45

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THE BULLETIN
TH ULEI

BIOLOGICAL OXIDATION AND VITAMINS

know, the ultimate source of all energy, sustaining life, is the radiant energy of the sun. The energy of the A Msun's rays is captured by the green dyestuff of plants, the chlorophyl. This radiating energy cannot, as such, support life, for if it were essential, life would fail at night. Thus the energy is used to build up carbohydrate molecules from carbon dioxide and water, the excess of 0 being sent back into the atmosphere as 02. In essence these carbohydrate molecules represent small parcels of energy, which can be stored and released by the cell according to needs. The "unpacking" of these parcels is the reverse reaction, in which the carbohydrate molecule is united again with oxygen to form carbon dioxide and water. This process we call oxidation or combustion. This oxidation is thus the ultimate source of all our energy. Since all cellular functions consume energy, oxidation is intimately connected with all activities of the cell and stands, so to speak, at the center of life. During the last decades our conceptions regarding biological oxidation have been revolutionized and today oxidation represents one of the widest and most fruitful fields of biological research. When speaking of oxidation we must always bear in mind one thing, i.e. that its purpose is to liberate energy and to liberate it in a form which can be used to maintain cellular functions. Misled by the analogy of the steam engine, it was thought a few decades ago that in this process the molecule of foodstuff was simply attacked by oxygen, with the liberation of its energy in the form of heat. But there is this fundamental difference between the steam engine and our body: the latter is unable to use heat as a source of energy. Moreover, at the relatively low temperature of our body, oxygen does not attack the foodstuff molecule. It is evident that the process has to be activated in some way to take place at 37oC. It is natural that investigators looked first for an activation of oxygen, several active forms of oxygen being known to the chemist.
s YOU

21

Biological Oxidation and Vitamins

4 57

0. Warburg proved first that the oxygen is "activated" in cells. He studied the effects of cyanide, a poison which stops biological oxidation even at minimal concentrations. This substance has one peculiar property, and this is to form very easily coordination complexes with metal-atoms. Thus Warburg arrived at the conclusion that cyanide stops respiration by inactivating certain metal-atoms responsible for the activation of oxygen. These metal-atoms are linked to protein and these metal-proteids have the properties of an enzyme, the metal acting as the active prosthetic group. Warburg called this metal-proteid the "respiratory enzyme." Somewhat later, H. Wieland, studying hydrogenation processes, arrived at another theory of respiration. It is known that organic molecules, having a double bond, can be made to take up hydrogen if treated with hydrogen in the presence of a catalyst, such as colloidal palladium. The metal combines with hydrogen and "activates" it. Wieland found that this process is reversible and H can also be detached from organic molecules in the presence of a suitable catalyst, if, instead of adding H2 to the system we add a substance that is capable of binding H, i.e. an "H acceptor." Detaching H from an organic molecule is an oxidation. Wieland arrived at the conclusion that biological oxidations proceed in a similar way in oxidizing the organic foodstuffs, their H's being "activated" and detached, provided there is a suitable "H acceptor" present. Molecular oxygen, in Wieland's conception, is such an "H acceptor," while the foodstuff molecule itself acts as an "H donator." The activating enzyme we call "dehydrogenase" according to Thunberg's nomenclature. The place of the lost H's in the foodstuff molecule is taken by H-OH, water. In this way the molecule, by repeated dehydrogenation and hydration, becomes richer and richer in oxygen, until it is completely oxidized. But how does all this work from the energy standpoint? We must remember that the aim of oxidation is in the first place the liberation of energy. The detaching of hydrogen from the organic molecule does not liberate energy. Neither does the introduction of water yield energy. The whole energy of oxidation is liberated in the oxidation of the hydrogen. It is well known that the oxidation of hydrogen is one of the greatest energy yielding reactions of chemistry. These are quite simple statements but their consequences are rather far-reaching. They tell us that Nature knows, in essence, but one fuel and this is hydrogen. All our food is essentially but a fixed form of hydrogen and all the energy which supports life is derived from its oxidation,

