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A Rapid and Simple Determination of Histamine and Polyamines
SHORT COMMUNICATIONS
A RAPID
AND
SIMPLE
HISTAMINE
AND
Japan. J. Pharmacol. 25, 610 (1975)
DETERMINATION
OF
POLYAMINES
Yasuo ENDO and Yasumi OGURA Department of Pharmacology, School of Dentistry, Tohoku University,Sendai 980, Japan Accepted June 30, 1975
The authors have already reported a method to separate catecholamines, serotonin, histamine and polyamines by a single P-cellulose column chromatography adsorption
(1, 2). As the
of histamine and polyamines to the column is significantly strong, the weak
acidic cation exchanger, CM-cellulose, was utilized to separate these amines and the method was successfully applied to rat tissues. CM-Cellulose (capacity: 0.70 meq/g) was obtained from Brown Co., U.S.A. Phosphate buffer (pH 6.2 and 7.5) was prepared from 0.1 M NaH2PO4 and 0.1 M Na2HPO4.
Borate
buffer (pH 9.0) was prepared from 0.2 M H3B03 (containing 0.2 M KCI) and 0.2 M Na2CO3. These buffers were diluted and NaCl was added at the time of application. buffers were used in the separation procedure.
The following
1 : 0.01 M phosphate buffer (pH 6.2), 2:
0.03 M phosphate buffer (pH 6.2), 3: 0.03 M phosphate buffer (pH 7.5), 4: 0.02 M borate buffer containing 0.05 M NaCI, 5: 0.02 M borate buffer containing 0.075 M NaCl, 6: 0.02 M borate buffer containing 0.15 M NaCl.
Wistar female rats (350-380 g) were decapitated
and each tissue was homogenized in 5 volumes of ice-cold 0.4 N HCIO4. was centrifuged at 3000 rpm for 5 min.
The homogenate
The supernatant was neutralized to pH 5-6 with
2 N KOH through a microtube with stirring in an ice bath, and the precipitate was removed by centrifugation (3000 rpm, 5 min).
The neutralized tissue extract (1-4 nil) was applied
to a CM-cellulose column (0.6 x 10 cm) equilibrated with buffer-l.
The elution was per
formed at room temperature (20-25'C) by increasing stepwise both concentration and pH of the buffer (buffer-l to 6).
Histamine and polyamines were determined by OPT-reaction
and TNBS-reaction, respectively, as described previously (1). The standard substances showed the following separation in the CM-cellulose column chromatography. Fraction
Buffer-1 Fraction
(15 ml): adrenaline,
metanephrine,
tryptamine,
(15 ml): tryptophan,
noradrenaline,
serotonin,
dopamine,
tyrosine, and dopa, Buffer-2 tyramine,
metanephrine,
nor
lysine, histidine and arginine, Buffer-3 Fraction
(10 nil): histamine, Buffer-4 Fraction (15 in]): cadaverine, putrescine and agmatine, Buffer-5 Fraction (18 ml): spermidine, Buffer-6 Fraction (15 nil): spermine. The separation and the recovery of histamine, spermidine and spermine were complete (recovery: 95-100%). The substances interfering with the fluorometric determination of histamine such as arginine, agmatine and spermidine (3) were removed from the histamine fraction.
In the determi
nation of polyamine only, tissue extract was applied to a column equilibrated with 0.02 M
SHORT
COMMUNICATIONS
Japan. J. Pharmacol. 25, 611 (1975)
FIG. 1.
Elution patterns of histamine, spermidine and spermine in rat tissues Each neutralized tissue extract of brain (4 ml), liver (1 ml) and kidney (2 ml) was applied to a CM-cellulose column (0.6 >: 10 cm) equilibrated with buffer-1. After washing with buffer-1 (15 ml) and buffer-2 (15 ml), histamine, spermidine and spermine were eluted stepwise by exchanging the elution buffers. Fractions were subjected to OPT-reaction (-®-, histamine) or TNBS-reaction spermidine and spermine). a: brain, b: liver, c: kidney F.I.: fluorescence intensity in OPT-reaction A420:absorbance at 420 nm in TNBS-reaction I fraction-3 ml, flow rate= l ml/min * : buffer number and buffer change position
TABLE1.
borate
buffer.
Contents of histamine, spermidine and spermine in rat tissues
After washing
in the same manner Fig. 1 shows
with 15 ml of buffer-4, spermidine
as described
the chromatographic
rat tissue extracts.
