- Email: [email protected]
Reaction of catecholamines with hydroxylamine and its application to the assay of catechol O-methyl transferase
ANALYTICAL
Reaction Application
BIOCHEMISTRY
4fj8-475 (1969)
29,
of Catecholamines to the Assay
with
Hydroxylamine
of Catechol
O-Methyl
and
Its
Transferase
ATA A. ABDEL-LATIF Department
of Biochemistry,
Medical
College of Georgia, Augusta, Georgia 50909!
Received October 30, 1968
In the course of studies in our laboratory on the effect of catecholamines on the activity of acetylcholinesterase and Na+-K+ activated ATPase, it was observed that norepinephrine, epinephrine, and serotonin react with hydroxylamine at alkaline or acidic pH to give a colorless complex which is converted to a colored complex upon the addition of the ferric chloride reagent. Since acetylcholine is determined by the ferric-acethydroxamic acid complex (1) it was of interest to compare the stoichiometry and absorption spectra of the catecholamine complexes with that of acetylcholine. Further studies on the interaction of various catecholamines with the hydroxylamine reagent revealed the disappearance of the colored complex upon the methylation of the neurohormones. This prompted us to apply the latter observation to the assay of the enzyme catechol 0-met,hyl transferase calorimetrically. MATERIALS
Acetylcholine chloride; L-epinephrine, bitartrate; n-norepinephrine, bitartrate; serotonin; and DL-metanephrine hydrochloride were purchased from Sigma Chemical Company. Adenosylmethionine (AMe) was prepared according to the procedure of Stekol (2). Catechol Omethyl transferase was prepared from rat liver and purified according to the method of Axelrod and Tomchick (3). REAGENTS
Reagents were prepared as described by Hestrin 1. 2. 3. 4. 5. 6.
(1) :
2 M hydroxylamine hydrochloride. 3.5 N sodium hydroxide. 1 M phosphate buffer, pH 7.5. 4N hydrochloric acid. 0.37 M ferric chloride in 0.1 N hydrochloric acid. 0.01 M solutions of acetylcholine and catecholamines. 468
REACTION
OF
CATECHOLAMINES
WITH
469
HYDROXYAMINE
7. Alkaline hydroxylamine reagent is prepared mixing equal volumes of reagents 1 and 2.
just before use by
METHODS
Assay
of Catechol
O-Methyl
Transferase Calorimetrically
The O-methyl transferase assay is based upon the disappearance of the catecholamine as determined by the “ferric-hydroxylamine-catecholamine complex.” It consisted of: 2 ,umoles catecholamine; 100 pmoles phosphate buffer, pH 7.5 ; 12 pmoles magnesium chloride; 1.8 @moles AMe; and 0.35 mg protein of the enzyme preparation in a final volume of 1 ml. After 30 min of incubation at 37”, 2 ml alkaline hydroxylamine, 1 ml HCl, and 1 ml ferric chloride were added, respectively, with shaking after each addition. The denatured proteins were removed by centrifuging the tubes at 2500 rpm for 10 min. The supernatant was read in the Beckman DB-G spectrophotometer at 540 mp. Although hydroxylamine reacts similarly with CatechoIamines in the absence of NaOH and HCI, alkaIi.ne hydroxylamine was added routinely to the reaction mixture in order to denature the enzyme. RESULTS
Observations on CoEorsFormed between Hydroxylamine and Catecholamines
Except when specified, conditions for the hydroxylamine test were as described under “Methods.” For comparative studies acetylcholine, which is known to form a ferric-acethydroxamic acid complex in the hydroxylamine test (1) was included in the present work (Table 1). While serotonin, norepinephrine, TABLE 1 Results of Colors Formed with Different Compounds Compound
Acetylcholine Serotonin GNorepinephrine L-Epinephrine nL-Metanephrine L-Tyrosine L-Tryptophan
Color
in Hydroxylamine
Test
formed
Brown Brown Olive-green Olive green None None None
To 1 ml of the solution containing 4 &moles of the compound were added 2 ml alkaline hydroxylamine, 1 ml HCl, and 1 ml ferric chloride reagent. Colors observed are those of the soluble complexes. No precipitates are formed under the present experimental conditions.
470
A.
A.