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THE BULLETIN H BLEI

with the formation of water. We thus have the whole energy cycle: the plant cell, with its chlorophyl stores the energy of the sun, separating the elements of water, fixing the hydrogen to a solid carbon chain and sending the oxygen back to the atmosphere. All cells cover their energy need by reversing the process; taking the hydrogen from the organic molecule and uniting it again with the oxygen from the air. It was thought at one time that the two theories, the theory of Wieland and the theory of Warburg, were contradictory. My first modest step in this domain was to show that there was no contradiction and that in the animal cell "activated oxygen oxidized activated hydrogen." The next step of development is linked with the name of D. Keilin, who rediscovered certain hemin derivatives, found many years before by MacMunn. Keilin called these substances "cytochromes" and showed in classic experiments that they are substances involved in respiration. As Keilin taught us, there are three cytochromes, A, B and C. Cytochrome C has been isolated and shown to contain an iron atom. Probably the other two likewise contain iron. The metal atom is the active constituent of the molecule. In respiration this metal is alternately oxidized and reduced from its higher to its lower valency (ferric-ferrous). The three cytochromes are shunted in series: A is oxidized by Warburg's enzyme, A oxidizes C and C oxidizes B (Ball, Laki). Then cytochrome B oxidizes the hydrogen of the foodstuff, the "H donator." The H's of the foodstuff thus do not react immediately with the oxygen. Oxygen and hydrogen are separated by the series of cytochromes and Warburg's enzyme. The latter I will refer to as "cytochrome-oxidase," for its function is to oxidize cytochrome. It is worthwhile to consider for a moment the implications of these findings. Of what does the oxidation and the reduction of a metal-atom consist? What is the difference, let us say, between ferrous and ferric iron? The difference is that the ferrous atom contains one more electron than ferric. When the hydrogen is oxidized by the Fe of cytochrome B it gives over its electron to the metal. The hydrogen itself acquires thereby a positive charge and becomes a hydrogen ion and unites with OH ions present in water. The electron added to Fe is then transferred from metal to metal, i.e. from cytochrome to cytochrome, then to the cytochrome-oxidase, to be transferred eventually to oxygen. The oxygen, after having taken up four electrons, can unite with four hydrogen

Biological Oxidation and Vitamins

45 9

COOH
Cytochromes ~

COOH

HCH
HCH

-2H +2H
Fig. 1

CH CH

Foodstuff

COOH

COOH

ions of the water to become two molecules of water. There is thus in the cell a whole chain of electron transfers in which the hydrogen of the foodstuff serves as an "electron donator," the oxygen serves as an "electron acceptor," while the cytochromes with their oxidase serve as "electron transmitters." The role of oxygen in respiration is thus limited to accepting the electrons. At every step the electron itself liberates a certain quantity of its energy. On this series of electron transfers about two-thirds of the total energy of oxidation is liberated. To sum up, the hydrogen of the foodstuff is activated by the specific enzymatic activators, the "dehydrogenases," yields its electron to cytochrome and is transferred through the series of metals to oxygen. From the investigations of Thunberg we know a number of such dehydrogenases, which could activate the hydrogen of different foodstuff molecules. But is it really the sole function of dehydrogenases to split off the hydrogen of the foodstuff molecule and make its electron available to cytochrome? This was the question I asked myself a number of years ago. Studying the enzymes of muscle I was struck by the fact that there was one among these dehydrogenases which had specific qualities. Its resistance and kinetics (O type) were extraordinary and its activity amazing. This dehydrogenase was the succinodehydrogenase, the dehydrogenase of succinic acid, discovered long ago by Thunberg. How were we to explain that Nature should produce such an extraordinary enzyme for succinate, when we had no reason to believe that succinic acid itself was one of the principal foodstuffs? Aided by my faithful collaborators, Banga, Gozsy, Huszak, Laki, Straub and others, I arrived at the conclusion that the function of this enzyme was not to split off H from a foodstuff but to transfer H from the foodstuff to cytochrome. Succinic acid is activated and dehydrated by this enzyme to fumaric acid, the two free valencies being joined in the form of a double bond (Figure I). Fumaric acid is likewise activated by the same enzyme, the "succino-

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THE BULLETIN

dehydrogenase," so that it can bind by its double bond two new H atoms. The function of the acid and its enzyme would thus be to give off and take up H atoms alternately. The idea that dehydrogenases could also act as H transmitters was then somewhat revolutionary and needed support. We have made a few observations which I think admit of no doubt, at least for muscle, of this function. First we looked for a substance that would inactivate the succinodehydrogenase in a selective way. If the succinodehydrogenase is a member of the chain of oxidation, as we supposed, the whole respiration would have to stop on its inactivation. J. H. Quastel's studies opened the way for the demonstration of such a specific inactivation. Quastel found that the succinodehydrogenase can be inactivated specifically by malonate. Malonic acid is the same substance as succinic acid, except it is one C atom shorter (Figure 2 ). It Will thus take the place of succinate on the enzyme without being able to take on its function. By adding a small amount of malonate to respiring muscle we found that the whole oxygen uptake was stopped. We also found that, if the small normal store of succinate is washed out from the muscle, respiration stops and it starts again if the succinate is restored.
COOH HCH COOH
Fig. 2

