Thiamine and Peripheral Neurophysiology

Thiamine and Peripheral Neurophysiology BY ALEXANDER VON MURALT-(Bern) COXTENTS

I. Introduction. . . . . . . . . . . . . . . . . . . . . . 11. Liberation of Thiamine in Nervous Excitation . . . . . . 111. Thiamine Content of Peripheral Nerves . . . . . . . . IV. Liberation of Thiamine on Vagus Stimulation in the Heart. V. Discussion and Summary . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . .

Paqe

. . . . . . 93 . . . . . . . 93 . . . . . . . 106 . . . . . . . 110 . . . . . . 114 . . . . . .

116

I. ISTRODUCTION Strange as it may seem, direct evidence that thiamine plays an important r81e in the chemical mechanism of nervous excitation was not presented until 1938. Indirect evidence of such a correlation, however, goes back to the work of Eijkman (1897) and Grijns (1901) in the Dutch East Indies, a t a time when nobody had even thought of “vitamins.” They recognized that it was the lack of a certain dietary factor(s) present in rice bran, which produced avian polyneuritis and beri-beri. We would say today that the lack of thiamine produces a fundamental change in the chemical mechanism of each neurone, by which its function is dangerously affected. As soon as the factor is replaced, the chemical equilibrium‘ of the neurone is restored and, with this assumption, one is entitled to call the factor an “antineuritic principle,” as Eijkman (1897) has done. We would say today: thiamine is essential for the maintenance of the internal chemical equilibrium of the neurone (nerve cell and peripheral nerve). The store of thiamine in the cell is continuously used up in maintaining this equilibrium, and, as the cell is not able to synthesize this vitamin, a constant requirement from other sources is the result. What is the r8le of thiamine in the chemical equilibrium of the neurone? Why is thiamine permanently used up in maintaining this equilibrium? What is the requirement of a neurone at rest and during activity? These questions seem so simple, and yet it is so hard to give an answer which corresponds to a first approximation of the problem! This article has been written in an attempt to show the methods and procedures which have been employed since 1938 in an attempt t o answer these three questions. 11. LIBERATIONOF THIAMINE IS NERVOUS KXCITATIOK

In 1938 Minz (1938a,b) published a few experiments with excised ox nerves, which were placed in a physiological solution after the surfaces 93

94

ALEXANDER VON MURALT

had been cut. Electrical stimulation was applied t o the nerves while their ends were dipped in the bath. The solution was tested for thiamine content by the Flagellatae test. Polytomella caeca, which was used in one group of experiments can only grow if thiamine, or the two fractions of the thiamine molecule, pyrimidine and thiazole, are present in the medium, i.e., this organism can synthesize thiamine from its two fractions. Glaucoma piriformis, which was used in another experiment, requires the whole molecule for growth and cannot use the fractions alone. The solution was collected and tested after stimulating the nerve-trunks for 10 minutes (40 stimuli/sec.), a second portion of the solution, corresponding to an identical period of immersion of the nerves without stimulation, being similarly treated. The rcsulfs are shown in Table I. TABLE I Number of organelles grown in 1 mm.3 Specimen Polytomella . . . . . . , , . . , . . . . .

Glaucoma. . . . . . . . . . . . . . . . .

The growth effect of the solution, S, from stimulated nerves corresponds to about 1/100 y of thiamine. The figures show that about 4-8 times more thiamine diffuses from the cut surface while the nerve is stimulated, than when at rest. In one experiment stimulation was tried aftcr 30 minutes, (Sso in Table I), but without any further liberation of thiamine. Either the cut surface had lost its permeability or no thiamine had been formed due to inexcitability of the nerve. These few, but important, observations were of the greatest interest to the group in Berne, who had been working since 1936 on acetylcholine liberation in excited peripheral nerves. Liberation of Acelglcholine in Ne~vous Excitation. Nachmansohn (1945) has reviewed the rBle of acetylcholine in the mechanism of nerve activity. In 1936 it was a well established fact that cholinergic nerves (motor nerves, pregangconic sympathetic, parasympathetic, and postganglionic parasympathetic nerves) liberate acetylcholine a t their tcrminals on stimulation (Loewi, 1937; Dale, 1935). At that time it was also evident, but not generally known arid accepted, that the cut surftrces of nerves liberated acetylcholine into bathing fluids on stimulation (Calabro, 1933; Bergami, 1936a,b; Binct and Minz, 1934; Rossine, 1936a, 1936b). From this observation the conclusion could be drawn that acetylcholine is formed along the entire length of the nerve during

