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Physiologically active amines in common fruits and vegetables
ARCHIVES
OF
BIOCHEMISTRY
AND
86, 487-490
BIOPHYSICS
(19%)
Physiologically Active Amines in Common Fruits and Vegetables Sidney Udenfriend, Walter Lovenberg’ and Albert Sjoerdsma From the Laboratory of Clinical Biochemistry and the Section on Experimental I’herapeutics, National Heart Institute, National Institutes of Health, Public Health Service, United States Department of Health, Education, and Welfare, Bethesda, Maryland Received
June 19, 1959
Following the demonstration of 5-hydroxytryptamine (serotonin), 3,4dihydroxyphenylethylamine (dopamine), and norepinephrine in bananas (l), it became of interest to determine just how widespread was the distribution of these physiologically active arylalkylamines among the edible plants. Studies were therefore extended to many of the more common fruits and vegetables. While these studies were under way, West (2), in a communication in which he corroborated the findings in the banana, also reported the presence of serotonin and tryptamine in the tomato. As will be shown below, not only is this true for the tomato, but several amine metabolites of the aromatic amino acids are found in varying amounts in a large number of edible plants. EXPERIMENTAL Generous supplies of plantains and bananas were made available by the United Fruit Company. All other fresh fruits and vegetables were obtained from the local markets and homogenized in 2 vol. of 0.1 N HCl using a mechanical blendor. The homogenates were then centrifuged to remove debris, and the various amines were isolated, identified, and assayed as described below.
5-Hydroxytryptamine Aliquots of the above supernatant solutions were adjusted to pH 10 and extracted and assayed for serotonin according to the procedure of Udenfriend et al. (3). The serotonin in the extracts was identified by its characteristic fluorescence properties, 295.rnr activation and 540.rnp fluorescence. In the experiments with the tomato, banana, and plantain, the isolated serotonin was also identified by chromatography on Whatman 11 paper using n-butanol-acetic acid-water (12:3:5) (ascending) and spraying with p-dimethylaminobenxaldehyde; RI = 0.56. 1 Fellowship
supported
by the United
Fruit 487
Company.
488
UDENFRIEND,
LOVENBERG
AND SJOERDSMA
Tryptamine Aliquots of the supernatant solutions were adjusted to pH 12-13 and extracted with benzene. After twice washing with 0.1 N NaOH, the tryptamine was returned to a small volume of 0.1 N HCl and adjusted to pH 7. The tryptamine in the extracts was assayed and identified in the spectrophotofluorometer, activation maximum 280 mp and fluorescence maximum 360 mp (4). In the experiments with the tomato, plum, and egg plant, the tryptamine was also identified by chromatography on paper, using the system as described for serotonin; R/ = 0.77.
Tyramine Tyramine was extracted from the supernatant solutions with ether according to the procedure of Mitoma et al. (5). The extract thus obtained was then treated with or-nitroso-@-naphthol and measured fluorometrically as described by Waalkes and Udenfriend (6). The tyramine was identified by chromatography on paper using n-butanol-acetic acid-water (12:3:5). After spraying with a-nitroso+3-naphthol (0.1% in alcohol) followed by 3 N HNOI containing 0.05% NaNOz , the tyramine appeared as a red spot (Rf 0.65) which upon heating became highly fluorescent (yellow) when illuminated with a Woods lamp (360 mp). The tyramine was also identified by the characteristic fluorescence of its cu-nitroso-@-naphthol derivative, activation 460 mp and fluorescence 570 mp.
