds the fish egg. For such directly exposed living tissues or animals, it is a rule that a pure NaCl solution of sufficient concentration requires, besides the CaCL,, a trace of KC1, in order to become harmless, as was shown in the above-mentioned case of marine Gammarus. Besides, it is not possible to substitute in that case for the Ca any bivalent cation; only Sr can serve as a substitute for Ca in these cases. These limitations become intelligible on the assumption that the surrounding salts diffuse slowly into the cells. As long as this diffusion is so slow that the secretory activity of the cells or glands of an animal may remove them as fast as they enter, the cell or the animal may live in such a solution. I consider

this the reason why a Fundulus may live in a — NaCl solution, while it cannot live in a - • NaCl solution. A second condition for the main- tenance of life is, according to this hypothesis, the continuation of the action of the secretory mechanism. If the latter depends on the con- tractile power of the protoplasm, as I believe it does, we can understand that, in order to make a NaCl solution harmless, not only Ca but also K are required. We shall see in a later lecture that apparently Na, Ca, and K are required for the contractile phenomena of protoplasm. Hober and Gordon* have pointed out the existence of an antago-

* Hober und Gordon, Hofmeisttr's Beitr'dge zur chemischen Physiologic und Pathologic, Vol. 5, p. 432, 1904. nism between the precipitating effects of salts of univalent and bivalent metals, which Linder and Picton had already found. If arsenic sulphide is precipitated with a mixture of two salts with a univalent cation, or of two salts with a bivalent cation, the effects of the two salts are added to each other. If a mixture of a salt with a univalent cation and a salt with a bivalent cation is used, however, for the precipitation, the result is an inhibition instead of a summation of the effects. Hober and Gordon have repeated and confirmed this observation. In addition, they have found that, just as in my own experiments, the valency of the anion plays no role. I am not able to state whether this explains the observations made on Fundulus.

It is rather remarkable that many authors have found distilled water to be poisonous for fresh-water animals. Locke showed that some authors had been deceived by the fact that their distilled water contained traces of copper salts, owing to the fact that the water had been distilled in copper vessels. But Bullot* found that for fresh-water Gammarus distilled water is toxic even if distilled with all necessary precautions in Jena glass or quartz or platinum vessels, and if care is taken that it is free from ammonia. He found that if a trace of NaCl is added to the distilled water (so that the concentration of the latter was 0.00008 N) fresh-water Gammarus could live indefinitely in the distilled water. The presence of a trace of NaCl in the distilled water possibly preserves the membrane better, or maintains better the secretory activity of the cells so that the animal can be freed from the excess of water which diffuses into it.

Dr. Wolfgang Ostwaldf investigated the duration of life of the same fresh-water Gammarus in solutions of higher concentration. He found that these animals live longer in a mixture of one hundred mole- cules NaCl, two molecules KC1, and two molecules CaCl2, than in a pure sugar or NaCl solution of the same concentration. This is in harmony with the assumption that the absorption as well as the secretive action of the cells requires the presence of Na, Ca, and K in definite proportions, as we shall see more fully later.

In connection with these experiments I made an observation which possibly may become of some use in the study of the phenomena of adap- tation. When marine Gammarus is put into sea water, which has been diluted with various quantities of distilled water, one notices that, with increasing dilution of the sea water, the duration of life of the Gammarus at first diminishes but little ; that, however, at a certain degree of dilu- tion (about ten times that of the normal sea water) the duration of life

decreases quite suddenly. It is obvious that the discontinuity in the curve of duration of life means that here a new condition, or a group of new conditions, enters which before that time were not noticeable. What are these conditions? Experiments which I have recently made on the eggs of sea urchins showed that up to a certain degree the dilution of the sea water with fresh water killed the eggs only slowly, but that beyond a certain degree of dilution death was rather sudden. This sudden death was due to a process of cytolysis in which the eggs were transformed into "shadows." I am inclined to believe that some- thing similar occurs in certain cells of marine Gammarus and of marine animals in general, when the dilution of the sea water falls below a certain limit.