THIAMINE AND PERIPHERAL NEUROPHYSIOLOGY

95

excitation. However, Gaddum et d.(1937) published a paper in which they came to the conclusion: “The release of the active substance is surprising, but does not reveal a physiological mechanism. The main object of this communication is to draw attention to this local phenomenon a t the electrodes. The methods used by the workers mentioned above (Calabro, Bergami, Binet and Minz) would not discriminate between effects due to the passage of impulses, and effects due to local changes at the electrodes.” It was, therefore, necessary to prove that acetylcholine is formed in excited nerves by using a method which leaves no doubt of its nature. If the excitation wave is associated with, or followed by, a chemical wave, it must be possible t o determine the amount of acetylcholine by freezing excited nerves in liquid air and thereby fixing the state of excitation. The first results obtained by a special technique were published 3 months after Gaddum’s paper by Muralt (1937). By this technique the chemical waves were not only frozen, but also accumulated in the nerve. The acetylcholine content of nerves frozen by this technique were compared with the content of their controls, taken from the same animal, but frozen a t rest. Special precautions had t o be taken because liquid air itself acts as a stimulus. The “resting” nerves were made unexcitable by the slow action of cold, by cocaine orbyether vapors. These experiments showed (Muralt, 1937; Muralt el al., 1938; Muralt, 1939a,b,c; 1942; 1946) that excited frog nerves contain 0.24 y 0.01 of acetylcholine, whereas unexcited nerves had only 0.15 y 5- 0.01 (average of 50 independent determinations with calculated errors). The difference of less than 0.1 y seems to be small and it is questionable whether such a difference is real. The mean error Z ( D ) for a difference D between two mean values M I and M ) was calculated Z(D) = f d Z ( M l ) * + Z ( M z ) 2 and the quotient D / Z ( D ) determined. A difference must be regarded as insignificant if D / Z ( D ) is smaller than 2, “probable” if greater than 2 but smaller than 3, and “certain” if greater than 3 (Norberg, 1942). In the case of the acetylcholine determination the quotient D / Z ( D ) was 7, i.e., the difference is reliable and real. With each excitation wave traveling down a nerve, 0.1 y of acetylcholine is “liberated” per g. of nerve in a sort of “chemical wave” and almost as quickly inactivated during the refractory period following each excitation. The determinations of acetylcholine were made with: (a) the leech preparation and ( b ) the response of the frog-heart. To the Bernese group it was obvious in 1938 that another unknown factor was interferini with the determinations of acetylcholine, especially in nerve extracts. Extracts made from excited nerves showed a very considerable difference between method

96

ALEXANDEB VON MURALT

( a ) and ( b ) , the leech method yielding higher values than did the frog heart method. I t was also surprising that these differences were greater in summer than in winter months. As soon as hlinz published his results it became clear that thiamine might be interfering in the determination of acetylcholine. Thiamine has a sensitizing action for acetylcholine on eserinized leech muscle (I3inet and hlinz, 1934; Brucke and Sarkander, 1940; Muralt, 1942), so that an excessive amount of acetylcholine is found in extracts containing both. On the frog heart, thiamine has an inhibiting action for acetylcholine (Agid and Balkanyi, 1938; Kaiser, 1030; Moniz de Bettencourt, and Paes, 193Ga,b), i e . , the negative chronotropic and inotropic effects of acetylcholine are decreased if thiamine is present a t the same time and too low nn umonnt of acetylcholine is determined. I t became evident that thiamine is liberated a t the same time as acetylcholine and that the amount liberated had t o be determined. The figures given above for acetylcholine liberation have been corrected for the “thiamine effect.” Determination of Thiamine Liberation an Excited Nerves. Reginning in 1939 the Bernese group applied the technique of freezing nerves in the excited state t o the determination of thiamine liberated during or shortly after excitation. Since thc. tmounts to be determined are of the order of 0.01 y, very refined methods had t o be used. The evidence so far obtained is based on: (a) direct determination using specific thiamine methods: bradycardia test (Iiechti ~1 aZ., 1943), Yhycomycev test (Muralt and Zemp, 1943), yeast fermentation test (Wyss and Wyss, 1945a,b) und the yeast micro test by the diver method (Went, 1946); ( h ) direct determination with nonspecific thiamine methods: thiochromc method using a highly sensitive electric fluorometer (Wyss, 1943 ; 1914), sensitizing effect on the leech muscle (Muralt, 1942), arid polarograplic determination (Muralt, 1941; 1942; Weidmann, 1945; 194G) ; and ( c ) indirect evidence gathered from experiments with ultraviolet light, making use of the specific absorption of thiamine, of its transformation into thiochrome, and of the photochemical decomposition of thiamine. A description of the freezing method and the methods used for thiamine determination has been given in full detail elsewhere (Muralt, 1946). The results obtained with these methods are interesting and in one point unexpected. Fig. 1 is an example of experiments with the bradycardia test. The extracts of 50 frozen nerves (50 excited and 50 unexcited) were fed to various groups of rats in a state of avitaminosis. The average increase of pulse rate after having fed the extracts on day 0 is plotted as a function of time, together with controls of standard thiamine preparations and a blank. It is obvious that the extracts from stimulated nerves contain a measurable ?mount of thiamine, whereas the

THIAMINE AND PERIPHERAL NEUROPHYSIOLOQY

97

extracts of unstimulated nerves contain only a subthreshold amount of thiamine, if any is present. At this point of the discussion a very important point in procedure must be mcntioncd to clarify the meaning of the words “extract” and “liberated.” The frozen nerves are ground in a mortar until a very fine powder is obtained and then brought to the melting point. At this moment, t o 1 CC. of Ringer solution, 10 mg. of nerve-powder is added and left in contact with th e powder for only 10 minutes. The extract is then filtered off or separated from the powder by centrifuging in such a way that the contact between powder and solution is never longer than 15 minutes. The amount of thiamine found in the solution is, thqrefore, a measure of the soluble fraction rather than of the actual concentration in the nerve powder. If we say “thiamine is liberated” it really means t h a t thiamine in the powder of excited nerves i s in such a state, that more can be extracted b y Ringer solution in 10 minutes than can be obtained f r o m a corresponding sample of unexcited nerves. 100 Fig. 2 shows a corresponding 0 1 2 3 4 experiment with the Phycomyces test. DAYS The growth curves of Phycomyces are FIG. 1.-Thiamine in excited and plotted for varying standard thia- unexcited nerves, determined by the mine concentrations and also for bradycardia test. Ordinate: average extracts of excited and unexcited pulse rate increase and decrease reduced nerves. The same difference was to starting pulse rate. Abscissa: time obtained and the determinations in days. Ordinate: pulse rate. R = extract of cxcited nerves; U-extract of gave a n average “liberation” of thia- unexcited nerves; S = blank; B, = mine of 1-2 y / g . of excited nerve. thiamine tests.‘ This is surprising, considering that the acetylcholine “liberation” only yielded 0.1 y / g . Thiamine is “liberated ” in 10-20-fold greater amounts than acetylcholine. The same “Note: Figs. 1, 2, 6 and 7 are taken from Muralt’s “Signaliibermittlung im Nerven,” pp. 295, 297, 106 and 107, respectively.