Catecholamines The supernatant solutions were made 5% with respect to trichloroacetic acid and centrifuged to remove proteins, and the deproteinized solutions were extracted twice with ether to remove organic acids. Catecholamines were then adsorbed on alumina in a manner similar to that described by von Euler and Floding (7) and eluted with 0.2 N acetic acid. Aliquots of the eluates were assayed for norepinephrine and epinephrine as described by Udenfriend and Wyngaarden (8). An aliquot of the eluate was also assayed for dopamine by the highly specific method of Carlsson and Waldeck (9). The catecholamines were also identified by procedures described previously (1). RESULTS The
data
presented
large number
in Table
of ordinary
AND
DISCUSSION
I summarize
edible
plants.
are more widespread
in the plant kingdom
be emphasized
each
samples
and
variability age
that that
there
of the values is great
must be due to many
conditions,
use of different
etc. Of all the fruits
studied,
amounts
of these substances.
is unique
in that it contains
compared
It would
or toxic plants. many
varieties
important
that amino
The
plant
of amines
applications
inasmuch
sible for some types
of browning
and
the average
It must
of several
from
sample
to sample.
This
degree
of ripening,
stor-
of plants,
conditions
of growth,
obviously
contained
the largest
the skin of the banana
of the amines materials in fruits
which
thus far.
is not limited
and vegetables
as these substances discoloration
which
are enormous
investigated
acid decarboxylation
finding
on a
including
It is, however,
to all the other edible appear
variability
the banana
of analyses
that arylalkylamines
than has been imagined. represents
factors
quantities
the results
It is apparent
to exotic may
have
may be respon-
of fruits.
Preliminary
AMINES
IN
FRUITS
AND
TABLE Amine
489
VEGETABLES
I
of Fruits and Vegetables
Content
Whole fruit was homogenized unless otherwise indicated. Serotonin
Tryptamine
Tyramine
Dopamine
Norepinephrine
M./P.
M./g.
Pg./g.
pg./g.
Pg./P.
5&150 28 45 12 10 8 0 10 0 0 0 0
0 0
65 7
700 8
4 6
0 0
122 2 0 + 0 0.1-2.0 0 0 +
Banana (peel) Banana (pulp) Plantain (pulp) Tomato Red plum Blue-red plum Blue plum Avocado Potato Spinach Grape Orange (pulp) Eggplant
2
studies have indicated
4 o-2 2 5 0 0 0 0 0.1
23 1 1 0 10
4-5 0 0 0 0
0.5-3.0
3
0
0
that some of the inhibitors
of amine oxidation, now so widely used in medicine, can prevent the browning of banana skin. Fur-
ther studies may help elucidate exactly how important amine metabolism is in the discoloration of edible plants. An obvious consideration is whether ingestion of these potent pharmacologic agents can in any way produce toxic manifestations in man. Apserotonin and parently this is not so. Large amounts of norepinephrine, tryptamine can be ingested without producing any ill effects. Metabolism is so rapid during absorption that when as much as 60 mg. serotonin was administered orally to humans it did not cause a detectable increase in the blood level nor produce any detectable effects on blood pressure, heart rate, etc. In rats it was necessary to administer 10 mg./kg. serotonin to obtain detectable blood levels of the amine. Apparently the major factor to consider with respect to amines in the diet is their contribution to urinary TABLE Serotonin
Content oj Bananas
II as a Function
of Ripening
Individual fruits from a single bunch were taken at each stage of ripening. The values represent the averages of two separate experiments. Condition of fruit
Hard Green Ripe Over Ripe
Serotonin content, pg./g. outer peel Inner peel
74
96 161
13 38
170
Pulp
24 36 35
490
UDENFRIEND,
LOVENBERG AND SJOERDSMA
amines and their metabolites when these are of importance for diagnostic purposes. The serotonin and norepinephrine in bananas may be sufficient to cause erroneous diagnosis of either carcinoid or pheochromocytoma (10). However, one would have to eat enormous amounts of most other fruits and vegetables to produce significant effects on urinary excretion of amines. Of course, the introduction of inhibitors of amine metabolism as therapeutic agents may make it necessary to re-examine this problem. It is possible that some precursors of amines may be present in amounts larger than the amines themselves. This may be the case during ripening. The serotonin content of bananas was measured during the ripening process. As shown in Table II, ripening produces a large increase in the serotonin content of the peel. Somewhat similar results have been reported by West (2). The increase in serotonin content with ripening suggested that enzymes involved in its formation were operative during the ripening process. In animal tissues serotonin formation involves the hydroxylation of tryptophan to 5-hydroxytryptophan which is then decarboxylated to 5-hydroxytryptamine. It was expected, therefore, that an active 5-hydroxytryptophan decarboxylase and a large depot of 5-hydroxytryptophan would be present in the green banana. However, preliminary experimentation has failed to demonstrate either of these. It will be of interest to study the mechanisms involved in amine formation and metabolism in these plants and to compare them with the systems found in animal tissues. SUMMARY
5-Hydroxytryptamine, tryptamine, tyramine, 3,4-dihydroxyphenylethylamine and norepinephrine have been identified and assayed in a large number of fruits and vegetables. The significance of these findings with respect to plant physiology and to dietary effects in animals is discussed. REFERENCES 1.
