Principles of General Physiology

Now we must remember that the diagrams used on paper to represent chemical structure are in one plane, whereas the compounds themselves are in space of three dimensions. In order, therefore, to understand the relationship of these " optical isomers," as they are called, on account of the identity of their chemical composition, we must endeavour to represent them in space, remembering that we can only use conventional diagrams. The simplest way is to place the four groups at the points of a tetrahedron, represented in perspective, for the case of lactic acid, in Fig. 74. It will be found impossible to turn these figures in any position so as to make them coincide, in fact one is the image of the other seen in a mirror. The photograph on p. 180 of Wade's book (1905) and Fig. 75 (page 282) represent this fact.

Since, however, it is awkward to use these perspective figures, it is customary to represent them by their projections on a plane surface, thus : — bearing always in mind that such formulae are not to be supposed to be removed out of the plane of the paper, so that, although one can be slid over the other, it must not be taken up and placed face downwards. This is, of course, merely a convenient arrangement, in order to avoid the inconvenience of using solid figures. The real existence of asymmetry of such a kind that one of the isomers is the mirror-image of the other may, perhaps, be made clearer by Fig. 76, which represents crystals of the d- and I- forms of ammonium hydrogen malate. These would be said to have the same shape, but they cannot be made to coincide, and are, in fact, mirror-images of one another. They are sometimes called " enantiomorphic " forms of hemihedral crystals.

Optical isomers are also called "optical antipodes," but a mild protest must be made against the use of "antipode" in the singular, as if "antipodes" were the plural of an English word. When there are two asymmetric carbon atoms, as in tartaric acid, we have the possibility of a further complication ; thus, to begin with : — MALATE.— Although the geometrical form is the same, they cannot be made to coincide, and are, in fact, mirrorimages of one another.

A mixture of these is found in grapes and is sometimes called racemic acid. But there is another possibility of an inactive acid, in which the asymmetry, represented in the above figures, mutually compensates itself in one single molecule : — this is known as meso-tartaric, or internally compensated tartaric acid. In the representation of the formula1 of the tartaric acids, it is to be understood that the two asymmetrical carbon atoms are supposed to be looked at from opposite ends ; they will then appear alike and rotate in the same direction.

There is another class of optical isomers which must not be confused with those which are real mirror-images of one another. For example, in the a- and /3-glucosides of d glucose it is only the aldehyde ends that are mirror images of one another ; the other parts of the molecules are identical. The properties of isomers of this kind are therefore different, and they can be separated by solubility, etc. The rotatory power of the one is also not equal and opposite to that of the other. The real mirror-image of a-methyl-c?-glucoside is a-methyl- /-glucoside. These have the same numerical values of optical rotation, but in opposite directions, they have the same melting point, the same solubility and the same external form of crystals, as shown by Fischer (1909, pp. 741-742). The following formulas will make the relationship clear : —

It is a remarkable fact that the structures of living organisms are composed only of the one series of optical isomers, when there is the possibility of both. The amino-acids are all of the c?-series, the starches and sugars also of the c?-series, and so on. Not only so, but in the use of food-stuffs even for energy purposes, there is a decided preference for the same series. Much has been made of this fact in connection with that of the inability of pure chemical methods alone to produce anything but optically inactive mixtures of the two optical isomers. It is true that all the ordinary means used, for convenience, to separate the two components involve the use of vital agency at some stage or other, but there are several considerations which seem to me to indicate that it is possible to lay too much stress on the argument. We will mention them briefly : —

1. When an optically active substance is synthesised under the agency of an optically inactive catalyst, such as hydrochloric acid, a mixture of both isomers is formed. On the other hand, if the catalyst is an optically active one, only the one isomer is formed ; or, at all events, it is in great preponderance. For example, from glucose and methyl alcohol, by the action of hydrochloric acid, the two optically isomeric a- and (3-methyl-glucosides are produced. When the enzyme, emulsin, is used, the /3-glucoside is formed, and by another enzyme, maltase, the a-glucoside. The reader is recommended to consult the paper by Fajans (1910) with respect to the question of asymmetrical catalysis, both by enzymes and other optically active substances. Of course, it may be said that all optically active catalysts were originally produced by vital agency, but the point here is that a chemical substance, not actually living, is able to form new optically active material, provided that it is itself optically active.