98

ALEXANDER VON MURALT

1

-

FIG.2.-Thiamine in excited and unexcited nerves, determined by the Phycomyces teat. Ordinate: weight of mycclia in g.; abscissa: amount of tested solution; R cxtract of excited nerves, U = extract of uncxcited nervee. The curves combhe the values obtained with the same concentration but in different amounts. To show the reproducibility, two sets of curves from independent experiments are presented (Muralt, 1946).

99

THIAMINE AND PERIPHERAL NEUROPHYBIOLOQY

result was obtained in the experiments with the 3 unspecific methods mentioned previously and it seems to be well established that a considerable amount of thiamine is “liberated” during the process of excitation. The first surprise was the observation of Wyss and Wyss (194513) that, after poisoning a nerve with monoiodoacetic acid, more thiamine is obtained in the extract of resting nerves than in the extract of excited nerves. In the poisoned nerve thiamine is “liberated” in the resting metabolism and (‘fixed” in the process of excitation, ie., under conditions in which the sequence of chemical reactions has been altered by the poisoning, the poisoned nerve shows a behavior opposite to that of the normal nerve. The second surprise was the fact that the yeast fermentation method used, either according to Atkin, Schultz and Frey (1939) or in the highly refined form of the micro diver method (Holter and Linderstrom-Lang, 1943; Went, 194G), gave no difference between “excited” and “unexcited” extracts and much lower values for the thiamine content of the solutions. Addition of thiamine t o nerve extracts, however, always yielded correct values, so that an “inhibitory” effect can be excluded. The fact that the yeast method does not show any signs of thiamine liberation in excited nerves is a very important finding and throws some doubt on the previous observations by 6 independent, and more or less specific, methods. No explanation for this discrepancy can be offered today. Only experimental work can show whether yeast has special properties with regard to thiamine derived from nerves, or whether the extracts contain other factors which cause the abnormal reaction. Keeping this in mind, we should now consider the indirect evidence for the important role of thiamine in nervous conduction. Thiamine has a very characteristic ultraviolet absorption band near 260 mp. At the same time thiamine is photochemically destroyed by the action of shortwave ultraviolet, but not by long-wave ultraviolet (Ruehle, 1939; Uber and Verbrugge, 1940; Stlbmpfli, 1942.) Under the influence of alkali and potassium ferricyanide, thiamine is transformed into thiochrome (Kuhn et al., 1935; Jansen, 1936) and this substance shows a very strong fluorescence. These facts were the starting points for various investigations. Caspersson (1940) was the first to apply methods of ultraviolet absorption to cytological problems. An apparatus for measuring the ultraviolet absorption of living, single nerve fibers was built according to his design, but with certain improvements (Muralt, 1946; Luthy, 1946a). Fig. 3 shows the absorption curve of a living nerve fiber in ultraviolet light. There is a first maximum between 280-285 mp which

.

100

ALEXANDER VON MURALT

is due to the protein of the nerve, especially the tyrosine in the protein. The second maximum at 265 mp is due t o nuclein components and, in part, to the thiamine content of nerve. It can be shown that treating the nerve with potassium ferricyanide and thereby transforming thiamine into thiochrome reduces this band considerably. (Thiochrome has no specific band a t 265 mp.) A very interesting fact was brought out by the experiments of Liithy (1946b) who found in the living nerve a very 0.4

E

0.3

042

I

260

280

300

340

320

mP

FIG.3.-Ultraviolet absorption of single nerve fibers of the frog. Ordinate: extinction coefficient (E) ; abscisfia:wave length of light (mp).

marked dichroism in the ultraviolet region! Fig. 4 shows the absorption curve of medullated nerve-fibers (frog) under polarized light. If the clectric vector is parallel to the fiber, the absorption at 265 m p is greater than if it is perpendicular. At 280 mp exactly the reverse effect is observcd. If we say that by and large the 280 mp absorption is due to protein structures and a t 265 mp to lipoid structures, it would follow that the absorbing elements in these structures are oriented a t right angles. And that is cxactly what Schmitt’s (1939) studies of double refraction and X-ray patterns of the myelin sheath of medullated nerves have shown! If the interpretation is correct, then a non-medullatcd nerve should

THIAMINE AND PERIPHERAL NEUROPHYSIOLOOY

101

produce a different picture. This is shown in Fig. 5 which was obtained from the olfactorius of the pike. This nerve consists of nerve fibers (axones) immersed in a myelin-like environment, without protein-myelin structure. The axones have a definite protein structure, but parallel to the fiber. The protein leaflets which alternate with lipoid layers in the sheath of medullated nerves are not developed in these nerves. The ultraviolet absorption of the lipoid component is, therefore, unchanged

E 0.3

0.2

0.1

?60

28 0

300

340

320

9 FIG.4.-Llltraviolet absorption of single nerve fibers of the frog in polarized light. Ordinate: extinction coefficient (E); absclsa: wave length of light (mp). The orientation of the electrical vector with respect to the fiber axis is indicated by signs.

with regard to the character of dichroism, the protein component however is reversed, just as must be expected, because the dominating effect of the protein leaflets is missing and the axone structure shows its dichroism exposed. With electric excitation very specific effects can be observed in these living nerve fibers with regard to their dichroism.

102

ALEXANDER VON MURALT

These experiments are in progress but i t is still too early t o say much about them. They seem to show in a very conclusive way that, with and after excitation, a molecular regrouping takes place in the protein and in the lipoid system, together with the thiamine component. The “libcration” of thiamine is not only a question of a change of solubility but also in its fundamental nature in the nerve. From this point of view the experiments described previously take on increasing importance.

mP

.

