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Oxidation of reduced nicotinamide adenine dinucleotide by melanin
Life Sciences Vol . 6 pp . 2605-2612, 1967 . Printed in Great Britain .
Pergamon Press Ltd.
OXIDATION OF REDUCED NICOTINAMIDE ADENINE DINQCLEOTID$
BY MEIANIN M . H . Van woert** Medical Division, Oak Ridge Associated Universities Oak Ridge, Tennessee
(Received 11 July 1967 ; in final form 9 October 1967) MELANIN has been demonstrated to be physiologically important because of its protective action against photon irradiation .
However, whether melanin has any
functional significance in non-cutaneous tissues such as the substantia nigra of the brain (1) and the melanocytes in various internal organs of auphibia (2) has not been investigated . Melanins are pigments of high molecular weight formed by the enzymatic ozidation of polyphenols (3) .
The polymer melanin contains stable semiquinone
monomer units which probably explains its paramagnetic properties .
Commoner,
et al . (4), using electron spin resonance spectroscopy, observed that natural and synthetic melanins are stable free radicals .
The concentration
of
these free
radicals or unpaired electrons increases upon irradiation with visible light (5) . Pullman and Pullman (6) have suggested that melanin can act as a one-dimensional semiconductor and theoretically it should have exceptional electron-acceptor properties .
Since free radicals can occur as intermediates in biological elec-
tron transfer processes, melanin could also participate as an electron acceptor in certain metabolic pathways . This paper reports a study of the ability of
melanin
to oxidize the coen-
zyme, reduced nicotinamide adenine dinucleotide (NADH), in vitro .
Numerous re-
actions catalyzed by dehydrogenases produce NADH and its predominant mode of Present Address ;
Departments of Medicine and Pharmacology, Yale University
School of Medicine . New Haven . Connecticu t, U .S .A . *This work was supported by the U . S . Atomic Energy Commission .
2605
OXIDATION OF NADH
2606
Vol. 6, No . 24
oxidation is by NADH dehydrogenase, which is a component of the electron transport chain of the mitochondrion.
This investigation showed that melanin can also
oxidize NADH and that this reaction may be light sensitive . Materials and Methods Melanin was prepared from 100 mg of L-dihydroxyphenlalanine (Dope) and 10 mg of purified mushroom tyrosinase according to a method described in an earlier paper,(1) .
Its similarity to natural melanins has been demonstrated by electron
paramagnetic resonance (EPR), infrared and ultraviolet spectroscopy (1,7) . The final preparation of melanin was boiled for 15 minutes to destroy any residual enzymatic activity . The oxidation of NADH was measured manometrically in a Warburg apparatus at 370C., with duplicate 15-ml flasks with side arms .
Each flask contained in the
main compartment 1 ml (1 mg) of aqueous melanin solution and 1.3 ml of buffer . The side arm contained 0.3 ml aqueous solution of NADH . 0.2 ml of 10% NaOH and the gas phase was 100% oxygen . for a blank without NADH .
The inner cup contained Readings were corrected
The upper half of the Warburg apparatus was covered
with a opaque hood so that the determination of the oxygen consumption could be made in the dark .
The effect of light was determined by exposing the flasks to
visible light from two 200-watt bulbs positioned 50 cm above the Warburg apparatus .
Mirrors were used to reflect the light up through the bottom of the War-
burg flasks . The pH was controlled with the following buffer solutions: acetate 0.15èî (pH 5 .0, 5 .5) ; phosphate 0 .15M (pH 6 .0, 6.5, 7 .0) ; Trio 0 .07514 (pH 7 .5, 8.0, 8.5) ; glycine NaOH . 0.075M (pH 9.0, 9 .5, 10 .0) .
Tyrosinase, NADH, alcohol dehy
drogenase, catalase, and L Dopa were purchased from the Nutritional Biochemicals Corporation .
Spectrophotometric measurements were made with the Cary model 14PH
double-beam recording spectrophotometer .
The Beckman - model G pH meter was used
for all pH measurements . Results Oxidation of NADH by Melanin
Vol .
OXIDATION OF NADH
6, No . 24 When
NADH
2607
was incubated with melanin the reduced coenzyme was rapidly
oxidized (FIG . 1) .