In connection with the production of other asymmetrical compounds by the aid of those already existent, the work of Erlenmeyer (1914) is of importance. By the action of rf-tartarip acid on benzaldehyde in alcoholic solution, a Itevo-benzaldehyde was obtained. Other papers by the same worker may be found in Biochemische Zeitschrift (Band 64). 2. Although organisms show a preference for one isomer, they are not incapable of using the other one. In the classical experiment of Pasteur (1858) of separating the two tartaric acids by the action of moulds, the o?-acid is used up first, so that the 1-a.cid can be separated from the culture after the rf-acid is used up. But, if the experiment be allowed to continue, it was found that the rotation began again to diminish, owing to the consumption of the 1-a.cid. This fact is sometimes forgotten. Similar cases have been described in the utilisation of amino-acids by fungi.

These and other cases will be found given in my monograph on Enzyme Action (1913, 2, pp. 152 and 153). It may be mentioned here that this capacity of utilising both isomers is not confined to fungi. Parnas (1911) has shown that the rabbit can utilise /-lactic acid when the inactive mixture is given, although, given alone, this acid itself is toxic and is excreted. 3. In such cases, the question arises as to how far the use of what we may call the " foreign " isomer is merely for energy purposes, or whether new tissue is formed from it. If the latter, it must obviously be converted into the opposite isomer in some way. This is not an impossible occurrence. If /-leucine be heated with baryta water at 180°, it is converted into cK-leucine, or racemised. That is, half of it is changed from the I- to the d- form. As we shall see in the next chapter, there are present in organisms, in the form of enzymes, more active catalytic agents than alkalies or acids.

4. When we produce artificially, in the laboratory, an inactive mixture, it is not that the chemical reaction is unable to produce the substance that the " vital " reaction does, but that the former produces the opposite isomer in addition. 5. There is considerable evidence to show that, when an enzyme appears to deal with one optical isomer alone, it is not absolutely inactive with respect to the opposite one ; there are many differences of degree in this respect (see the paper by Fajans, 1910, and the monograph by myself, 1913, 2, pp. 151-155).

6. It is quite conceivable that optically active products might be obtained by allowing a reaction to proceed under the influence of some asymmetrical external force, say, for example, a photo-chemical reaction under polarised light ; although attempts to do so have not yet met with success, It is not easy to see what is the advantage to the organism of this undoubted preference for particular optical isomers. We have to remember that the existence of asymmetric carbon atoms is geometrically unavoidable. The enzymes which act on such compounds will probably also be themselves optically active, and the rate of action on one kind of optical isomer will no doubt be greater than that on the opposite one. A certain economy in the number of enzymes necessary is effected by limiting them to those required for one set of optical isomers, but it is scarcely to be supposed that this can be of much consequence, and it would seem indeed that more is lost than gained in the process. The replacement of an enzyme acting on one isomer by that acting on the opposite one, moreover, does not appear to be of much difficulty. Currie (1911) found that, of various pure cultures of Bacillus bulgaricus (the vigorous Bulgarian lactic acid organism), obtained from different sources, some formed d-lactic acid alone, others a mixture of d- and I- forms, and one culture produced /-lactic acid alone.

We must suppose that the external forces, under which the asymmetric carbon compounds forming the basis of living protoplasm were produced, were in some way or other themselves asymmetric. At that geological period, the synthesis may have received a bias in one direction, which has naturally been adhered to, as more and more elaborate compounds were evolved. On this view, the preference of one isomer over the other is, as it were, a matter of chance as to which happened to be first produced by the particular direction of the asymmetrical force.

Recent work by Emil Erlenmeyer (1913), however, suggests a possible way of separating the constituents of a racemic mixture without the aid of optically active substances. Since the properties of isomers depend only on the relative position and distance from one another of atomic groups in the molecule, it is clear that molecules, which are mirror-images of one another, must be identical in all those properties which depend on molecular dimensions and attractions. So that their unlikeness can only be expressed in the shape of their crystals, their behaviour to polarised light, or to other asymmetrical forces or substances, such as those in living organisms. This fact was pointed out by Pasteur and by van't Hoff (1901, 1'ti-s Heft, p. 98). On the other hand, isomers will have different chemical and physical properties when their atomic structure is neither the same as, nor the mirror-image of, each other.

Thus, when a compound of a d-acid with a d-base is compared with that of the same acid with an /-base, the two salts are neither of the same structure nor mirror-images, so that they are chemically and physically separable. This is, of course, the usual means adopted for the purpose ; the racemic mixture is caused to form salts with an optically active base or acid and it is found that the salt of one isomer has different solubilities from that of the other, so that they can be separated by fractional crystallisation. The lactic acids, for example, can be isolated by combination with the optically active base, brucine. Similarly the two isomeric forms of glucosides, as pointed out above, are not mirror-images and can be separated l>y crystallisation, etc.