FIQ. 5.-Ultraviolet absorption of the olfactory nerve of the pike in polarized light. Ordinate: extinction coefficient (E); abscissa; wave length of light (mp).

The photochemical sensitivity of thiamine led to another group of experiments of considerable theoretical interest (Hutton-Rudolph, 1913; 1944). Single living nerve fibers were irradiated with ultraviolet light. At the same time their excitability was measured by determining the threshold and chronaxy of these nerves. Fig. 6 shows one experiment

103

THIAMINE AND PERIPHERAL KEUROPHYSIOLOGY

in which the light was filtered by only a quartz cuvette containing distilled water. The very constant values for the rheobase show that the single nerve fiber was in an excellent .state up to the time of irradiation. Two phases of the irradiation effect were always observed. We called them phase of increased excitability and phase of destruction. The fact that only ultraviolet light of less than 300 mp produces these specific effects, showed at once that they are due to a specific absorption in the nerve. This can be shown by a very interesting effect. If a cuvette containing a substance with a specific absorption is used as a light filter, the best protective action is produced by that substance which has the same absorption a5 the photochemically sensitive substance in the nerve, i.e., by the substance itself. We found, among all substances investi-

U.V. I R R A D I A T I O N CUVETTE W I T H WATER

I

I

0

I

I

10

I

I

20

I

I

30

I

I

40

I

I

50

I ! I

I'

60

MINUTES Fro. 6.-Photochemica1 action of short wave ultraviolet on the excitability of a single nerve fiber. Ordinate: rheobase in millivolts; abscissa; time in minutes. The irradiation begins after 40 minutes; typical results of 32 experiments (Muralt, 1946).

gated, that thiamine has an absolutely ideal protective action for, as long as the ultraviolet is passed through a 1% thiamine filter, all those wavelengths which have photochemical action on the nerve fiber (Fig. 7) are absorbed before reaching the fiber and cannot excite its action. Those wave-lengths which are not absorbed by a 1% thiamine filter 0.5 cm. in thickness pass without any decrease in intensity, but have no photochem-

104

ALEXANDER VON MURALT

ical action on the nerve fiber. As soon as the filter is replaced by a filter with distilled water the photochemical effect begins. The conclusions from a great number of such experiments were: the first phase of the photochemical reaction (overexcitability) is due to the destruction of a substance with an absorption spectrum similar to, or identical with, that of thiamine. This substance is localized in the sheath of medullatcd nerves. The phase of overexcitability corresponds to the effect produced in nerve i l L uiuo by the initial phase of thiamine avitaminosis. The phase of destruction corresponds to denaturation of the nerve proteins. Irradiation of the node of Ranvier alone, where there is only protein and no

J ~

0

,

,

10

,

1

20

OUARTZCUVETTE ! W I T H THIAMINE I : I O O ,

30

1

I

I

40

I

I

CUVETTE

I WITH WATER I I

50

I

I

I

60

MINUTES FIG. 7.--Protective action of a thiamine filter against the photochemical action of short wave ultraviolet on the shgle nerve fiber. Ordinate: rhcobase in millivolts; abscissa: time in minutes. Tho light passed through a thiamine solution before falling on the nerve fiber from 27th47th minute (Muralt, 1946).

lipoid or thiamine produces only the second phase. One should not draw too far-reaching conclusions, but it is obvious to those who have been working with single nerve fibers and ultraviolet that there is u close relationship between thiamine destruction and change of excitability of the nerve. If histological preparations of nerves are made with alkali and potassium ferricyanide and neutralized, a definite blue fluorescence is observed (Muralt, 1943). To a great degree this is due to the trans-

T H I m I N E AND PERIPHERAL NEUROPHYBIOLOGY

105

formation of nerve thiamine into thiochrome. Fig. 8 shows a preparation of a single fiber in the fluorescent microscope. The node of Ranvier has no fluorescence a t all and the fluorescent substance is localized in the medullary sheath. This can be interpreted as showing that thiamine is stored in the nerve sheath and not in the axone and corresponds to the finding of a specific ultraviolet dichroism at 265 mp. Nachmansohn and Steinbach (1942) studied the giant axone of the squid and found that cocarboxylase is concentrated on the surface of the axone. If the nerves are in electrotonus at the moment of fixation, an accumulation of thiamine is observed at the site of the anode, which is an indication that thiamine is liberated in relation to anabolic, rather than to catabolic, processes. All these findings support the view that thiamine plays an important role in nerve 8.-fluorescence of a metabolism and that during the state of excitation or shortly afterwards a certain single nerve fiber in which, by “liberation” of thiamine takes place which Muralt’s technique, the thiadisappears almost as quickly as it is pro- mine was PreviOUSb’ oxidized to duced. Such is the experimental evidence thiochrome. Photomicrograph taken in fluorescent light, 5 today. It must be stressed that the find- minutes exposure. ings are fragmentary and their interpretation might be influenced by further experimental work.

111. THIAMIKE CONTENT OF PERIPHERAL NERVES Thus far no distinction has been made between thiamine and the various forms in which thiamine can exist in the cell, such as thiamine diphosphate (cocarboxylase), thiamine monophosphate, thiamine sulfide and disulfide, elc. There is also a t least one form, and possibly more, of bound thiamine. The content of nerves with regard to free and bound thiamine is of special interest. As far as could be gathered these findings have been summarized in Table TI. The body is unable to store thiamine to any extent, so that the figures given correspond to the normal and, a t the same time, nearly the maximum amount in each nerve. “Free thiamine” is the moiety which can be extracted in aqueous solutions without special methods, “bound thiamine” is the amount (total minus free thiamine) which can be determined only after acid extraction, or by the Phycomyces method, in which case it is assumed that Phycomyces is able to make use of the total amount of vitamin present. The data are

106

ALEXASDER VON MURALT

TABLE I I a Thiamine, Free and Bound, i n Nerves and Brain Animal Frog. . . . . . .