ABDEL-LA!MF
and epinephrine gave characteristic colors in the hydroxylamine teat, tyrosine and tryptophan gave none, thus indicating that the reagents of the test react with the more reactive hydroxyl groups of the catecholamines. When a hydroxyl group of the catecholamine is blocked by methylation as in metanephrine (Table l), no color is formed between the latter and the hydroxylamine reagent. This observation suggested to us the possibility of assaying for O-methyl transferase colorimetrically (see below). To show which reagent reacts with the hydroxyl groups, different reagents were omitted from the hydroxylamine test (Table 2). No color was formed when hydroxylamine was omitted and, interestingly, this reagent reacted with the catecholamines in neutral medium as well as in basic or acidic medium (Table 2). The reaction was slowest in the acidic medium. Effect
of Omitting
TABLE Different Reagents from “L-Norepinephrine-Hydroxylamine
2 Hydroxylamine
Test
on Formation
OD at 540 mr After 10 min
Conditions
Complete system” - hydroxylamine -NaOH and HCI -NaOH -HCl
Brown
of
Complex” After
0.415 0.05.5 0.480 0.125 precipitate
2 hr
0.510 0.055 0.490 0.440 --
5 Conditions
for developing
Visible Light
the color
are as described
under
Spectra of the Colored
Table
1.
Complexes
The visible light spectra of the ferric complex of hydroxylamine derivatives of various neurohormones are shown in Figure 1. In general the serotonin, acetylcholine, and norepinephrine complexes were found to be stable with time and highest in concentration after 25, 10, and 30-60 min, respectively (Fig. 2). Results relating the neurohormone concentrations to color intensity are shown in Figure 3. The yield of the complex is shown to be a direct function of the neurohormone concentration (Fig. 3). Furthermore the color intensities of the complexes are independent of one another and are additive upon combining the various neurohormones (Table 3). Thus the presence of a catecholamine, for example, could interfere with the calorimetric assay of acetylcholinesterase (1). of Hydroxylamine Test to Catechol O-Methyl Transferuse Assay While Axelrod and Tomchick assayed for the methylated product of the catecholamines fluorometrically at 335 rnp after activation at 285 Application
REACTION
OF
CATECHOLAMINES
700
650 WAVE
WITH
600
550
LENGTH
471
HYDROXYLAMINE
500
450
(mu)
FIG. 1. Absorption curve of product of hydroxylamine reagents-neurohormone reaction obtained with 4 &moles of each neurohormone, read against water.
rnp (3), the present method is based on the disappearance of the catecholamine and the quantitative determination of the catecholaminehydroxylamine derivative calorimetrically. Upon methylation of the catecholamine by AMe in the presence of O-methyl transferase almost 5 pmole n-epinephrine/mg protein/hr is metabolized, and no reaction was observed in the absence of AMe (Table 4). TABLE 3 Effect of Serotonin and Norepinephrine on Calorimetric Determination Acetylcholine by Ferric-Acethyldroxamic Acid Complex Expt.
I II III IV, V VI VII
No.
Ac;t ylc~,$ne P
+ + + +
Serotonin (2 pmoles)
+ + + -I-
Norepinephrine (2 &moles)
+ + + +
O.D.
of
at 540 rnr
0.270 0.310 0.218 0.570 0.477 0.522 0.790
472
A. A. ABDEL-LATIF
Acetylcholine 0.6-
X-----X---+ Norepmephrme
E‘
0.5-
s LO z
0.4-
d 6
0.3-
0.2-
O.IControl Ott
l-t-i IO
0
20 TIME
FIG. 2. Effect neurohormones.
of time
I 40
30
on stability
I 50
I 60
(MIN)
of colored
complex
formed
by different
lSaratonin
/
i
0
I
2
CONCENTRATION
3 IN
4 or MOLES
FIQ. 3. Density of color as function of neurohormone
concentration.