The next step was to ascertain the position of the succinate in the mechanism of oxidation. We have found, and have been corroborated by D. E. Green, that succinate, activated by its dehydrogenase, could reduce cytochrome, but no other substance could do this. Other substances and dehydrogenases could reduce cytochrome only if succinate and succinodehydrogenase were present to transfer the hydrogen. Thus it became evident that the place of the succinate in the oxidation system was close to cytochrome, as shown in Figure i. But this was not all. H. Einbeck found more than twenty years ago that all animal tissues contained a very powerful enzyme which had the function of hydrating fumaric acid, that is to say, to add one molecule of water to its double bond and transform it into l-malic acid (Figure 3).

Biological Oxidation and Vitamins

46 I

COOH CH
CH COOH Fumaric acid

+H20

COOH HCH

-2H ---4

COOH HCH

-H20

HC0H
COOH Malic acid
Fig. 3

+2H

CO COOH Oxaloacetic acid

Not the whole fumarate is transformed in this way; the reaction stops when three-fourths of it is transformed. If we start out with l-malic acid, water is taken off until the same In3 equilibrium mixture is reached. Now what is the function of malic acid in the cell? There is a very powerful dehydrogenase present in muscle which will dehydrogenate malic acid, splitting off two hydrogen atoms, thus oxidizing malate into oxaloacetate. Oxaloacetate in turn will take up again any hydrogen coming from the foodstuffs. The series of events will thus be as follows: the foodstuff molecule is activated by dehydrogenase and passes over two of its H's to oxaloacetate, which is activated by the malicodehydrogenase. In this way the oxaloacetate becomes malate, which again passes on the H to fumarate. The fumarate then becomes succinate through the oxidation of the two newly acquired H atoms by the Fe of cytochrome. Then starts the chain of electron transport by the three cytochromes and their oxidase towards 02. To sum this up we can say that the H mobilized goes through a series of reactions, is transferred from substance to substance, and yields energy at every step. Then, reaching cytochrome the H turns into an H-ion and its electron continues the journey. At the very end of the reaction we find both the respired 02 and the H of the foodstuff in the form of water and it looks as if they had united directly. But this is, as we have seen, only an appearance. The H joined to the inspired 02 is taken from water and so is the 0 which is eventually linked to the H derived from the foodstuff. It is amazing to think that these simple four C atoms containing dicarboxylic acids should play such an important catalytic role in respiration. These substances seem to be one of the most important tools of living Nature. It was found recently by A. I. Virtanen that the same acids, in the form of oxaloacetate, play an important role also in N-fixation which

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THE BULLETIN

is one of the most fundamental chemical processes in Nature. As I will show you later, the same dicarboxylic acids probably also play a fundamental role in the respiration of plants.* Further development in this field is linked with the discovery and analysis of the codehydrogenases. Some fifteen years ago I observed that certain dehydrogenases, as the dehydrogenase of lactic acid, acted only in the presence of a thermostable substance. This coenzyme was later purified in my laboratory and found to be a nucleotide. von Euler and Nilsson showed that Euler's "cozymase," likewise a nucleotide, was capable of acting as codehydrogenase. But real progress was made when Warburg discovered that this codehydrogenase contained a pyridine derivative, nicotinic acid amide. This pyridine acted as an H acceptor, one of its double bonds being hydrogenated. Apart from the succinodehydrogenase, all dehydrogenases acting in the main respiration cycle seem to need codehydrogenase. These findings not only lengthen the known chain of oxidations but also throw a new light on the nature of dehydrogenases. We must now look upon a dehydrogenase, like the malic-, lactic- or triose-phosphoricdehydrogenase, as upon a specific protein which is capable of adsorbing and activating simultaneously two substances, and of causing two H atoms to pass from one to the other. If we take the oxidation of triosephosphate, the first thing that happens is that this sugar is adsorbed and activated by a specific protein, the dehydrogenase. At the same time the coenzyme is also adsorbed and activated, two H's pass from the sugar to the coenzyme and we obtain oxidized sugar and dihydro-coenzyme. Now the dihydro-coenzyme is adsorbed by the malicodehydrogenase together with oxaloacetate and the two H's pass from the dihydrocoenzyme on to the oxaloacetate and we obtain coenzyme and dihydrooxaloacetate, i.e. malate. Now malate again gives its two H's to the coenzyme, and the coenzyme transmits them to fumarate. At this point we get into difficulty, for the dehydrogenase that activates fumarate (the succinodehydrogenase) does not activate coenzyme.
Lately the role of H transporters of the C4 dicarboxylic acids has been denied by H. A. Krebs who believes that these acids act only as members in a more complicated cycle of carbohydrate breakdown. Krebs is of the opinion that oxaloacetate combines in muscle with an oxide of triose to citric acid, and the citric acid oxidizes to succinate. The succinate is oxidized to oxaloacetate, and the cycle begins again. I am unable to accept Krebs' generalization. Even if this cycle exists, it represents, as shown by Thomas, only an additional function of the C4 dicarboxylic acids.