Ilat . . . . . . . .

Tissue

Thiamine

sciatic

(frcc) y / g . I .2

Sciatic

..........

Rrain

3.3

Brain

3 .&l

Braiii

D.3

Brain

1.9

Brain

0.8

Sciatic Ccwhrllum Medulla ob longata Spinal cord

0.7 0.2 0.5

~-

.o

Thiamine Author Mothod (hound) r/g. 2.5 Thiocliroine Wyss (1944) (mnnomctric) D. G (unstiiu- Phycomyces Muralt and Zemp ulated ( 1943) nerves) to 2 . 7 (stimulated nervm) Thiochromc Westenbrink 6.0 and Goudsmit (1938) Thiochroirie Ritscrt 2.5-3.5 (1939) Thiochrolue Wicdenbauer 2.3 (1939) (modificd) Thiochrome dc Caro and 3.5 Bu tturni (1 940) Thioohromc Muralt jun. 3.1 ( 1946) and PhyC@myce3

1 .o

1.6 3.4 3.6

id. id.

id. id.

id.

. . . . . . . . . . Thiochronie Ititscrt

Pig. . . . . . . , . Obturatorir Dove. . . . . . . Brain

0.28 0.1

0.39 2.6

(hiinca pig, ,

Brain

0.2

3.2

Sciatic

0.1

1.0

Sciatic

0.8

1.1

Vagus Phrenic Spinal cord Medullti ot longata

0.4 0.4 0.5 0.7

0.9 1.9

Dog. . . . . . . .

id.

0.5 1.7

(1938) Thiorhroine Sanz (1943) T1iioc:hromc de Car0 and Ihtturini (1940) Thiochrolue Wicdcnbauer (1939) (modificd) Thiochrome Muralt and (manomet- wyst3 ( 1944) ric) Thioclirorne Muralt jun. (1946) and Phycornyces id. id, id. id. id. id. id. id.

107

THIAMINE AND PERIPHERAL NEUROPHYSIOLOBY

TABLE Ha.-(Continued) Animals

Calf. . . . . . . .

Tissue Brain (COI tcx, occip tal lobe) Thalamus Corpus ca losum Corpora quadrigemina Cerebellum (vcrmis) Splanchnic Thoracic sympathetic chai Preganglionic sympr thetic Sciatic Phrenic Splanchnic Vagus Spinal cord Ventral roots Dorsal root (without ganglion) spinal gan glion (with out roots) rhoracic sympathetic chai (without ganglion) Sympathetii ganglion 3rain (cor. tex) :orpus cal. losum iupcrior cor pora quad. rigemina dcdulla oblongata .'halamus krebellum (vermis)

Thiamine (free) r l g . 0.6

Thiamine (bound) r l g 2.0

id.

id.

0.4 0.6

1.4 0.8

id. id.

id. id.

0.5

0.8

id.

id.

0.7

1.4

id.

id.

0.5 0.4

0.7 0.4

id. id.

id. id.

10-7

1 .o

id.

id.

10-7

id. id. id. id. id. id.

id. id. id. id.

<1x

0.7 0.7 D.9 0.5 D.6 0.4

<1x

10-7

D.5

id.

:d.

1.5

D .0

id.

Id.

1.2

1.3

id.

'd.

1.4

1.6

id.

'd.

1.7

1.8

id.

d.

I .3

:d.

d.

1.8

1.2

:d.

d.

1.6

.4

'd.

d.

1.9 1.7

.2

<1 x D.5 0.4 D.4 0.3 3.6

1.2

P

.8

~

Method

d.