REACTION
OF
CATECHOLAMINES
Ensymic O-Methyl&ion
WITH
TABLE 4 of Epinephrine L-Epinephrine
No. of expts.
n-Epinephrine : complete system AMe omitted n-Norepinephrine: complete system AMe omitted
and Norepinephrine
metabolized cJbmole)
4 2
0.86 f 0.05 0
5 2
-
473
HYDROXYLAMINE
L-Norepinephrine lized
metabo(~mole)
0.97 * 0.04 0
tiEpinephrine n-bit&rate or n-norepinephrine n-bitartrate, 2 rmoles, was incubated at 37” with purified liver O-methyl transferase (0.35 mg), 100 rmoles phosphate buffer at pH 7.5, 1.8 rmoles AMe, and 12 pmoles magnesium chloride, in a final volume of 1 ml. After incubation for 30 min, the reaction mixture was assayed for disappearance of the catecholamine calorimetrically (see under Table 1). DISCUSSION The reaction of catecholamines with hydroxylamine described here bears some similarity to the reaction of catechol derivatives with amines and amino acids in the presence of oxidizing agents observed several
years ago by Hackman and Todd (4). The hydroxyl group of serotonin and one or other of the 3,bdihydroxyl groups of norepinephrine and epinephrine could be oxidized in the presence of acid or base to a quinone (Fig. 4, II), followed by the condensation of the latter with hydroxylamine (Fig. 4, III), which in turn complexes with the ferric chloride reagent to give a characteristic color (Fig. 4, IV). The structure of the colored complex (Fig. 4, IV) is similar to that proposed by Soloway and Wilen for ferric chloride and salicylaldehyde (5). The fact that neither tryptophan nor tyrosine reacts with hydroxylamine indicates that the hydroxyl group of serotonin and the additional hydroxyl group on epinephrine and norepinephrine react with this reagent. This is supported by the fact that, when the additional hydroxyl groups on the latter neurohormones were methylated with AMe, no reaction was observed with the hydroxylamine reagent. This observation constituted the basis for the calorimetric assay of catechol O-methyl transferase described in the present paper. Although more sensitive fluorometric techniques are used to assay the concentrations of these neurohormones in tissue (6-S) as well as the formation of methylated products (3)) the calorimetric assay described for catechol O-methyl transferase in the present work is also simple, sensitive, reliable (Table 4) and the colored complex formed is rather stable (Fig. 2). The complexes formed with acetylcholine and serotonin are both brown in color (Table 1) and their spectra are similar (Fig. 1). Further studies on the chemical and bio-
474
A.
A.
ABDEL-LATIF
OH I CH-CH,NH, NH2 OH Ii+ OH (I) Norepinephrine
++
OH
Colored
FIG. 4. Possible stepwise reactions of norepinephrine
Complex
with hydroxylamine
reagents.
chemical properties of the hydroxyl groups of these neurohormones throw more light on their physiological function.
could
SUMMARY
Serotonin, norepinephrine, and epinephrine react rapidly with hydroxylamine in acidic or basic medium and form characteristic colored complexes upon addition of the ferric chloride reagent. These complexes were found to be stable with time, they absorb visible light maximally in the range X 520 to 540 rnp, where little or no absorption is shown with ferric chloride, and the optical density of their color is a function of the neurohormone concentration. A simple and sensitive assay is described for the enzyme catechol O-methyl transferase, based on disappearance of the colored complex upon methylation of the reactive hydroxyl group of the catecholamine. ACKNOWLEDGMENTS This work was supported by U. S. Public Health Service Research Grant NB 0787602. Thanks are due to Mr. John R. McRae for technical assistance, to Mrs. Julia F. Agee for the S-adenosylmethionine preparation, and to Dr. John C. Howard for fruitful discussions. REFERENCES S., J. Bid. Chem. 180, 249 (1949). J. A., in “Methods in Enzymology” (S. P. Colowick eds.), Vol. VI, p. 566. Academic Press, New York, 1963. 3. AXELROD, J., AND TOMCHICK, R., J. Biol. Chem. 233, 702 (1958). 4. HACKMAN, R. H.! AND TODD, A. R., B&hem. J. 55,631 (1953) 5. SOLOWAY, S., AND WILEN, S. H., Anal. Chem. 24, 979 (1952). 1. HESTRIN,
2. STEKOL,
and N. 0. Kaplan,
REACTION
6. UDENFRIEND,
S.,
OF
CATECHOLAMINES
WEISSBACH,
H., AND CLARK,
WITH
HYDROXYLAMINE
475
C. T., J. Biol. Chem. 215, 337 (1955).
(1955). 7. MAICKEL, R. P., Cox, R. H., SAILLANT, J., AND MILLER, F. P., Intern. pharmacol. 7, 275 (1968). 8. ANSELL, G. B., .~ND BEESON, M. F., Anal. Biockem. 23, 196 (1968).