This idea receives some support from the fact that Wolfgang Ostwald found that a rise in the concentration of the sea water above a certain limit also caused a sudden decline in the vitality curve of fresh-water Gammarus. If the concentration of the sea water be raised above a certain point, the eggs of the sea urchin also undergo cytolysis.* THE phenomena which allow us to discriminate between dead and living matter are physical processes, e.g. in higher animals, the contraction of the heart, the respiratory and other muscular motions. If the chemi- cal processes in living matter and the physical changes they bring about in the colloids were entirely known, the physical manifestations of life would also be clear to us. The periodic character of many of the mani- festations of life suggests the idea that these processes occur in several phases which are probably connected, partially at least, in a catenary way, so that the preceding process has effects which cause the subse- quent phase of the process.

These catenary mechanisms are for the most part still unknown. Inasmuch as the number of possible changes in the condition of colloids seems limited, the impression might be gathered that by a guess the whole secret of the physical manifestations of life might be unraveled. Such surmises find their way occasionally into print. As a rule, those who are familiar with the specific case for which the guess is made are not helped by it. It is not worth while to devote any time to the point- ing out of the futility if not open absurdity of most of these attempts.

The origin of animal heat from chemical energy offers no further mystery. We know that a kilo of sugar yields about four thousand calories of heat, if burned in the laboratory, and that it gives the same heat if oxidized in the body. In our modern theory of nutrition, the heat value of the various kinds of food is justly used as the basis for the calculation of their nutritive value. The times are gone when physi- cians and biologists dared to raise the objection — as they did against Robert Mayer — that our body inherits its heat.

As far as the transformation of chemical energy into mechanical energy in the muscle is concerned, Robert Mayer and Helmholtz con- sidered the muscle as a thermodynamical machine. They assumed that in the muscle the heat produced by chemical processes is partly transformed into mechanical energy; but they refrained from stating how this transformation occurs. Engelmann tried to fill this gap.* Striped muscle consists of alternating stripes of optically isotropic and anisotropic substance. Engelmann observed that in the contraction of the muscle the anisotropic substance increases in volume, while the isotropic substance decreases. As the total volume of the muscle does not change during the contraction, Engelmann concluded that part of the liquid of the isojtropic substance diffused into the anisotropic during contraction. He showed by experiments on violin strings (made of catgut) that such a process of absorption can be produced by heat. The violin strings show the same double refraction as the aniso- tropic stripes in the muscle. When a violin string is suspended in water, and the latter suddenly heated, the string contracts, and is able to lift a weight in this contraction. This shortening of the string is caused by an absorption of water by the string; and this imbibition is caused by the increase in temperature. Like the contracting muscle, the violin string, in this case, becomes shorter and thicker. The process is reversible as the string elongates again upon cooling.

In the case of the muscle, Engelmann assumes that through the stimulus which causes muscular contraction, heat is produced (through the oxidation of carbohydrates); and that the increase in temperature causes the anisotropic substance to absorb water from the isotropic substance. This causes the change of form in the muscle - - the thicken- ing and shortening - - by which it is able to lift a weight. For such a shortening of the violin string through heating, an increase of about 10° in the temperature of the water is necessary, while the temperature of a frog's muscle during a single contraction increases only by 0.001°. Engelmann points out, however, that the increase in the temperature of the whole muscle does not indicate the rise in tem- perature in individual spots in the muscle, which may be considerably higher. The foci of combustion heat the whole mass of the muscle, and we measure only the latter increase of temperature, which may of course be quite small. Provided we grant this, it is necessary to assume that the heat is sufficiently rapidly dissipated by conduction to allow the rapid succession of relaxation and contraction of the muscle in tetanus. We know that the muscles can contract and relax many times a second, e.g. the muscles of the wings of insects contract and relax more than a hundred times a second. I do not believe that the process of dissipation of heat in liquids is rapid enough to make Engel- mann's hypothesis probable, or even possible. It is, however, conceiv- able that with a slight modification his hypothesis may be rendered free