Biological Oxidation and Vitamins

46 3

As I pointed out two years ago a member of the chain was missing at this point which had to transmit H from the dihydro-coenzyme on to fumarate. I suggested that this "something" might be a "yellow enzyme." These "yellow enzymes" are specific proteins which hold adsorbed a yellow alloxasine dye, which was observed first in my laboratory. Later it was shown by Warburg that these dyes acted in conjunction with specific proteins. Recently F. B. Straub, working in Keilin's laboratory, has isolated such an alloxasine protein which seems to answer all the requirements. Its exact place in the oxidation chain is not yet ascertained, but probably it is identical with the alloxasine protein, tentatively postulated two years ago between malate, the dihydro-coenzyme and fumarate, respectively. Straub's substance is also identical with the substance described by von Euler and Adler as "Diaphorase" and by Green and Dewan as "coenzyme factor." Unfortunately, time is too short to enter into detailed discussion of these catalysts, but I hope that this incomplete description brings out the point that the mechanism of biological oxidation represents a very complicated chemical apparatus of high complexity, specificity and precision. This mechanism liberates the energy of the H piecemeal, shifting the H atoms of the foodstuff molecule from substance to substance, then detaches the electron of H, and sends it through a series of metals to oxygen, liberating in this way the energy of the electron in appropriate small quantities fit to be used by the cell for its

different functions. I am hoping that you will forgive me if I do not present on this occasion an impartial review of the problems of oxidation, but instead present the development of the field from the narrow angle of my personal experience, giving undue prominence to my own work. To take this wholly personal outlook I must confess that in starting in this field some fifteen years ago my primary interest was not oxidation at all. What I really wanted to know was the function of the adrenal cortex. At that time we knew next to nothing about it. We only knew that man could not live without adrenals and turned brown before dying. You all know that one of the typical symptoms of adrenal cortical deficiency, or Addison's disease, is the brown pigmentation of the skin and mucous membranes. Now about half of the plants, including potatoes, apples, pears and bananas, do the same thing: they turn brown when dying. I have always been convinced of the fundamental chemical unity of living Nature and I thought that the brown pigmentation in dying plants and

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THE BULLETIN BULLETIN

in patients with Addison's disease might have a common chemical foundation. In regard to the pigmentation of plants we knew from Palladin's work that it is connected with the disturbance of biological oxidation systems. Consequently, I decided to take up the study of oxidation, hoping that if I understood oxidation better I also would understand the adrenal gland. It was known that the brown pigmentation of dying plants was connected with the activity of a specific enzyme, a polyphenoloxidase which oxidized polyphenols, mostly catechol derivatives. Different theories were presented concerning the mechanism of this action. I was able to show that the enzyme simply oxidized the polyphenol into the corresponding diquinon, taking off its two H's (Figure 4). The diquinon, if not reduced with sufficient rapidity, combines with proteins or amino acids to form highly colored melanoid substances.
OH