J. Neuro-
Reaction Application
BIOCHEMISTRY
4fj8-475 (1969)
29,
of Catecholamines to the Assay
with
Hydroxylamine
of Catechol
O-Methyl
and
Its
Transferase
ATA A. ABDEL-LATIF Department
of Biochemistry,
Medical
College of Georgia, Augusta, Georgia 50909!
Received October 30, 1968
In the course of studies in our laboratory on the effect of catecholamines on the activity of acetylcholinesterase and Na+-K+ activated ATPase, it was observed that norepinephrine, epinephrine, and serotonin react with hydroxylamine at alkaline or acidic pH to give a colorless complex which is converted to a colored complex upon the addition of the ferric chloride reagent. Since acetylcholine is determined by the ferric-acethydroxamic acid complex (1) it was of interest to compare the stoichiometry and absorption spectra of the catecholamine complexes with that of acetylcholine. Further studies on the interaction of various catecholamines with the hydroxylamine reagent revealed the disappearance of the colored complex upon the methylation of the neurohormones. This prompted us to apply the latter observation to the assay of the enzyme catechol 0-met,hyl transferase calorimetrically. MATERIALS
Acetylcholine chloride; L-epinephrine, bitartrate; n-norepinephrine, bitartrate; serotonin; and DL-metanephrine hydrochloride were purchased from Sigma Chemical Company. Adenosylmethionine (AMe) was prepared according to the procedure of Stekol (2). Catechol Omethyl transferase was prepared from rat liver and purified according to the method of Axelrod and Tomchick (3). REAGENTS
Reagents were prepared as described by Hestrin 1. 2. 3. 4. 5. 6.
(1) :
2 M hydroxylamine hydrochloride. 3.5 N sodium hydroxide. 1 M phosphate buffer, pH 7.5. 4N hydrochloric acid. 0.37 M ferric chloride in 0.1 N hydrochloric acid. 0.01 M solutions of acetylcholine and catecholamines. 468
REACTION
OF
CATECHOLAMINES
WITH
469
HYDROXYAMINE
7. Alkaline hydroxylamine reagent is prepared mixing equal volumes of reagents 1 and 2.
just before use by
METHODS
Assay
of Catechol
O-Methyl
Transferase Calorimetrically
The O-methyl transferase assay is based upon the disappearance of the catecholamine as determined by the “ferric-hydroxylamine-catecholamine complex.” It consisted of: 2 ,umoles catecholamine; 100 pmoles phosphate buffer, pH 7.5 ; 12 pmoles magnesium chloride; 1.8 @moles AMe; and 0.35 mg protein of the enzyme preparation in a final volume of 1 ml. After 30 min of incubation at 37”, 2 ml alkaline hydroxylamine, 1 ml HCl, and 1 ml ferric chloride were added, respectively, with shaking after each addition. The denatured proteins were removed by centrifuging the tubes at 2500 rpm for 10 min. The supernatant was read in the Beckman DB-G spectrophotometer at 540 mp. Although hydroxylamine reacts similarly with CatechoIamines in the absence of NaOH and HCI, alkaIi.ne hydroxylamine was added routinely to the reaction mixture in order to denature the enzyme. RESULTS
Observations on CoEorsFormed between Hydroxylamine and Catecholamines
Except when specified, conditions for the hydroxylamine test were as described under “Methods.” For comparative studies acetylcholine, which is known to form a ferric-acethydroxamic acid complex in the hydroxylamine test (1) was included in the present work (Table 1). While serotonin, norepinephrine, TABLE 1 Results of Colors Formed with Different Compounds Compound
Acetylcholine Serotonin GNorepinephrine L-Epinephrine nL-Metanephrine L-Tyrosine L-Tryptophan
Color
in Hydroxylamine
Test
formed
Brown Brown Olive-green Olive green None None None
To 1 ml of the solution containing 4 &moles of the compound were added 2 ml alkaline hydroxylamine, 1 ml HCl, and 1 ml ferric chloride reagent. Colors observed are those of the soluble complexes. No precipitates are formed under the present experimental conditions.
470
A.
A.