The reaction showed a linear relationship with time over
the first 60 minutes ; the rate of oxygen consumption was determined at two con FIG . 1
Z
FIG . 2
150
0
10
20
30 40
80
60
MINUTES
FIG. 1
Oxidation of 52 micromoles (upper 2 curves) and 13 micromoles (lower 2 curves) of NADH versus time in the presence and absence of visible light . Melanin - 1 mg, tris buffer 0 .75M pH 8 .7, Tamp . - 3700 .
FIG. 2
Effect of substrate concentration on rate of oxidation of melanin .
NADH
by
centrations of substrate (52 and 13 micromoles of RUM both in the dark and in the presence of visible light .
The oxygen uptake was consistently some
what higher when the reaction was exposed to light . was 69X of the rate at 370C . 0 .2 cc, 10%
Na0H
The oxygen uptake at 230C .
The reaction rate was identical with and without
in the center well ; this indicates that C02 was not produced .
Figure 2 shows that the rata of oxidation of
was directly propor-
NADH
tional to the concentration of the substrate in the range tested . subsequent experiments 13 micromoles of As shown in FIG . 3 the rate of tration of melanin .
NADH
NADH
In the
were used .
oxidation is dependent on the concen-
When'13 micromoles of
NADH
were incubated with increasing
concentrations of melanin, the rate of oxygen consumption showed a linear in-
OXIDATION OF NADH
2608
Vol . 6, No . 24
crease up to 1 mg of melanin. FIG. 3 200
Z
Ô i
50
.5 FIG . 3
1 .0
MELANIN (MG)
1.5
Effect of melanin concentration on rate of oxidation of 13 micromoles of NADH in trio buffer 0 .0754 pH 7 .6 . Each point is the mean of three determinations and the perpendicular line the range.
Properties of NADH Oxidation by Melanin Figure 4 shows that the optimal pH for the oxidation of NADH was 7 .2 .
How-
ever, a correction of the rates at the lower pH is necessary because of the in stability of NADH at pH values below neutrality .
The NADH stability was deter-
mined by measuring the decrease in U .V . absorption at 340 millimicrons in the Cary spectrophotometer under conditions identical with those used in the previously mentioned experiments .
The acid catalyzed decomposition of NADH was 17 .3%
at pH 5 .0, 5.6% at pH 6 .0, and no significant loss of NADH at pH 7.0 and above . Thus the true rate of oxygen consumption was slightly higher in the acid range than plotted in FIG. 4.
The slightly greater rate of oxidation in the presence
of visible light was again seen at all pH's except 9 .2 . The total oxygen uptake corresponded to 0 .5 mole of 02 per mole of NADH utilized .
This molar ratio of NADH oxidized to 02 consumed did not suggest the
formation of hydrogen peroxide .
Nevirtheless, catalase was added to the reac-
tion mixture to rule out the production of H202 .
Catalase (0 .3 mg/ml) reduced
Vol . 6, No . 24
2609
OXIDATION OF NADH FIG . 4
M
Z Q
J W i i W
K
W N i
pH FM . 4
Rate-of oxidation of NADH (13 micromoles) by melanin (1 mg) versus pH . Points and perpendicular lines are the mean of 4 to 7 determinations t SHi .
the oxygen uptake in the Warburg apparatus by 58 .4% at pH 7 .2 and 3700 .
At
the optimum temperature for catalass, 200 C ., the rate of oxygen consumption was reduced by 92 .6% when catalase was added to the NAM - melanin reaction mixture .
Therefore, hydrogen peroxide is most likely produced during the
oxidation . Identification of NAD+ formed from NAM during the reaction was determined by adding 0 .5 ml of 2 M ethanol and 1 .5 mg of alcohol dehydrogenase after the NADH was completely oxidized and the oxygen uptake had terminated .
In this ez-
Vol . 6, No . 24
OXIDATION OF NADH
2610
periment it was observed that the oxygen uptake resumed owing to the regeneration of NADH from NAD+ .
The oxidation of NADH by melanin was also observed in
the Cary double-beam spectrophotometer with the use of the same concentration of melanin in both the sample and blank cuvettes .