Putrescine,
low, were not detected
and spermine
patterns
cadaverine
of histamine,
and agmatine,
spermidine
obtained
these values for histamine
by Beaven than
column
chromatography
the isolated
those
et al. (4).
higher
and spermine
of which contents
in
in tissue are
in the present method.
Table 1 shows the content of each amine in rat tissues determined Except for kidneys,
were eluted
above.
obtained
content
As our histamine
by Beaven
values
ileum of guinea-pig.
The result obtained
Polyamine
for kidneys
et al., the histamine
on kidney extract was subjected
with the level in Table 1.
by the present method.
are in good agreement
were several
fraction
from
to the bio-assay
in agreement
times
CM-cellulose
of histamine
from the bio-assay
levels are generally
with those
using
was in agreement
with those obtained
SHORT COMMUNICATIONS by Janne et al. (5).
Japan. J. Pharmacol. 25, 612 (1975)
The present values of polyamines for the brain are higher than those
in our previous experiment using a P-cellulose column (1).
This can be explained by the
fact that there was a miss in calculation in so far as one molar spermidine phosphate con taining two molars of spermidine was accounted for as one molar spermidine. value of absorbance of spermidine at 420 mn in the TNBS-reaction
Thus, the
reported previously
(2) is double the true value. Another reason is that the cerebellum had been removed from the brain in the previous experiment. The cerebellum contains the highest polyamine level of all brain regions (6).
When using the whole brain, both methods (CM-cellulose and
P-cellulose column chromatography)
showed the same levels of polyamines.
In comparison with P-cellulose column chromatography,
the present method has the
advantage of a rapid separation of histamine, spermidine and spermine. REFERENCES 1) ENDO,Y. ANDOGURA,Y.: Europ. J. Pharmacol., 21, 293 (1973); 2) ENDO,Y. ANDOGURA, Y.: Japan. J. Pharmacol., 23, 491 (1973); 3) KREMZNER, L.T. ANDPFEIFFER, C.C.: Biochem. Pharmacol., 15, 197 (1966); 4) BEAVEN, M.A., JACOBSEN, S. ANDHORAKOVA, Z.: Clin. Chini. Acta, 37, 91 (1972); 5) JANNE, J., RAINA,A. ANDSIIMES, M.:A cta physiol. scand., 62, 352 (1964); 6) SHASKAN, E.G., HARASZTI, J.H. ANDSNYDER, S.H.: J. Neurochem., 20, 1443 (1973)
A RAPID
AND
SIMPLE
HISTAMINE
AND
Japan. J. Pharmacol. 25, 610 (1975)
DETERMINATION
OF
POLYAMINES
Yasuo ENDO and Yasumi OGURA Department of Pharmacology, School of Dentistry, Tohoku University,Sendai 980, Japan Accepted June 30, 1975
The authors have already reported a method to separate catecholamines, serotonin, histamine and polyamines by a single P-cellulose column chromatography adsorption
(1, 2). As the
of histamine and polyamines to the column is significantly strong, the weak
acidic cation exchanger, CM-cellulose, was utilized to separate these amines and the method was successfully applied to rat tissues. CM-Cellulose (capacity: 0.70 meq/g) was obtained from Brown Co., U.S.A. Phosphate buffer (pH 6.2 and 7.5) was prepared from 0.1 M NaH2PO4 and 0.1 M Na2HPO4.
Borate
buffer (pH 9.0) was prepared from 0.2 M H3B03 (containing 0.2 M KCI) and 0.2 M Na2CO3. These buffers were diluted and NaCl was added at the time of application. buffers were used in the separation procedure.
The following
1 : 0.01 M phosphate buffer (pH 6.2), 2:
0.03 M phosphate buffer (pH 6.2), 3: 0.03 M phosphate buffer (pH 7.5), 4: 0.02 M borate buffer containing 0.05 M NaCI, 5: 0.02 M borate buffer containing 0.075 M NaCl, 6: 0.02 M borate buffer containing 0.15 M NaCl.
Wistar female rats (350-380 g) were decapitated
and each tissue was homogenized in 5 volumes of ice-cold 0.4 N HCIO4. was centrifuged at 3000 rpm for 5 min.
The homogenate
The supernatant was neutralized to pH 5-6 with
2 N KOH through a microtube with stirring in an ice bath, and the precipitate was removed by centrifugation (3000 rpm, 5 min).
The neutralized tissue extract (1-4 nil) was applied
to a CM-cellulose column (0.6 x 10 cm) equilibrated with buffer-l.
The elution was per
formed at room temperature (20-25'C) by increasing stepwise both concentration and pH of the buffer (buffer-l to 6).