But, if these considerations were invariably and unconditionally true, it would be for ever impossible to separate the components of a racemic mixture without the aid of another optically active compound. Further, unless experiments of producing asymmetric compounds by the action of asymmetric external forces, such as polarised light, are rewarded with more success than hitherto, we are apparently compelled to assume the intervention of unknown, supernatural forces in the origin of life.

Now Emil Erlenmeyer (1913, p. 442) points out that van't Hoff himself shows the possibility of the occurrence of a form of isomerism of a different kind, which may be called relative. Thus, when two carbon atoms are united together, there are six free affinities and when these are satisfied by six different univalent groups, twelve different arrangements are possible. But eight of these are derived from the other four by mere rotation, without change of combination. The four different ones are shown diagrammatically in the scheme below, where the two carbon atoms are represented by discs, supposed to be white on one side, black on the other, and the letters, A, B, C, D, E, F, are six different chemical groups.

It will be seen that one or the other carbon atom is supposed to be turned by 180° around the longitudinal axis. See further description by van't Hoff (1901. 2tes Heft, p. 110). How far all these isomers are capable of existence in a given case is a matter for experiment, but the optical rotations of the four different substances may be understood better if we call the effect of the one carbon atom, A, and that of the other, B. Then we have the four following results :—

Out of these relative isomers, it is to be presumed that one will be the most stable and that the different relative positions of the various groups will entail different properties. We may take lactic acid and brucine as A and B, for the sake of illustration, so that there are : — rf-brucine d-lactate, rf-brucine /-lactate, /-brucine d-lactate, and /-brucine Mactate. These facts are of importance in connection with the variety of processes in the living organism. It appears that there is evidence of the existence of malic acids in greater number than usually supposed. Erlenmeyer shows further (p. 447) that, if the d- and I- forms of a substance are not absolutely fixed " point systems," but convertible by external forces into relative isomers by rotation around an axis of combination, then a force acting in the same direction on the two mirror-images will have a different effect on them, so that they may be converted into modifications which are no longer mere mirror-images of one another, and can therefore be separated by means which do not involve the use of optically active reagents.

The experimental evidence is as follows : — If a solution of rf-/-asparagine (inactive) be boiled for twenty minutes, /-asparagine separates out first on cooling. If d- and /-asparagine are dissolved cold in the same solution, no separation is possible, since their solubilities are identical. One of the two is altered by heating, since the relative solubility is changed. By further fractionation, Erlenmeyer succeeded in preparing specimens of d- and /-asparagine in nearly pure condition. It appears to be impossible to predict whether d- or /-asparagine will be obtained in any particular experiment. Further details and also the formation of two different copper salts will be found described in the original paper. The same phenomena were found to exist in the case of the tartrates.

Should these results be confirmed, it is clear that we have a possibility of the production in the plant of optically active substances from racemic mixtures. One of the "relative isomers" would probably be more easily oxidised for energy purposes, leaving in excess the other mirror-image, which had not been changed. Having once obtained asymmetrical compounds, further production is comparatively simple, as we have seen. It will be noticed that the change from one relative isomer to the other is much more readily brought about than that from one optical isomer to the other. The asparagine which has been changed by boiling goes back into its original form when redissolved in cold water.

An important point is that the method is not a general one, and it is very suggestive that these two substances, asparagine and tartaric acid, which have been shown to manifest the property, and especially asparagine, are of such common occurrence in plants that it is by no means unlikely that optical activity commenced in this way. The work of Horace Brown (1906), on the part played by asparagine in the protein metabolism of the plant, is also to the point here.

The experiments of Ross Harrison and of Carrel have been referred to previously (page 23). A few remarks with regard to the chemistry of the process are of interest in the present connection. It is plain that in these experiments, granting that new tissue was actually formed, which appears to be satisfactorily shown by the presence of dividing nuclei, the proteins of the blood plasma, used as the culture medium, must have been utilised for the purpose. In the adult animal the serum proteins are not used as food-stuffs. We have seen that injection of such proteins does not increase the nitrogen output, although that of amino-acids does (Quagliariello, 1912). On the other hand, tissue protein can be used in starvation, so that we must admit the presence of enzymes in the cells able to hydrolyse the proteins. It may well be that, in the growth experiments referred to, these enzymes are called upon to hydrolyse the proteins of the plasma culture medium before they are made use of by the growing tissue.