As the NADH optical density
at 340 millimicrons decreased, the absorption at 260 millimicrons increased, confirming the formation of NAD+ . The suggested equations for the oxidation of NADH by melanin are : NADH + Ff+ Melanin
-s NAD+
+
Melanin
Melanin .2H + 02-0-Melanin + H202
.2H
(1) (2)
The ratio of 0 .5 M 02/1M NADH may be due to the peroxidation of half the NADH by H202
(Eq . 3), or the partial decomposition of H202 during the reaction . NADH + H+ +
H202 - i NAD+ + 2H20
The autoxidation of reduced melanin (Eq . 2)
(3) is probably the rate-limiting
step, since the rate of oxygen uptake in atmospheric oxygen is 64% of the reaction rate in the presence of 100% oxygen . Heating the melanin for 15 minutes at 1000C . had no effect on the oxygen consumption,
indicating that this is a catalytic but not an enzymatic reaction . Discussion
It has been shown that melanin in the presence of oxygen promotes the nonenzymatic oxidation of NADH to NAD+ .
Hydrogen is transferred directly from the
dihydropyridine compound to melanin.
The reduced melanin is autoxidized by the
dissolved 02 producing hydrogen peroxide and reforming melanin .
The formation
of peroxide suggests that a two-electron reduction step of the melanin is likely . It is proposed that the oxidation of NADH is due to melanin's stable free radical property inherent in its conjugated semiquinone structure .
The slight
increase in oxidation in the light is compatible with this hypothesis .
This
melanin had been examined previously by electron spin resonance spectroscopy (1) and found to have approximately a 10% increase in concentration of free radicals after irradiation with visible light .
Still, we cannot neglect the possibility
Vol .
6,
No .
24
OXIDATION OF NADH
2611
that light may also have an effect on the susceptibility of NADH to oxidation . Other investigators have reported the oxidation of NADH by transitory free radicals produced in the presence of various oxidases .
Free radicals formed
from noradrenaline by ceruloplasmin oxidize WADE by acting as "one electron acceptor" agents
(8) .
Akazawa and Conn (9) reported that certain phenols in the
presence of peroxidases are'also able to oxidize NADH .
Unlike these unstable
free radicals, which require the presence of oxidases for their action, melanin is a stable free radical and is therefore persistently ractive . In the mammalian skin and eyes, melanin is synthesized by specialized cells known as meLanocytes .
Melanin pigment has also been found in other cells .
Lipofuscin, the brown pigment that accumulates in numerous tissues with aging, has recently been shown to contain a melanin component in addition to its lipid fraction (1) .
In man, the neurons of the substantia niera of the brain also
accumulate melanin with aging (10) . The physiologic significance of this oxidation of NADH by melanin is unknown.
It is conceivable, however, that due to non-mitochoodrial oxidative
pathways involving melanin,
the melanin-containing cells may have a WAD to NADH
ratio or oxidation-reduction state different from that of the nonpigmented cell . Summary Reduced nicotinamide adenine dinucleotide is rapidly oxidized in the presence of melanin.
One atom of oxygen is consumed per mole of coenzyme oxidized .
The reduced melanin is subsequently autoxidized producing melanin and H202 .
The
stable free radical property of melanins may serve a metabolic function in the melanocyte . References 1. 2.
M. H. VAN WOERT, R. N. PBASAD, and D . C. BORG, J . Neurochem. 14, 707, 1967 . mm M. H. VAN WOERT, A . R. NICHOISON, and G. C . COTZIAS, Comp . Biochem. Physiol. 22, 477, 1967 .
3.
H. S . MASON, In: Pi Academic Press (1959
t Cell Biology, M . Gordon, Ed ., p . 563, Wow York,
4.
B. COMMONER, J . TOWNSEND, and G. E. PARE, Nature 174, 689 (1954) .
2612
5.
OXIDATION OF NADH
Vol .
6,
No . 24
F. W. COPE, R. J. SEM, and B. D. POLIS, Arch . Bioehen. Biophys. 100, 171 (1963)
6,
A. PQLLiM and B. PMI«, Bioehen. Biophys. Acta 54, 384 (1964) .
7.
M. S . BLOIS, A. B . ZANIAN, and J. E. MALING, Biophys . J. 4, 471 (1964) .
8.
E. WAIAAS and 0 . WALLAB, Arch . Biochem. Biophys . 95, 151 (1961) .
9.
T. ARAZAWA and E . E . CONS, J. Biol . Chan . 232, 403 (1958) .
10 .