WAALKEG, T. P., SJOERDSMA, A., CREVELING, C. R., WEISSBACH, FRIEND, S., ~%ience 137, 648 (19%). 2. WEST, G. B., J. Pharm. and Pharmacol. 10, 589 (1958). 3. UDENFRIEND, S., WEISSBACH, H., AND CLARK, C. T., J. Biol.
H., AND UDEN-
Chem. 216, 337
(1955). 4. SJOERDSMA, A., OATES, J., ZALTZMAN, P., AND UDENFRIEND, S., J. Pharmacol. Exptl. Therap. (in press). 5. MITOMA, C., POSNER, H. S., BOGDANSKI, D. F. AND UDENFRIEND, S., J. Pharmacol. Exptl. Therap. 120, 188 (1957). 6. WAALKES, T. P., AND UDENFRIEND, S., J. Lab. Clin. Med. 60, 733 (1957). 7. EULER, U. S. VON, AND FLODING, I., Stand. J. Clin. & Lab. Invest. 8, 288 (1956). 8. UDENFRIEND, S., AND WYNGAARDEN, J. B., Biochim. et Biophys. Acta 20,48 (1956). 9. CARLSSON, A., AND WALDECK, B., Acta Physiol. Stand. 44, 293 (1958). 10. CROUT, J. R., AND SJOERDSMA, A., New Engl. J. Med. 261, 23 (1959).
OF
BIOCHEMISTRY
AND
86, 487-490
BIOPHYSICS
(19%)
Physiologically Active Amines in Common Fruits and Vegetables Sidney Udenfriend, Walter Lovenberg’ and Albert Sjoerdsma From the Laboratory of Clinical Biochemistry and the Section on Experimental I’herapeutics, National Heart Institute, National Institutes of Health, Public Health Service, United States Department of Health, Education, and Welfare, Bethesda, Maryland Received
June 19, 1959
Following the demonstration of 5-hydroxytryptamine (serotonin), 3,4dihydroxyphenylethylamine (dopamine), and norepinephrine in bananas (l), it became of interest to determine just how widespread was the distribution of these physiologically active arylalkylamines among the edible plants. Studies were therefore extended to many of the more common fruits and vegetables. While these studies were under way, West (2), in a communication in which he corroborated the findings in the banana, also reported the presence of serotonin and tryptamine in the tomato. As will be shown below, not only is this true for the tomato, but several amine metabolites of the aromatic amino acids are found in varying amounts in a large number of edible plants. EXPERIMENTAL Generous supplies of plantains and bananas were made available by the United Fruit Company. All other fresh fruits and vegetables were obtained from the local markets and homogenized in 2 vol. of 0.1 N HCl using a mechanical blendor. The homogenates were then centrifuged to remove debris, and the various amines were isolated, identified, and assayed as described below.
5-Hydroxytryptamine Aliquots of the above supernatant solutions were adjusted to pH 10 and extracted and assayed for serotonin according to the procedure of Udenfriend et al. (3). The serotonin in the extracts was identified by its characteristic fluorescence properties, 295.rnr activation and 540.rnp fluorescence. In the experiments with the tomato, banana, and plantain, the isolated serotonin was also identified by chromatography on Whatman 11 paper using n-butanol-acetic acid-water (12:3:5) (ascending) and spraying with p-dimethylaminobenxaldehyde; RI = 0.56. 1 Fellowship
supported
by the United
Fruit 487
Company.
