Agents Actions 36 (1992)
0065-4299/92/02001%05 $1.50+0.20/0 9 1992 Birkh/iuser Verlag,Basel
Simultaneous fluorometric determination of intracellular polyamines separated by reversed-phase high-performance liquid chromatography J. M. Mat6s 1, j. M/trquez t, M. Garcia-Caballero z, I. Nfifiez de Castro 1 and F. Sfinchez-Jim6nez 1,3 1Departamentode Bioquimica y Biologia Molecular, Facultad de Ciencias, 2 Cfitedra de Cirugia, Facultad de Medicina, Universidad de M/tlaga, 29071 Mfilaga,Spain
Abstract
A reversed-phase H P L C technique in combination with fluorescent detection is described for simultaneous quantification of the precolumn Dansyl derivatives of intracellular amines. The derivatives were stable for at least one week, kept protected from the light at - 20 ~ The detection limit was between 1 and 5 pmol for all tested polyamines. Serotonin coeluted with tryptamine. The method has a very good reproducibility for both, retention times and chromatographic peak areas. The average recovery of standard amine solutions added to cellular extracts was estimated to be higher than 90%. The described method enables a rapid, reliable and reproducible quantification of biogenic and related polyamines in biological fluids and tissues. Introduction
and simultaneous determination of them. Several methods have been proposed to analyze polyamines in biological tissues. High-performance liquid chromatography procedures have been used to separate fluorescent derivatives of polyamines [6-9]. As the K. Munich Consensus Development Conference of Histamine Determination advises, any histamine assay must be validated for each particular situation [113]. The present work describes a reversed-phase H P L C method in combination with fluorescent detection, useful to separate and quantify intracellular histamine, serotonin and other polyamines in Ehrlich ascites tumor ceils continuously perifused in the presence of different amines.
There is evidence supporting a relationship between metabolism of histamine and other polyamines, Bartholeyns and Bouclier [1] postulate that histamine could play a similar role to the biogenic polyamines in rapidly growing tissues. Histamine may interact with DNA like polyamines do. Scolnik et al. [2] report an increased uptake (3H) histaniine by tumor nuclear fraction, somehow modulating genomic expression of DNA [3]. Recently, Mat6s et al. [4] have reported the suppression by histamine of the ornithine decarboxylase activity induced by ornithine in perifused Ehrlich ascites cells; different concentrations of histamine produce a clear dose-response effect. Since the intracellular concentrations of histamine and natural polyamines are usually low [5], a sensible and rapid method is needed for the routine
Material and methods
3Author for correspondence.
Chemicals. Histamine dihydrochloride was from Fluka Biochemika (Switzerland). Other chemicals
18 used were purchased from Sigma Chemical Co. (USA). HPLC reagents were used as described by Mfirquez et al. [11].
Ehrlich ascites cells. A hyperdiploid Lettr6 strain was maintained as previously reported [12]. The cells were washed once with 0.9% NaC1, and twice with phosphate saline buffer (6.16 mM KC1, 154 m M NaC1, 1.65 m M NaH/PO4, 9.35 mM Na2HPO 4 pH 7.4) and centrifuged at 500x 9 for 5rain. The final cell suspension was 4.2 x 108 cells/ml. Determination of free intracellular polyamines. Continuous incubations were performed as previously described by Segura et al. [13] and Mat6s et al. [4]. Immediately after perifusion, cells were collected, centrifuged at 2000 x 9 for 5 rain, and suspended in 1 ml of phosphate saline buffer, and counted; the whole cells were centrifuged at 2000 x 9 for 90 s through 1 ml of silicone oil mixture (AR 200 and AR 20, 2:4:1, w/w, Wacker-Chemie, Munich, FRG) in a 3 ml polypropylene tube, loaded with 0.5 ml of 0.25 M HC104 as the bottom layer. The calculated total volume of the cells was 0.60 + 0.03 gl/106 cells [14]. The Dansyl derivatives of the diamines were obtained as described by Smith and Davies [8] with minor modifications. Perchloric acid suspension (400 ~1) was supplemented with 100 ~tl of 1,8-diaminooctane (1 ~mol/3 x 108 cells) as internal standard. Then, the mixture was homogenized and centrifuged at 3000 x 9 for 20 min at 4 ~ The supernatant was mixed with 200 tll of saturated Na2COs (final pH 10.3) and 200 lal of acetone DnsC1 solution (10 mg/ml). The mixture was incubated overnight, protected from the light at room temperature. Afterwards, 10 ~tl of proline solution (100 mg/ml) was added to the mixture to remove the unreacted Dns-C1. After 30 min, the polyamine derivatives were extracted with 1.5 ml of toluene with vigorous vortexing, centrifuged at 2000 x 9 for 5 rain, and 1 m] of the organic phase was dried in a Speedvac Concentrator (Savant Instrument, Inc., New York, USA). The dried Dansyl derivatives were dissolved in 0.5 ml acetonitrile:water (70: 30), filtered through HVLP 013 Millipore membranes (0.45 Jam pore size; Bedford, MA, USA) and immediately assayed or stored at - 2 0 ~ A Beckman HPLC system (San Ram6n, CA, USA) was used, equipped with a 421A Controller and a 110-B Solvent Delivery Module doted with a 20 ~1
Agents Actions 36 (1992)
sample loop. A 420-AC Waters Millipore (Milford, MA, USA) fluorescent detector (excitation filter at 338 nm and emission cutoff filter at 425 nm) was used. Continuous on-line quantification of chromatographic peaks was carried out by a Spectra Physics Model SP 4290 computing integrator (San Jos~, CA, USA). The separation of Dansyl derivatives was performed on a 250 x 4 mm LiChrosorb | RP18 (5 g) reversed-phase column (Merck, FRG) at constant room temperature 22+ 1 ~ Two mobile phases were used: (A) water: acetonitrile: methanol (5: 3: 2) and (B) acetonitrile: methanol (3 : 2). A sigmoidal-like gradient described in the Results sections was used. Results and discussion
The aim of the present work was to devise a rapid, routine and reliable method to quantify free biogenic and related polyamines in tumor cells.