J . M. POLEY and D . BARTER, J . Neuropathol. Exp . Neurol . 17, 586 (1958) .
Pergamon Press Ltd.
OXIDATION OF REDUCED NICOTINAMIDE ADENINE DINQCLEOTID$
BY MEIANIN M . H . Van woert** Medical Division, Oak Ridge Associated Universities Oak Ridge, Tennessee
(Received 11 July 1967 ; in final form 9 October 1967) MELANIN has been demonstrated to be physiologically important because of its protective action against photon irradiation .
However, whether melanin has any
functional significance in non-cutaneous tissues such as the substantia nigra of the brain (1) and the melanocytes in various internal organs of auphibia (2) has not been investigated . Melanins are pigments of high molecular weight formed by the enzymatic ozidation of polyphenols (3) .
The polymer melanin contains stable semiquinone
monomer units which probably explains its paramagnetic properties .
Commoner,
et al . (4), using electron spin resonance spectroscopy, observed that natural and synthetic melanins are stable free radicals .
The concentration
of
these free
radicals or unpaired electrons increases upon irradiation with visible light (5) . Pullman and Pullman (6) have suggested that melanin can act as a one-dimensional semiconductor and theoretically it should have exceptional electron-acceptor properties .
Since free radicals can occur as intermediates in biological elec-
tron transfer processes, melanin could also participate as an electron acceptor in certain metabolic pathways . This paper reports a study of the ability of
melanin
to oxidize the coen-
zyme, reduced nicotinamide adenine dinucleotide (NADH), in vitro .
Numerous re-
actions catalyzed by dehydrogenases produce NADH and its predominant mode of Present Address ;
Departments of Medicine and Pharmacology, Yale University
School of Medicine . New Haven . Connecticu t, U .S .A . *This work was supported by the U . S . Atomic Energy Commission .
2605
OXIDATION OF NADH
2606
Vol. 6, No . 24
oxidation is by NADH dehydrogenase, which is a component of the electron transport chain of the mitochondrion.
This investigation showed that melanin can also
oxidize NADH and that this reaction may be light sensitive . Materials and Methods Melanin was prepared from 100 mg of L-dihydroxyphenlalanine (Dope) and 10 mg of purified mushroom tyrosinase according to a method described in an earlier paper,(1) .
Its similarity to natural melanins has been demonstrated by electron
paramagnetic resonance (EPR), infrared and ultraviolet spectroscopy (1,7) . The final preparation of melanin was boiled for 15 minutes to destroy any residual enzymatic activity . The oxidation of NADH was measured manometrically in a Warburg apparatus at 370C., with duplicate 15-ml flasks with side arms .
Each flask contained in the
main compartment 1 ml (1 mg) of aqueous melanin solution and 1.3 ml of buffer . The side arm contained 0.3 ml aqueous solution of NADH . 0.2 ml of 10% NaOH and the gas phase was 100% oxygen . for a blank without NADH .
The inner cup contained Readings were corrected
The upper half of the Warburg apparatus was covered
with a opaque hood so that the determination of the oxygen consumption could be made in the dark .
The effect of light was determined by exposing the flasks to
visible light from two 200-watt bulbs positioned 50 cm above the Warburg apparatus .
Mirrors were used to reflect the light up through the bottom of the War-
burg flasks . The pH was controlled with the following buffer solutions: acetate 0.15èî (pH 5 .0, 5 .5) ; phosphate 0 .15M (pH 6 .0, 6.5, 7 .0) ; Trio 0 .07514 (pH 7 .5, 8.0, 8.5) ; glycine NaOH . 0.075M (pH 9.0, 9 .5, 10 .0) .
Tyrosinase, NADH, alcohol dehy
drogenase, catalase, and L Dopa were purchased from the Nutritional Biochemicals Corporation .
Spectrophotometric measurements were made with the Cary model 14PH
double-beam recording spectrophotometer .
The Beckman - model G pH meter was used
for all pH measurements . Results Oxidation of NADH by Melanin
Vol .
OXIDATION OF NADH
6, No . 24 When
NADH
2607
was incubated with melanin the reduced coenzyme was rapidly
oxidized (FIG . 1) .