ABDEL-LA!MF
and epinephrine gave characteristic colors in the hydroxylamine teat, tyrosine and tryptophan gave none, thus indicating that the reagents of the test react with the more reactive hydroxyl groups of the catecholamines. When a hydroxyl group of the catecholamine is blocked by methylation as in metanephrine (Table l), no color is formed between the latter and the hydroxylamine reagent. This observation suggested to us the possibility of assaying for O-methyl transferase colorimetrically (see below). To show which reagent reacts with the hydroxyl groups, different reagents were omitted from the hydroxylamine test (Table 2). No color was formed when hydroxylamine was omitted and, interestingly, this reagent reacted with the catecholamines in neutral medium as well as in basic or acidic medium (Table 2). The reaction was slowest in the acidic medium. Effect
of Omitting
TABLE Different Reagents from “L-Norepinephrine-Hydroxylamine
2 Hydroxylamine
Test
on Formation
OD at 540 mr After 10 min
Conditions
Complete system” - hydroxylamine -NaOH and HCI -NaOH -HCl
Brown
of
Complex” After
0.415 0.05.5 0.480 0.125 precipitate
2 hr
0.510 0.055 0.490 0.440 --
5 Conditions
for developing
Visible Light
the color
are as described
under
Spectra of the Colored
Table
1.
Complexes
The visible light spectra of the ferric complex of hydroxylamine derivatives of various neurohormones are shown in Figure 1. In general the serotonin, acetylcholine, and norepinephrine complexes were found to be stable with time and highest in concentration after 25, 10, and 30-60 min, respectively (Fig. 2). Results relating the neurohormone concentrations to color intensity are shown in Figure 3. The yield of the complex is shown to be a direct function of the neurohormone concentration (Fig. 3). Furthermore the color intensities of the complexes are independent of one another and are additive upon combining the various neurohormones (Table 3). Thus the presence of a catecholamine, for example, could interfere with the calorimetric assay of acetylcholinesterase (1). of Hydroxylamine Test to Catechol O-Methyl Transferuse Assay While Axelrod and Tomchick assayed for the methylated product of the catecholamines fluorometrically at 335 rnp after activation at 285 Application
REACTION
OF
CATECHOLAMINES
700
650 WAVE
WITH
600
550
LENGTH
471
HYDROXYLAMINE
500
450
(mu)
FIG. 1. Absorption curve of product of hydroxylamine reagents-neurohormone reaction obtained with 4 &moles of each neurohormone, read against water.
rnp (3), the present method is based on the disappearance of the catecholamine and the quantitative determination of the catecholaminehydroxylamine derivative calorimetrically. Upon methylation of the catecholamine by AMe in the presence of O-methyl transferase almost 5 pmole n-epinephrine/mg protein/hr is metabolized, and no reaction was observed in the absence of AMe (Table 4). TABLE 3 Effect of Serotonin and Norepinephrine on Calorimetric Determination Acetylcholine by Ferric-Acethyldroxamic Acid Complex Expt.
I II III IV, V VI VII
No.
Ac;t ylc~,$ne P
+ + + +
Serotonin (2 pmoles)
+ + + -I-
Norepinephrine (2 &moles)
+ + + +
O.D.
of
at 540 rnr
0.270 0.310 0.218 0.570 0.477 0.522 0.790
472
A. A. ABDEL-LATIF
Acetylcholine 0.6-
X-----X---+ Norepmephrme
E‘
0.5-
s LO z
0.4-
d 6
0.3-
0.2-
O.IControl Ott
l-t-i IO
0
20 TIME
FIG. 2. Effect neurohormones.
of time
I 40
30
on stability
I 50
I 60
(MIN)
of colored
complex
formed
by different
lSaratonin
/
i
0
I
2
CONCENTRATION
3 IN
4 or MOLES
FIQ. 3. Density of color as function of neurohormone
concentration.