d.

~~~

Author

a.

id.

.

d.

J.

108

ALEXANDER VON MURALT

instructive inasfur as they show that in peripherd nerveR of warmblooded animals less than 0.5 y of free thiamine, and not much more than 1 7 of bound thiamine, is present on the average. In the spinal cord and brain the corresponding figures are at least twice as great. The extent to which seasonal differences influence the values is shown in Table IIb where the same determinations were made on 6 rabbits in winter and on 6 other animals in summer. It is obvious that, as soon as the food provides more vitamin, the storage is on the “bound” side and not on the ;I free’’ side of thiamine in brain and nerve. TABLE IIb Free and Bound Thiamine i n Nerves and Brain of Rabbits I. Six rabbits of the mme agc and fed the eame diet for three weckR hcfore determination of thiamine. Determinations mndc in winter. Frcc thiamine (Thiochromr)

0.7

Bound thiamine (I’hyeomyces) r /g. 0.9

0.5

1.3 0.9

rlg.

..

. . -

Brain (cortex) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mid-brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Medulla oblongata. . . . . . . . . . . . . . . . . . . . . . . . . . . Spinal cord.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sciatic. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vagus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phrenic. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

0.6 0.8 0.4

1.4

0.3

0.6 0.5

0.2

0.5

11. Six rabbite of different agee rrnd on diffcrent diets bcfore determination of thiamine. Detcrminations made in summer. Free thiamine (Thiochrome) -

Brain (cortex). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mid-brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Medulla ohlangnt n . . . . . . . . . . . . . . . . . . . . . . . . . . . Spinal c o r d . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sciatic. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vague. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phrenic. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

rlR* 0.6 0.4

0.5 0.4 0.5

0.3 0.3

Bound thiamine (Phycomyces) rlg. 2 .o 2.4 2.9 2.1 0.7

1 .o

0.8

G. v. Murrrlt, unpublished.

During degeneration the nerves loose their thiamine eoritcnt rather quickiy, but more slowly than their acetylcholine, which shows that. this valuable chemical component is sufegiiardcd quite well in the nerve. In the blood the level is much lower and corresponds to 0.1 y/g. and

THIAMINE AND PERIPHERAL NEUROPHYBIOLOCIY

109

practically all is in the form of cocarboxylase (Westenbrink, 1940), so that there is always a gradient of concentration between blood and tissue. In the degenerating nerve the trophic influence of the nerve cell has been eliminated by cutting or crushing and, therefore, the “protective organization” is lost and the thiamine diffuses out. The thiamine content of the sciatic nerve in the guinea pig has been investigated by Muralt and Wyss (1944) and Fig. 9 shows their results. The operation was always effected on one side of the animal and the thiamine content of the nerve of the operated side is expressed in per cent of the content of the unoperated symmetrical nerve. Very striking is the fact, not visible in Fig. 9, that 10 hours after operation no free thiamine was found in the degenerating nerve, whereas in the normal nerve there was always

a measurable amount of free thiamine. The rapid loss of thiamine, together with the very rapid loss of acetylcholine, as Brown and Feldberg (1936), McIntosh (1938, 1941), Feldberg (1943) and Muralt and Schulthess (1944) have found, is one of the first signs of the start of degeneration. The relationship between acetylcholine content and thiamine

110

A L F a N D E R VON MUILALT

content is shifted in degeneration. Table I11 represents the shift in this relation (Muralt, 1946). When the relation reaches the value of 0.2, TABLE I11 Relafion of Acetylcholine to Thiamine i n the Degenerating Nerve

Acetylcholine . - in per Thiamine cent before cutting nerve

Relation of

1 .o

I- _-

-

5hr.

Hours aftcr cutting nerve -

10 hr.

20hr.

30hr.

40hr.

60 hr.

0.4

0.2

-----_-___._-

0.9

0.8

0.7

0.5

excitation of the nerve is no longer possible. Thiamine is considered as a storage substance in the nerve, closely related to excitation. If thiamine is administered to the animals during degeneration of the nerve, then it takes more time for the relation acetylcholine: thiamine to fall to the limiting value of 0.2 and the loss of excitability is reduced. The administration of thiamine has been recommended (Muralt, 1943b) in all cases where the risk of nerve degeneration is impending, with good results. Degeneration is inevitable if a nerve is cut and the administration of thiamine is not recommended because it does not favorably affect regeneration. The fact that thiamine and acetylcholine are disappearing so quickly when a nerve starts degenerating is regarded as additional proof of the importance of these two substances in the chemical mechanism of excitation. In muscle physiology it is well known that the important substances in the chemistry of contraction (phosphagen, glycogen, elc.) are the first ones to be lost with degeneration (Tower, 1939). It is not dangerous t o draw the same conclusion with regard to the nerve, although its chemical mechanism is known to a lesscr extent.

IV. LIBERATIONOF THIAMINE WHEN THE VAGWSIN STIMULATED

THE;

HEARTIs

The first step in the discovery of chemical factors connected with nervous excitation was Loewi's finding (1937) of the liberation of acetylcholine on vagus stimulation of the heart. The second step was the extension of this observation to apply to all endinga of cholinergic nerves. The third step was the establishment of the general importance of acetylcholine liberation in the excitation of the whole length of a cholinergic nerve. The role of thiamine was discovered in the reverse order; first the liberation of thiamine along the nerve was discovered and then it was only logical to look for the liberation of thiamine on vagus stimulation of the heart. It has been mentioned that thiamine inhibits the action of acetyl-

THIAMISE AND PERIPHERAL NEUROPHYSIOLOOY

111