488
UDENFRIEND,
LOVENBERG
AND SJOERDSMA
Tryptamine Aliquots of the supernatant solutions were adjusted to pH 12-13 and extracted with benzene. After twice washing with 0.1 N NaOH, the tryptamine was returned to a small volume of 0.1 N HCl and adjusted to pH 7. The tryptamine in the extracts was assayed and identified in the spectrophotofluorometer, activation maximum 280 mp and fluorescence maximum 360 mp (4). In the experiments with the tomato, plum, and egg plant, the tryptamine was also identified by chromatography on paper, using the system as described for serotonin; R/ = 0.77.
Tyramine Tyramine was extracted from the supernatant solutions with ether according to the procedure of Mitoma et al. (5). The extract thus obtained was then treated with or-nitroso-@-naphthol and measured fluorometrically as described by Waalkes and Udenfriend (6). The tyramine was identified by chromatography on paper using n-butanol-acetic acid-water (12:3:5). After spraying with a-nitroso+3-naphthol (0.1% in alcohol) followed by 3 N HNOI containing 0.05% NaNOz , the tyramine appeared as a red spot (Rf 0.65) which upon heating became highly fluorescent (yellow) when illuminated with a Woods lamp (360 mp). The tyramine was also identified by the characteristic fluorescence of its cu-nitroso-@-naphthol derivative, activation 460 mp and fluorescence 570 mp.
Catecholamines The supernatant solutions were made 5% with respect to trichloroacetic acid and centrifuged to remove proteins, and the deproteinized solutions were extracted twice with ether to remove organic acids. Catecholamines were then adsorbed on alumina in a manner similar to that described by von Euler and Floding (7) and eluted with 0.2 N acetic acid. Aliquots of the eluates were assayed for norepinephrine and epinephrine as described by Udenfriend and Wyngaarden (8). An aliquot of the eluate was also assayed for dopamine by the highly specific method of Carlsson and Waldeck (9). The catecholamines were also identified by procedures described previously (1). RESULTS The
data
presented
large number
in Table
of ordinary
AND
DISCUSSION
I summarize
edible
plants.
are more widespread
in the plant kingdom
be emphasized
each
samples
and
variability age
that that
there
of the values is great
must be due to many
conditions,
use of different
etc. Of all the fruits
studied,
amounts
of these substances.
is unique
in that it contains
compared
It would
or toxic plants. many
varieties
important
that amino
The
plant
of amines
applications
inasmuch
sible for some types
of browning
and
the average
It must
of several
from
sample
to sample.
This
degree
of ripening,
stor-
of plants,
conditions
of growth,
obviously
contained
the largest
the skin of the banana
of the amines materials in fruits
which
thus far.
is not limited
and vegetables
as these substances discoloration
which
are enormous
investigated
acid decarboxylation
finding
on a
including
It is, however,
to all the other edible appear
variability
the banana
of analyses
that arylalkylamines
than has been imagined. represents
factors
quantities
the results
It is apparent
to exotic may
have
may be respon-
of fruits.
Preliminary
AMINES
IN
FRUITS
AND
TABLE Amine
489
VEGETABLES
I
of Fruits and Vegetables
Content
Whole fruit was homogenized unless otherwise indicated. Serotonin
Tryptamine
Tyramine
Dopamine
Norepinephrine
M./P.
M./g.
Pg./g.
pg./g.
Pg./P.