Fig. 4

The enzyme itself, the polyphenol, was lately isolated by Kubowitz in Warburg's laboratory and shortly afterwards by Keilin and was found to be a metal-proteid of copper. This system, however, did not explain to me the function of the adrenal gland. So I turned the question around and did not ask why patients with Addison's disease turned brown, but why it was that we, who had normal adrenals, remained unpigmented. This could not be learned from the polyphenoloxidase plants, so I started to analyze the respiration of other plants which failed to turn brown when dying. About half of the plants, such as lemons and cabbages, do not turn brown when dying. I soon found that the juice of these plants not only failed to turn brown itself but also prevented the juice of the polyphenoloxidase plants from turning brown, when the two juices were mixed. The inhibition was not complete, lasting only a few seconds, but was distinct. The analysis showed that this inhibition was due to the presence of a substance with peculiar properties which greatly fascinated me. The most striking

Biological Oxidation and Vitamins

4 65

feature of this substance was its strong reducing power. It contained two very labile H atoms which explained the inhibition of pigment formation. By means of its two H's the substance reduced the quinols to phenol again, reversing the reaction shown in Figure 4. The substance was crystallized and analyzed to a certain extent. I will not dwell upon details of its history for you all know it today as ascorbic acid, and you also know that it is identical with vitamin C. One of the most exciting phases of that research was the discovery of a rich source of ascorbic acid in Hungarian red pepper, the discovery of which made the preparation of several pounds of the pure vitamin possible. This substance was distributed to all workers desirous of investigating it, and their collaboration led in a short time to the complete analysis and synthesis of the vitamin. That highly spirited international collaboration of those days is still one of my pleasantest scientific memories. Along with ascorbic acid I found in some plants an enzyme which could oxidize the acid in a reversible way and seemed to be involved, together with ascorbic acid, in respiration. The existence of this enzyme is denied in some quarters but I find it difficult to accept this conclusion. This ascorbic acid oxidase has not yet been isolated. It is probably a Cu protein, as is the polyphenoloxidase. To reduce 02 to water we need four electrons and four H ions. Thus to reduce oxygen to water the enzyme would have to hold the 02 molecule till it took up four electrons. Cu is unable to do this and will release the 02 after it has taken up two electrons and two H ions, and is thus reduced to H-O-O-HF hydrogen peroxide. Whenever the oxidation of a substance like a polyphenol or ascorbic acid is catalyzed by copper or a copper-proteid there is a formation of peroxide. Accordingly, in the presence of ascorbic acid oxidase and polyphenoloxidase we find a second enzyme, a peroxidase, which will react with the peroxide and utilize this peroxide for the oxidation of a second molecule of catechol or ascorbic acid. To sum up: catechol-oxidase will oxidize two atoms of H from a molecule of catechol in the presence of 02. The catechol is oxidized to quinon (Figure4),whiletheO2isreducedtoperoxide.02 + C6H4(OH)2 H202 + C6 H402. The H202 thus formed will react with peroxidase and oxidizes a second molecule of catechol: H202 + C6H4(OH)2 = 2H20 + C4H402. The reaction in the case of ascorbic acid oxidase is analogous, except that the peroxide with the peroxidase will oxidize a phenol and the oxidized phenol in turn will oxidize a second molecule of

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THE BULLETIN
THE

BULLETIN

ascorbic acid. These phenols are specific, are members of the group of the yellow benzopyrane dyes studied by Kostanecky and Perkins, and are called, according to their structure, flavones, flavanoles or flavanones. I have talked at such length about these reactions because we found that these dyes acting together with ascorbic acid also had a therapeutic potency which had certain characteristics of a vitamin action. We tentatively called these substances vitamin P. However, we were unable to demonstrate the vitamin nature conclusively in the animal experiments. Our own observations on guinea pigs were not corroborated. Beyond doubt the therapeutic action of these substances, which is capable of restoring the normal resistance of damaged capillaries, makes them available for the treatment of disease (vascular type of hemorrhagic purpura) for which medicine hitherto has had no remedy. But to come back to oxidation, there was something disquieting in these results. In all systems hitherto discussed the action of 02 was catalyzed by a metal or series of metals. The metal then oxidized reversibly two H atoms of an organic substance. In the muscle this substance was succinic acid. In certain plants it was catechol, in others ascorbic acid. It would be difficult to find three more heterogeneous substances and we were unable to give any reason why Nature selected these particular compounds for this end. Moreover, a greater uniformity was to be expected in such a fundamental process as oxidation. Another very disquieting matter was that in spite of all this work- we did not know how plants really respired. I do not want to lose myself in details and will limit myself to telling you that there were serious reasons for believing that neither the polyphenoloxidase, nor the ascorbic acid oxidase were really involved in respiration and so we had no idea how plants respired, what the enzyme was which interacted in plants with 02 and what the substance was on which this enzyme acted. It became evident that something of basic importance was still to be learned. To find out what this was, my faithful collaborators and I set out to study the catalytic action of metals which catalyze the dehydrogenating oxidation of organic molecules. Such oxidations are catalyzed by metals outside the cells, and when the cell used metals to catalyze oxidation it did not invent a new principle; it merely applied an age-old reaction, but applied it in a very clever way: linking the metal to a specific protein in the cell and thus giving it a chance to act at its best. There are two current theories to explain this catalytic activity of