The reaction showed a linear relationship with time over
the first 60 minutes ; the rate of oxygen consumption was determined at two con FIG . 1
Z
FIG . 2
150
0
10
20
30 40
80
60
MINUTES
FIG. 1
Oxidation of 52 micromoles (upper 2 curves) and 13 micromoles (lower 2 curves) of NADH versus time in the presence and absence of visible light . Melanin - 1 mg, tris buffer 0 .75M pH 8 .7, Tamp . - 3700 .
FIG. 2
Effect of substrate concentration on rate of oxidation of melanin .
NADH
by
centrations of substrate (52 and 13 micromoles of RUM both in the dark and in the presence of visible light .
The oxygen uptake was consistently some
what higher when the reaction was exposed to light . was 69X of the rate at 370C . 0 .2 cc, 10%
Na0H
The oxygen uptake at 230C .
The reaction rate was identical with and without
in the center well ; this indicates that C02 was not produced .
Figure 2 shows that the rata of oxidation of
was directly propor-
NADH
tional to the concentration of the substrate in the range tested . subsequent experiments 13 micromoles of As shown in FIG . 3 the rate of tration of melanin .
NADH
NADH
In the
were used .
oxidation is dependent on the concen-
When'13 micromoles of
NADH
were incubated with increasing
concentrations of melanin, the rate of oxygen consumption showed a linear in-
OXIDATION OF NADH
2608
Vol . 6, No . 24
crease up to 1 mg of melanin. FIG. 3 200
Z
Ô i
50
.5 FIG . 3
1 .0
MELANIN (MG)
1.5
Effect of melanin concentration on rate of oxidation of 13 micromoles of NADH in trio buffer 0 .0754 pH 7 .6 . Each point is the mean of three determinations and the perpendicular line the range.
Properties of NADH Oxidation by Melanin Figure 4 shows that the optimal pH for the oxidation of NADH was 7 .2 .
How-
ever, a correction of the rates at the lower pH is necessary because of the in stability of NADH at pH values below neutrality .
The NADH stability was deter-
mined by measuring the decrease in U .V . absorption at 340 millimicrons in the Cary spectrophotometer under conditions identical with those used in the previously mentioned experiments .
The acid catalyzed decomposition of NADH was 17 .3%
at pH 5 .0, 5.6% at pH 6 .0, and no significant loss of NADH at pH 7.0 and above . Thus the true rate of oxygen consumption was slightly higher in the acid range than plotted in FIG. 4.
The slightly greater rate of oxidation in the presence
of visible light was again seen at all pH's except 9 .2 . The total oxygen uptake corresponded to 0 .5 mole of 02 per mole of NADH utilized .
This molar ratio of NADH oxidized to 02 consumed did not suggest the
formation of hydrogen peroxide .
Nevirtheless, catalase was added to the reac-
tion mixture to rule out the production of H202 .
Catalase (0 .3 mg/ml) reduced
Vol . 6, No . 24
2609
OXIDATION OF NADH FIG . 4
M
Z Q
J W i i W
K
W N i
pH FM . 4
Rate-of oxidation of NADH (13 micromoles) by melanin (1 mg) versus pH . Points and perpendicular lines are the mean of 4 to 7 determinations t SHi .
the oxygen uptake in the Warburg apparatus by 58 .4% at pH 7 .2 and 3700 .
At
the optimum temperature for catalass, 200 C ., the rate of oxygen consumption was reduced by 92 .6% when catalase was added to the NAM - melanin reaction mixture .
Therefore, hydrogen peroxide is most likely produced during the
oxidation . Identification of NAD+ formed from NAM during the reaction was determined by adding 0 .5 ml of 2 M ethanol and 1 .5 mg of alcohol dehydrogenase after the NADH was completely oxidized and the oxygen uptake had terminated .
In this ez-
Vol . 6, No . 24
OXIDATION OF NADH
2610
periment it was observed that the oxygen uptake resumed owing to the regeneration of NADH from NAD+ .
The oxidation of NADH by melanin was also observed in
the Cary double-beam spectrophotometer with the use of the same concentration of melanin in both the sample and blank cuvettes .