The presence of autolytic enzymes in tissues can readily be shown by the use of Alxlerhalden's "silk peptone," a polypeptide containing a large percentage of tyros inc. If a small piece of tissue, say kidney, be immersed in a solution of this substance and kept at 40° the tissue will be covered in a few hours with crystals of tyrosine, from hydmlvsi* of the polypeptide. A question cognate to this is the growth of tissue transplanted from one part to another, investigated chiefly by Carrel (1910, 1912), Guthrie, and their co-workers. This has been effected with blood vessels which were removed from the body of the same animal some time previously. But it has been found extremely difficult to transplant the tissues of one animal into another animal, even of the same species. The transplanted tissue usually disappears sooner or later, although, if previously killed by formaldehyde, it seems capable of serving as a support for growth of new tissue on the part of the host.

This fact argues an extraordinary complexity of some kind or other on the part of the tissue protoplasm. The thyroid of one animal, for example, is distinguished by another animal of the same species from its own thyroid. Marshall and Jolly (1907), however, report success in two cases of transplantation of ovaries from one rat to another, apparently remaining functional. Guthrie (1908) also obtained fertile eggs from fowls whose ovaries had been replaced by those of other fowls. Carrel and Guthrie (1906) report a case in which they transplanted the kidneys of a dog into a bitch by vascular anastomosis and then removed the kidneys of the bitch. The transplanted kidneys continued to secrete normally for at least eight days, that is, up to the time at which the paper \\.IN written. The urine contained no abnormal constituent with the exception of a trace of protein.

Further facts bearing on the question will be found in Chapter XXIV. At an early period in the history of physiology it was supposed that the only kind of efferent nerves were those causing contraction of muscles. After the existence of nerves producing activity of the cells of secreting glands had been proved by Ludwig and that of nerves causing stoppage of the heart by the Webers, it was thought that there were nerve fibres presiding over growth and repair. After section of certain nerves, which, in point of fact, always contained sensory fibres, it was found that the skin, or other sensory surface supplied by them, became inflamed and that wounds on such denervated surfaces did not heal properly. Careful protection of these areas showed that there was no real difference between them and normal areas. The absence of warning on the part of the sensory nerves allowed the infliction of injuries, which would otherwise have been avoided.

Clara Jacobson (1910) made careful experiments on pigeons and on dogs and found that there was no difference whatever between the rate of healing of wounds in normal and in denervated areas. It is well known that organs grow in proportion to their use, but this is adequately accounted for by the increased blood supply always associated with the activity of any tissue. The manner in which this is ensured will be discussed in Chapter XXIII. After injury to certain parts of the central nervous system, in fever and after the administration of certain chemical substances, there is a rise of body temperature. Although this effect may be partly accounted for by diminished loss of heat, owing to constriction of the blood vessels of the skin, there appears to be evidence that, in some cases at all events, there is also

an increased production of heat. But it is not necessary to postulate special nerves presiding over this process, since the heat produced in muscular contraction is in itself sufficient to account for the fact. The reader may be reminded of the shivering in fever and in the waking up of animals from hibernation, in which latter process the heat produced by muscular activity serves to bring up the temperature of the animal to its normal height. According to Pembrey (1903) a dormouse raises its temperature by 19° in forty-two minutes.

Cathcartand Leathes (1907) found the amount of uric acid excreted to be different according to the manner in which . the muscular contraction was produced. If shivering was brought on by exposure to cold, a marked rise in uric acid output resulted ; whereas, if vigorous voluntary contraction was produced, there was a marked fall in the output. Although these experiments do not prove the existence of nerves directly affecting chemical changes, they * suggest that different kinds of innervation produce different chemical reactions in the cells affected.

When cells are known to be supplied with nerve fibres, it would be rash to deny the possibility of the cell processes being influenced by impulses passing from the fibres to the cells. All that we are justified in saying is that there is, as yet, B, One of the inflammatory foci in the third cervical ganglion of the left side. Both ganglia were cut in the same block, fixed on the same slide, and stained Note the multitude of small dark cells in B, and the effects on the ganglion cells.

no satisfactory evidence that the nutrition of the tissues is directly affected thereby. On the other hand, the work of Head and Campbell (1900) on Herpes Zoster requires consideration. This disease results in the formation of blisters on the skin in the area of distribution of particular nerves. It was shown by the investigators named that these changes in the skin are caused by irritative changes in the dorsal root ganglia (see Fig. 77). Owing to these changes, abnormal impulses are sent in an efferent direction along the sensory fibres to the skin. Although I have been able to show (1901, 2) that dilatation of blood vessels in the skin is produced by excitation of the sensory fibres of dorsal roots, it seems difficult to believe that mere vascular dilatation should cause the actual formation of blisters. At the same time, the possibility has not been disproved.