Histamine and polyamines were determined by OPT-reaction
and TNBS-reaction, respectively, as described previously (1). The standard substances showed the following separation in the CM-cellulose column chromatography. Fraction
Buffer-1 Fraction
(15 ml): adrenaline,
metanephrine,
tryptamine,
(15 ml): tryptophan,
noradrenaline,
serotonin,
dopamine,
tyrosine, and dopa, Buffer-2 tyramine,
metanephrine,
nor
lysine, histidine and arginine, Buffer-3 Fraction
(10 nil): histamine, Buffer-4 Fraction (15 in]): cadaverine, putrescine and agmatine, Buffer-5 Fraction (18 ml): spermidine, Buffer-6 Fraction (15 nil): spermine. The separation and the recovery of histamine, spermidine and spermine were complete (recovery: 95-100%). The substances interfering with the fluorometric determination of histamine such as arginine, agmatine and spermidine (3) were removed from the histamine fraction.
In the determi
nation of polyamine only, tissue extract was applied to a column equilibrated with 0.02 M
SHORT
COMMUNICATIONS
Japan. J. Pharmacol. 25, 611 (1975)
FIG. 1.
Elution patterns of histamine, spermidine and spermine in rat tissues Each neutralized tissue extract of brain (4 ml), liver (1 ml) and kidney (2 ml) was applied to a CM-cellulose column (0.6 >: 10 cm) equilibrated with buffer-1. After washing with buffer-1 (15 ml) and buffer-2 (15 ml), histamine, spermidine and spermine were eluted stepwise by exchanging the elution buffers. Fractions were subjected to OPT-reaction (-®-, histamine) or TNBS-reaction spermidine and spermine). a: brain, b: liver, c: kidney F.I.: fluorescence intensity in OPT-reaction A420:absorbance at 420 nm in TNBS-reaction I fraction-3 ml, flow rate= l ml/min * : buffer number and buffer change position
TABLE1.
borate
buffer.
Contents of histamine, spermidine and spermine in rat tissues
After washing
in the same manner Fig. 1 shows
with 15 ml of buffer-4, spermidine
as described
the chromatographic
rat tissue extracts.
Putrescine,
low, were not detected
and spermine
patterns
cadaverine
of histamine,
and agmatine,
spermidine
obtained
these values for histamine
by Beaven than
column
chromatography
the isolated
those
et al. (4).
higher
and spermine
of which contents
in
in tissue are
in the present method.
Table 1 shows the content of each amine in rat tissues determined Except for kidneys,
were eluted
above.
obtained
content
As our histamine
by Beaven
values
ileum of guinea-pig.
The result obtained
Polyamine
for kidneys
et al., the histamine
on kidney extract was subjected
with the level in Table 1.
by the present method.
are in good agreement
were several
fraction
from
to the bio-assay
in agreement
times
CM-cellulose
of histamine
from the bio-assay
levels are generally
with those
using
was in agreement
with those obtained
SHORT COMMUNICATIONS by Janne et al. (5).
Japan. J. Pharmacol. 25, 612 (1975)
The present values of polyamines for the brain are higher than those
in our previous experiment using a P-cellulose column (1).
This can be explained by the
fact that there was a miss in calculation in so far as one molar spermidine phosphate con taining two molars of spermidine was accounted for as one molar spermidine. value of absorbance of spermidine at 420 mn in the TNBS-reaction
Thus, the
reported previously
(2) is double the true value. Another reason is that the cerebellum had been removed from the brain in the previous experiment. The cerebellum contains the highest polyamine level of all brain regions (6).
When using the whole brain, both methods (CM-cellulose and
P-cellulose column chromatography)
showed the same levels of polyamines.
In comparison with P-cellulose column chromatography,
the present method has the
advantage of a rapid separation of histamine, spermidine and spermine. REFERENCES 1) ENDO,Y. ANDOGURA,Y.: Europ. J. Pharmacol., 21, 293 (1973); 2) ENDO,Y. ANDOGURA, Y.: Japan. J. Pharmacol., 23, 491 (1973); 3) KREMZNER, L.T. ANDPFEIFFER, C.C.: Biochem. Pharmacol., 15, 197 (1966); 4) BEAVEN, M.A., JACOBSEN, S. ANDHORAKOVA, Z.: Clin. Chini. Acta, 37, 91 (1972); 5) JANNE, J., RAINA,A. ANDSIIMES, M.:A cta physiol. scand., 62, 352 (1964); 6) SHASKAN, E.G., HARASZTI, J.H. ANDSNYDER, S.H.: J. Neurochem., 20, 1443 (1973)