REACTION
OF
CATECHOLAMINES
Ensymic O-Methyl&ion
WITH
TABLE 4 of Epinephrine L-Epinephrine
No. of expts.
n-Epinephrine : complete system AMe omitted n-Norepinephrine: complete system AMe omitted
and Norepinephrine
metabolized cJbmole)
4 2
0.86 f 0.05 0
5 2
-
473
HYDROXYLAMINE
L-Norepinephrine lized
metabo(~mole)
0.97 * 0.04 0
tiEpinephrine n-bit&rate or n-norepinephrine n-bitartrate, 2 rmoles, was incubated at 37” with purified liver O-methyl transferase (0.35 mg), 100 rmoles phosphate buffer at pH 7.5, 1.8 rmoles AMe, and 12 pmoles magnesium chloride, in a final volume of 1 ml. After incubation for 30 min, the reaction mixture was assayed for disappearance of the catecholamine calorimetrically (see under Table 1). DISCUSSION The reaction of catecholamines with hydroxylamine described here bears some similarity to the reaction of catechol derivatives with amines and amino acids in the presence of oxidizing agents observed several
years ago by Hackman and Todd (4). The hydroxyl group of serotonin and one or other of the 3,bdihydroxyl groups of norepinephrine and epinephrine could be oxidized in the presence of acid or base to a quinone (Fig. 4, II), followed by the condensation of the latter with hydroxylamine (Fig. 4, III), which in turn complexes with the ferric chloride reagent to give a characteristic color (Fig. 4, IV). The structure of the colored complex (Fig. 4, IV) is similar to that proposed by Soloway and Wilen for ferric chloride and salicylaldehyde (5). The fact that neither tryptophan nor tyrosine reacts with hydroxylamine indicates that the hydroxyl group of serotonin and the additional hydroxyl group on epinephrine and norepinephrine react with this reagent. This is supported by the fact that, when the additional hydroxyl groups on the latter neurohormones were methylated with AMe, no reaction was observed with the hydroxylamine reagent. This observation constituted the basis for the calorimetric assay of catechol O-methyl transferase described in the present paper. Although more sensitive fluorometric techniques are used to assay the concentrations of these neurohormones in tissue (6-S) as well as the formation of methylated products (3)) the calorimetric assay described for catechol O-methyl transferase in the present work is also simple, sensitive, reliable (Table 4) and the colored complex formed is rather stable (Fig. 2). The complexes formed with acetylcholine and serotonin are both brown in color (Table 1) and their spectra are similar (Fig. 1). Further studies on the chemical and bio-
474
A.
A.
ABDEL-LATIF
OH I CH-CH,NH, NH2 OH Ii+ OH (I) Norepinephrine
++
OH
Colored
FIG. 4. Possible stepwise reactions of norepinephrine
Complex
with hydroxylamine
reagents.
chemical properties of the hydroxyl groups of these neurohormones throw more light on their physiological function.
could
SUMMARY
Serotonin, norepinephrine, and epinephrine react rapidly with hydroxylamine in acidic or basic medium and form characteristic colored complexes upon addition of the ferric chloride reagent. These complexes were found to be stable with time, they absorb visible light maximally in the range X 520 to 540 rnp, where little or no absorption is shown with ferric chloride, and the optical density of their color is a function of the neurohormone concentration. A simple and sensitive assay is described for the enzyme catechol O-methyl transferase, based on disappearance of the colored complex upon methylation of the reactive hydroxyl group of the catecholamine. ACKNOWLEDGMENTS This work was supported by U. S. Public Health Service Research Grant NB 0787602. Thanks are due to Mr. John R. McRae for technical assistance, to Mrs. Julia F. Agee for the S-adenosylmethionine preparation, and to Dr. John C. Howard for fruitful discussions. REFERENCES S., J. Bid. Chem. 180, 249 (1949). J. A., in “Methods in Enzymology” (S. P. Colowick eds.), Vol. VI, p. 566. Academic Press, New York, 1963. 3. AXELROD, J., AND TOMCHICK, R., J. Biol. Chem. 233, 702 (1958). 4. HACKMAN, R. H.! AND TODD, A. R., B&hem. J. 55,631 (1953) 5. SOLOWAY, S., AND WILEN, S. H., Anal. Chem. 24, 979 (1952). 1. HESTRIN,
2. STEKOL,
and N. 0. Kaplan,
REACTION
6. UDENFRIEND,
S.,
OF
CATECHOLAMINES
WEISSBACH,
H., AND CLARK,
WITH
HYDROXYLAMINE
475
C. T., J. Biol. Chem. 215, 337 (1955).
(1955). 7. MAICKEL, R. P., Cox, R. H., SAILLANT, J., AND MILLER, F. P., Intern. pharmacol. 7, 275 (1968). 8. ANSELL, G. B., .~ND BEESON, M. F., Anal. Biockem. 23, 196 (1968).
J. Neuro-