choline on the heart (Agid and Ralkanyi, 1938; Kaiser, 1939; Moniz de Rettencourt, 1936) and thus acts as a brake. Assuming that normally this action counterbalances the action of acetylcholine which is liberated as a result of the natural action of the vagus, then a state of avitaminosis should produce bradycardia. This is exactly what Williams, Mason, Wilder and Smith (1940) observed in man a t rest a t the beginning of avitaminosis. In beri-beri, however, the tachycardia and arhythmia develop in the more severe cases and it is very probable that these disturbances are the consequence of the abnormal metabolism of the heart muscle tissue and have nothing to do with the disturbance of vagus action, which is noticeable only a t the beginning of avitaminosis and before the heart tissue is affected (a very comprehensive review has been given by Bicknell and Prescott, 1946). In the rat, the bradycardia is very obvious and is certainly to a large extent related to the disturbance of the normal vagus action by avitaminosis and to a smaller extent to the abnormal metabolism of the heart muscle. These theoretical considerations show that thiamine and vagus action are related. The experimental evidence for thiamine liberation on vagal stimulation of the frog heart was obtained by Wyss and Muralt (1944), Wyss and Wyss (1945), and lluralt (1945), and offered considerable technical difficulty. Thiochrome and Phycomycea test methods were used. Separated vagal and sympathetic stimulation on the same heart ‘preparation showed that only vagal stimulation yielded thiamine in the perfusion fluid. This perfusion fluid, transferred t o another frog heart (after inactivation of the acetylcholine by cholinesterase) showed an inhibiting effect on the action of vagus stimulation on the acetylcholine action of this heart. If the perfusion fluid was irradiated for only 5 minutes by short wave ultraviolet light, the inhibition disappeared because of the photochemical destruction of thiamine. Fig. 10 shows the result of such an experiment by Cottier (unpublished). The same disappearance of the thiamine effect was also observed after treatment of the perfusion fluid with a small amount of Frankonit (thiamine absorbent). Frog experiments are convincing only if confirmed by experiments on warm blooded animals. Gross (1947) has studied the effect of vagal stimulation in the rabbit with and without simultaneous action of thiamine. He found that the injection of thiamine a t once decreased the effect of the vagal stimulation and produced a shift in the balance between vagal and sympathetic effects toward the sympathetic side in the heart. Fig. 11 gives an example of the interesting results which he obtained. Without putting too much stress on an experiment which was performed by the members of the Berne institute, it may be mentioned here

112

ALEXANDER VON MURALT

It+ R U FIQ.lO.--Photochemical action of ultraviolet light on a perfusion fluid from vagal

-

stirnulation. R' = 1 cc. of a perfusion fluid from a vagal stimulation period, irradiated; R 1 cc. of a perfusion fluid from a vagal stimulation period, w m a t ; U = 1cc. of a perfusion fluid from an unatimulated heart. To all solutions equal amounts of acetylcholine were added.

A

B

TIM'E IN MINUTES Pro. 11.-Reeponse of blood pressure of the rabbit on vagus stimullttion before and after injection of thiamine. Ordinate: Relation of normal blood pressure to minimal blood pressure during vagus etimulation; abscissa: time. The arrow in experiment A indicates injection of 1mg. thiamine/kg. of weight; that in experiment B indicates injection of 2 mg. thiamine/kg. of weight. Kote inhibition of the effect of vagus stimulation after injection.

T H I A M I S E AND PERIPHERAL NEUROPHYSIOLOGY

113

that they made careful observations on the pulse rate at the moment of waking every morning. After having observed their normal average pulse rate for some time, they spent 36 hours without food. This fasting period produced in all subjects a more or less pronounced bradycardia. Fourteen days later the same experiment was performed after injection of thiamine and some subjects reacted with a less pronounced bradycardia on fasting, some without any change of the resting pulse rate, and one showed a small increase. The inhibiting effect of thiamine on the vagus or acetylcholine action in the heart is not specific: Table I V shows the extent to which thiamine derivatives are active if certain groups in the molecule are replaced by other groups. TABLE I V Inhibitory Effect of Thiamine Deriuatiues on the Action of Acetylcholine on the Heart

-

Pyrimidin 2

Thiazol

4

mr2 NHz

2 -

4 1

5

H H

ACtivity

t+S Thiamine chloride - (9 Cocarboxy1a.x

1 OH-

d 6,*

NHz NHz

O 'H H CHI CHzCIIzOCOCHa €1 CHa CHzCHzBr

KH* IVHz NIIz

H CH, CII2CONHz H CHs CHzCHzOH H ...

3-(4,5)-Methylimidaeolyl H CH3 CHzCH?OII 3-B- Acetoxyethyl H CH3 IT

CII ZCH 2013

0

++ + +0

."**--y;,

4,5

-

Substance

++ +

Acetylthiamine 5-8-Bromoethylestcr &Acetic acid amide 2-Ethylpyrinidil 4,bCyclopentenothiazoliumchloride

Acetoxyethylt hiazole . . . . Thiazole

It is most amazing that cocarboxylase has no action a t all. Gross (1946) found that the active water-soluble principle of adrenal cortex, (I percorten," has exactly the same action on the heart as thiamine. It is possible that tests on other substances will shorn them to have the same inhibitory action.

114

ALEXANDER VON MURALT

Before Loewi (1937) understood the exact nature of the chemical mediator in the heart, he called it “Vagusstoff .” Later it was proven to be acetylcholine. There is no doubt that on vagal stimulation a second substancc with inhibitory properties is liberated. This substance so far shows all the reactions of thiamine, but there is insufficient evidence to indicate whether it is thiamine or one of the possible thiamine compounds. As long as this uncertainty exists, Muralt (1945) proposed the substance be called “2. Vagusstoff.,’ It is worthwhile to mention that thiamine sensitizes the leech p r e p aration toward acetylcholine. It is not improbable that this is one of the reasons why the liberation of acetylcholine in synapses and motor nerve endings, elc., was SO easily detected with the leech preparation. This assumption includes the speculation that thiamine might be liberated not only a t the endings of the vagus, but a t the terminals of all cholinergic nerves. This far-reaching oonclusion remains to be proven by experiment.

v.

DIscussION

AND SUMMARY