5&150 28 45 12 10 8 0 10 0 0 0 0
0 0
65 7
700 8
4 6
0 0
122 2 0 + 0 0.1-2.0 0 0 +
Banana (peel) Banana (pulp) Plantain (pulp) Tomato Red plum Blue-red plum Blue plum Avocado Potato Spinach Grape Orange (pulp) Eggplant
2
studies have indicated
4 o-2 2 5 0 0 0 0 0.1
23 1 1 0 10
4-5 0 0 0 0
0.5-3.0
3
0
0
that some of the inhibitors
of amine oxidation, now so widely used in medicine, can prevent the browning of banana skin. Fur-
ther studies may help elucidate exactly how important amine metabolism is in the discoloration of edible plants. An obvious consideration is whether ingestion of these potent pharmacologic agents can in any way produce toxic manifestations in man. Apserotonin and parently this is not so. Large amounts of norepinephrine, tryptamine can be ingested without producing any ill effects. Metabolism is so rapid during absorption that when as much as 60 mg. serotonin was administered orally to humans it did not cause a detectable increase in the blood level nor produce any detectable effects on blood pressure, heart rate, etc. In rats it was necessary to administer 10 mg./kg. serotonin to obtain detectable blood levels of the amine. Apparently the major factor to consider with respect to amines in the diet is their contribution to urinary TABLE Serotonin
Content oj Bananas
II as a Function
of Ripening
Individual fruits from a single bunch were taken at each stage of ripening. The values represent the averages of two separate experiments. Condition of fruit
Hard Green Ripe Over Ripe
Serotonin content, pg./g. outer peel Inner peel
74
96 161
13 38
170
Pulp
24 36 35
490
UDENFRIEND,
LOVENBERG AND SJOERDSMA
amines and their metabolites when these are of importance for diagnostic purposes. The serotonin and norepinephrine in bananas may be sufficient to cause erroneous diagnosis of either carcinoid or pheochromocytoma (10). However, one would have to eat enormous amounts of most other fruits and vegetables to produce significant effects on urinary excretion of amines. Of course, the introduction of inhibitors of amine metabolism as therapeutic agents may make it necessary to re-examine this problem. It is possible that some precursors of amines may be present in amounts larger than the amines themselves. This may be the case during ripening. The serotonin content of bananas was measured during the ripening process. As shown in Table II, ripening produces a large increase in the serotonin content of the peel. Somewhat similar results have been reported by West (2). The increase in serotonin content with ripening suggested that enzymes involved in its formation were operative during the ripening process. In animal tissues serotonin formation involves the hydroxylation of tryptophan to 5-hydroxytryptophan which is then decarboxylated to 5-hydroxytryptamine. It was expected, therefore, that an active 5-hydroxytryptophan decarboxylase and a large depot of 5-hydroxytryptophan would be present in the green banana. However, preliminary experimentation has failed to demonstrate either of these. It will be of interest to study the mechanisms involved in amine formation and metabolism in these plants and to compare them with the systems found in animal tissues. SUMMARY
5-Hydroxytryptamine, tryptamine, tyramine, 3,4-dihydroxyphenylethylamine and norepinephrine have been identified and assayed in a large number of fruits and vegetables. The significance of these findings with respect to plant physiology and to dietary effects in animals is discussed. REFERENCES 1.
WAALKEG, T. P., SJOERDSMA, A., CREVELING, C. R., WEISSBACH, FRIEND, S., ~%ience 137, 648 (19%). 2. WEST, G. B., J. Pharm. and Pharmacol. 10, 589 (1958). 3. UDENFRIEND, S., WEISSBACH, H., AND CLARK, C. T., J. Biol.
H., AND UDEN-
Chem. 216, 337
(1955). 4. SJOERDSMA, A., OATES, J., ZALTZMAN, P., AND UDENFRIEND, S., J. Pharmacol. Exptl. Therap. (in press). 5. MITOMA, C., POSNER, H. S., BOGDANSKI, D. F. AND UDENFRIEND, S., J. Pharmacol. Exptl. Therap. 120, 188 (1957). 6. WAALKES, T. P., AND UDENFRIEND, S., J. Lab. Clin. Med. 60, 733 (1957). 7. EULER, U. S. VON, AND FLODING, I., Stand. J. Clin. & Lab. Invest. 8, 288 (1956). 8. UDENFRIEND, S., AND WYNGAARDEN, J. B., Biochim. et Biophys. Acta 20,48 (1956). 9. CARLSSON, A., AND WALDECK, B., Acta Physiol. Stand. 44, 293 (1958). 10. CROUT, J. R., AND SJOERDSMA, A., New Engl. J. Med. 261, 23 (1959).