Biological Oxidation and Vitamins

46 7

metals. The one is the very complicated radical theory of Haber. The other theory is the simpler one. According to the latter the metal simply oxidizes the H of the organic molecule, taking over its electron. The metal thus reduced gives this electron in a second reaction to oxygen. To use different and simpler words, the function of the metal is to be alternately oxidized by the 02 and reduced by the H. We found that none of these theories explained the changes and what really happened was that the metal combined, by its coordinate valencies, with the anion of the organic substance to be oxidized. By this combination both substances, the metal and the organic molecule, forming now a single molecule, are "activated." We obtain thus a complicated coordinative molecule in which the metal acts as a central atom. Now the valencies of this metal combine with a molecule of 02. Both the 02 and the organic molecule to be oxidized are built thus by the metal into a single molecule. Electrons are then transferred within this molecule via the metallic ccntral atom from the organic molecule to the 02. I will not go into the details of these reactions. The important matter is that the organic molecule, which is to be oxidized, has to answer very strict specifications. It must contain two OH groups at a certain distance, with a double bond between the C atoms holding these OH groups. The most suitable formation is the dienol grouping: -C-OH II - C-OH This formation is very unstable and thus very rare, and is found only in a very limited number of natural substances. One substance in which it is found is catechol, where it is stabilized by the aromatic cycle. Thus we are able to give an adequate reason why Nature applied catechol in this oxidation mechanism. Another substance, containing a dienol group, is ascorbic acid, in which the dienol is stabilized by the acid-lactone ring. Thus we can give the reason why Nature also applies this substance. At the same time the apparent heterogeneity of catechol and ascorbic acid disappears; in essence both are dienols. There is a third relatively common substance containing the dienol group, dioxymaleinic acid, in which the dienol formation is stabilized by the two neighboring COOH groups (Figure 5). If all our ideas were correct we would have to expect that this substance is also employed by Nature in oxidation mechanisms. I shall now describe the last phases

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THE BULLETIN
TH

BULEI

of my most recent research. Experiments of Banga, Philippot and myself show that of the twenty-five plants examined all contain an oxidase for dioxymaleinic acid. In some plants the enzyme has a very striking activity. It is the most active oxidase known. There is reason to believe that in this enzyme we have found the way in which oxygen enters into vegetable respiration. Furthermore, we hope that this enzyme will shortly give us the explanation why Nature in animal cells utilizes succinic acid. If you compare the formulae of succinic acid (Figure I) and dioxymaleinic acid you will observe that the two substances are closely related, dioxymaleinic acid being simply an oxide of succinic acid.
COOH COH I COH COOH
Fig. 5

I cannot cover my subject completely, nevertheless, I hope that I have made clear the great changes that have taken place during the last decades in our conceptions regarding biological oxidation. The field is hardly opened, but already it has given us a deeper insight into Nature's ways; it has yielded new substances, some of which are of vitamin nature and of great importance to health. I have discussed alloxasines, the dye component of yellow enzymes, substances connected with H transportation and dehydrogenation. The nicotinic acid amide of the codehydrogenases also acts as a member of the vitamin B group. Vitamin C was crystallized in the course of an analysis of vegetable respiration. The therapeutic action of Vitamin P was also discovered in the course of these experiments. I should also have mentioned Vitamin B1, a substance which is closely connected with oxidative processes. Oxidation studies have thus not only helped us to reveal the existence of new active substances but have also given us information concerning their exact function and have taught us to regard vitamins no longer as mysterious bearers of life and health but as simple little wheels in this wonderful chemical mechanism, the perfect functioning of which we call simply life and health. These studies have also given us weapons for fighting disease and reducing human suffering, weapons not less sharp than the

surgeon's knife.