As the NADH optical density
at 340 millimicrons decreased, the absorption at 260 millimicrons increased, confirming the formation of NAD+ . The suggested equations for the oxidation of NADH by melanin are : NADH + Ff+ Melanin
-s NAD+
+
Melanin
Melanin .2H + 02-0-Melanin + H202
.2H
(1) (2)
The ratio of 0 .5 M 02/1M NADH may be due to the peroxidation of half the NADH by H202
(Eq . 3), or the partial decomposition of H202 during the reaction . NADH + H+ +
H202 - i NAD+ + 2H20
The autoxidation of reduced melanin (Eq . 2)
(3) is probably the rate-limiting
step, since the rate of oxygen uptake in atmospheric oxygen is 64% of the reaction rate in the presence of 100% oxygen . Heating the melanin for 15 minutes at 1000C . had no effect on the oxygen consumption,
indicating that this is a catalytic but not an enzymatic reaction . Discussion
It has been shown that melanin in the presence of oxygen promotes the nonenzymatic oxidation of NADH to NAD+ .
Hydrogen is transferred directly from the
dihydropyridine compound to melanin.
The reduced melanin is autoxidized by the
dissolved 02 producing hydrogen peroxide and reforming melanin .
The formation
of peroxide suggests that a two-electron reduction step of the melanin is likely . It is proposed that the oxidation of NADH is due to melanin's stable free radical property inherent in its conjugated semiquinone structure .
The slight
increase in oxidation in the light is compatible with this hypothesis .
This
melanin had been examined previously by electron spin resonance spectroscopy (1) and found to have approximately a 10% increase in concentration of free radicals after irradiation with visible light .
Still, we cannot neglect the possibility
Vol .
6,
No .
24
OXIDATION OF NADH
2611
that light may also have an effect on the susceptibility of NADH to oxidation . Other investigators have reported the oxidation of NADH by transitory free radicals produced in the presence of various oxidases .
Free radicals formed
from noradrenaline by ceruloplasmin oxidize WADE by acting as "one electron acceptor" agents
(8) .
Akazawa and Conn (9) reported that certain phenols in the
presence of peroxidases are'also able to oxidize NADH .
Unlike these unstable
free radicals, which require the presence of oxidases for their action, melanin is a stable free radical and is therefore persistently ractive . In the mammalian skin and eyes, melanin is synthesized by specialized cells known as meLanocytes .
Melanin pigment has also been found in other cells .
Lipofuscin, the brown pigment that accumulates in numerous tissues with aging, has recently been shown to contain a melanin component in addition to its lipid fraction (1) .
In man, the neurons of the substantia niera of the brain also
accumulate melanin with aging (10) . The physiologic significance of this oxidation of NADH by melanin is unknown.
It is conceivable, however, that due to non-mitochoodrial oxidative
pathways involving melanin,
the melanin-containing cells may have a WAD to NADH
ratio or oxidation-reduction state different from that of the nonpigmented cell . Summary Reduced nicotinamide adenine dinucleotide is rapidly oxidized in the presence of melanin.
One atom of oxygen is consumed per mole of coenzyme oxidized .
The reduced melanin is subsequently autoxidized producing melanin and H202 .
The
stable free radical property of melanins may serve a metabolic function in the melanocyte . References 1. 2.
M. H. VAN WOERT, R. N. PBASAD, and D . C. BORG, J . Neurochem. 14, 707, 1967 . mm M. H. VAN WOERT, A . R. NICHOISON, and G. C . COTZIAS, Comp . Biochem. Physiol. 22, 477, 1967 .
3.
H. S . MASON, In: Pi Academic Press (1959
t Cell Biology, M . Gordon, Ed ., p . 563, Wow York,
4.
B. COMMONER, J . TOWNSEND, and G. E. PARE, Nature 174, 689 (1954) .
2612
5.
OXIDATION OF NADH
Vol .
6,
No . 24
F. W. COPE, R. J. SEM, and B. D. POLIS, Arch . Bioehen. Biophys. 100, 171 (1963)
6,
A. PQLLiM and B. PMI«, Bioehen. Biophys. Acta 54, 384 (1964) .
7.
M. S . BLOIS, A. B . ZANIAN, and J. E. MALING, Biophys . J. 4, 471 (1964) .
8.
E. WAIAAS and 0 . WALLAB, Arch . Biochem. Biophys . 95, 151 (1961) .
9.
T. ARAZAWA and E . E . CONS, J. Biol . Chan . 232, 403 (1958) .
10 .
J . M. POLEY and D . BARTER, J . Neuropathol. Exp . Neurol . 17, 586 (1958) .