The reader will have been surprised not to find any allusion to the enormous literature on vitamin deficiency effects in relation to the neurophysiological problem. I t is the conviction of the reviewer that such experiments cannot teach us more than the very obvious fact that thiamine is important for the mechanism of nerve function. Because it is of such importance, the nervous tissue is one of the last tissues to lose its thiamine content when a state of avitttminosis prevails. When it is losing its thiamine, the general disarrangement in the body is so far advanced that it is hard to distinguish between specific and general effects. There has been a tendency in recent years t o ascribe the nervous symptoms observed in avitaminosis t o starvation, rather than lack of thiamine. Starvation produces the same sort of peripheral degeneration as thiamine deficiency (Chamberlain el al., 1911). Engel and Phillips (1938) even went so far as to say that the nervous degeneration is due to lack of dietary factors other than thiamine! Swank and Prados (1942), however, have shown convincingly that there is a causative connection between the neurological lesions and thiamine deficiency. It is the impaired function which is the first sign, and only then degeneration begins in the nerves. It should be kept in mind, however, that lack of thiamine has a serious effect on the general metabolism of other tissues, and leads to the accumulation of pyruvic acid, and that many effects observed in avitaminosis must thus be ascribed to these changes. Lewy (1937) made chronaximetric determinations in cases of human avitaminosis and saw that the increased excitability (which corresponds to the effectsfound by Hutton-Rudolph (1944) on irradiating single nerve fibers

THIAMINE AND PERIPHERAL NEUROPHYSIOLOGY

115

of the frog) corresponded with the severity of the condition and that the improvement resulting from thiamine therapy could be followed directly by chronaximetric determinations. These indications are sufficient to prove that thiamine is important in nerve mctabolisrn. What is the function of thiamine in the mechanism of nervous excitation? Insight as to the possible action of thiamine is based on Peters’ (1940) classical experiments. From these we know that one mole of cocarboxylase is able to catalyze 1500 moles of oxygen/min. in the pyruvic acid cycle, and that cocarboxylase is a catalytic agent in a number of reactions in which pyruvic acid is the substrate (pyruvodehydrogenase, carboxylase). It is also essential in the resynthesis of carbohydrate from pyruvic acid and lactic acid (Barron and Lyman, 1940; 1941) and has, therefore, an important role in those reactions which in muscIe chemistry are known t o restore the energy spent in contraction. One must be careful in applying results which are valid in muscle chemistry to the peripheral nerve, but it is always helpful as long as we know so little about the chemistry of the nerve to reason from the known. Why thiamine is liberated in the normal excitation process and why it is accumulated at rest in monoiodoacetic acid-poisoned nerves and disappears on excitation, is a problem which needs further experimental work. It may be related to the reaction: adenosine triphosphate thiamine = adenylic acid cocarboxylase. It has been suggested that this mechanism is essential for the formation of acetylcholine (Muralt, 1943). The scheme which was visualized is the following: The breakdown of adenosine triphosphate activates thiamine to cocarboxylase, which catalyzes the anaerobic and aerobic decarboxylation of pyruvic acid, in the following two ways:

+

(1) anaerobic 2CHa.CO.COOH (2) aerobic 2CHa.CO.COOH

+

+ HzO = CHa.COOH + CHs.CHOH*COOH+ COI + = 2CH3.COOH + CO, 0 2

These reactions furnish the acetic acid necessary for the acetylation of choline, derived from the dephosphorylation of nerve phosphatides. Adenosine triphosphate andfcocarboxylase act as phosphate donor and acceptor, and are connected with the degradation of glucose, acting as energy transmitters. Nachmansohn (1945)) without knowledge of Muralt’s papers, because of the war, independently gave a scheme which is based on the same general idea and largely extended to the acetylcholine-phosphocreatinside, but did not include the thiamine-cocarboxylase-adenylicacid aspect. He mentions, however, that the acetyl portion of acetylcholine is derived

116

ALEXANDER VOX MUltALT

from pyruvic acid and that cocarboxylase plays an important role in the oxidation of pyruvic acid. Nachmansohn and Steinbach (1942) found that the vitamin, determined as diphosphothiamine, is concentrated a t the surface of the axoplasm and, therefore, is close t o the region where acetylcholine metabolism takes place. I t may be mentioned here that this quite independent and important approach, based on studies of cholinesterase in nerve, led Nachmansohn (1945) to propose the same concept of the role of acetycholine liberation along the axone and across the synapse, as was expressed by Muralt (1942) on the basis of his freezing experiments. Thus far thiamine and cocarboxylase have heen considered as catalysts. This view does not correspond t o the rather large daily requirement of thiamine in the body. Thiamine must also be considered as a metabolic substance. How thiamine or thiamine compounds could participate directly in the metabolism of the nerve, besides functioning as a catalyst, is a question which cannot easily be answercd on the basis of present knowledge. This review has given proof that thiamine plays an important role in neurophysiology, a role which can only be explained on the basis of further experimental work. REFERENCES 1. Agid, R.and Balkanyi, J. 1938. Compt. rend. soe. biol. 127, 680. 2. Atkin, L., Schultz, A. S., and Frey, C.N. 1939. J . Biol. Chem. 128, 471. 3. Rarron, E. 8.G., and Lyman, C. M. 1940. Science B2, 337. 4. Barron, E. 8.G., and Lyman, C.M. 1941. J . Biol. Chem. 141, 951, 957. 5. Bergami, G . 1936a. Boll. SOC. ital. biol. aper. 11, 275. 6. Bergami, G. 1936b. Arch. inat. biochim. ital. 2, 130. 7 . Bicknell, F.,and Prescott, F. 1946. The Vitamin8 in Medicine, 2nd ed., William Heinemann, London. 8. Binet, I,., and Minz, I3. 1934. Compt. rend. SOC. biol. 117, 1029. 9. Brown, G. L.,and Feldberg, W. 1936. J . Physiol. 88, 265. 10. Brtickc, F. v., and Sarkander, H. 1940. Arch. exptl. Path. Pharmakol. 196, 218. 11. Cdabro, Q. 1933. Riu. Biol. 16, 299. 12. Caclpcrmn, T. 1940s. J . Roy. Microscop. SOC.60, 8. 13. Caspersson, T. 1940b. Chronwsoma 1, 562. 14. Chamberlain, W.P. 1911. Cited by Bicknell and Preecott, 1946. 15. Dale, H. H. 1935. Rciztibertragung durch chemische Mittel im peripheren Nervensystcm. Urban and Schwarcenbcrg, Viennrr. 16. De Caro, I>.,and Butturini, 1,. 1940. Boll. SOC. itd. biol. sper. 16, 406. 17. Eijkman, C. 1897. Arch. path. Anat. (Virchotu’s) 148, 523; 14B, 187. 18. Engel, R. W., and Phillips, P. 11. 1938. J . Nutrition 16, 585. 19. Feldberg, W. 1943. J . Physiol. 101, 432. 20. Gaddum, J. H., Khayyal, M. A., and Rydin, H. 1937. J . Physiol. 89,9. 21. Grijna, G. 1901. Tijhchr. Ned. Znd. 41, 3. 22. Gram, F. 1946. Ezperientia 2, 191.

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