t~ Z no t/~ W rr" cr o c) W ~_1 a
3 1
2
10 11 4 5
8 9 6
Z W W 0s ~/
...3 ta_ 0 Figure1
I
10
J
|
20
30
min
Chromatogram of a standard mixture containing 11 Dansyl polyamine derivatives. The elution program is described in Table 1. Injection volume 20~tl; for experimental details see the Material and methods section. (1) N-Acetylputrescine, (2) tryptamine, (3) 1,4-benzenediamine, (4) 1,3-diaminopropane, (5) putrescine, (6) cadaverine, (7) histamine, (8) ornithine, (9) 1,8-diaminooctane, (i0) spermidine and (11) spermine.
19
Agents Actions 36 (1992)
Figure 1 shows a typical chromatogram of a mixture of 11 standard Dansyl polyamine derivatives separated by a Cls reversed-phase H P L C column under the chromatographic conditions described in Table 1. There was a good separation for all assayed polyamines. Because of the short retention time of Dns-C1, Dns-OH and Dns-NH 2, they did not interfere with the polyamine derivatives which left the column from the 9th rain onward. The total separation time was 40 rain. Under the experimental conditions serotonin coeluted with tryptamine. The reproducibility of the retention times of the Dansyl derivatives of tested polyamines was calculated from at least 20 analyses: except for the internal standard 1,8-diaminooctane and spermi-
dine, the variation coefficient (C.V.) was less than 5%. The reproducibility of the areas was also studied: all tested polyamine derivatives showed a linear response in the concentration range 10-3000 pmol in the sample. The Dansyl derivatives were stable for at least one week; this was confirmed by the reproducibility of the peak areas when samples were protected from the light and kept at - 20 ~ Dansyl histamine is less stable than other Dansyl polyamines; consequently, the recovery of histamine added was lower (Table 2); to get the best results in the histamine analyses the chromatographic run must be performed directly after Dansyl derivatization. The average recovery of standard polyamines added to different cellularfree extracts was 97%, showing the accuracy of the
Table 1 Chromatographic conditions for HPLC analysis of Dansyl polyamines.
Duration (rain)
Flow-rate (ml/min)
From ratio A: B
To ratio A: B
Gradient
0.0-0.2 0.2-3,0 3.0-13 13-15 15-17 17 18 18-20 20-24 24 29 29-35 35-40
0.2 1 1 1 1 1 1 1 1 1 1
72:28 72:28 72:28 60:40 47:53 40:60 28:72 20:80 15:85 10:90 5:95
72:28 72:28 60:40 47:53 40:60 28:72 20:80 15:85 10:90 5:95 0:100
Linear Linear Linear Linear Linear Linear Linear Linear Linear
Solvents: (A) water: acetonitrile: methanol (5 : 3 : 2); (B) acetonitrile: methanol (3 : 2).
Table 2 Recovery of standard polyamine added to cellular-free extracts.
Polyamine
1,3-Diaminopropane" Putrescine Cadaverine Spermidine Spermine Histamine Serotonin a 1,4-Benzenediamine a Acetylputrescine Ornithine
Polyamine in the sample (nmol) Cells (alone)
Cells + 3.0 nmol (actual)
Cells + 3.0 nmol (theoretical)
(%)
18.72 3.41 3.02 1.09 2.39 1.06 37.82 19.61 0.93 1.64
21.94 6.22 5.78 3.81 5.17 3.74 40.41 23.29 3.69 4.50
21.72 6.41 6.02 4.09 5.39 4.06 40.82 22.61 3.93 4.46
101 97 96 93 96 92 99 103 94 97
Cells perifused in the presence of 1,3-diaminopropane, serotonin or 1,4-benzenediamine.
Recovery
20
method. The detection limit obtained by this method was about 1 pmol for serotonin, 1,4benzenediamine, 1,3-diaminopropane, 1,6-diaminohexane, 1,8-diaminooctane, ornithine, putrescine and spermine, and 5 pmol for histamine, acetylputrescine, 1,5-diaminopentane and spermidine, although higher amounts are found in tumor cells (Fig. 2). The sensitivity of the determination of polyamines is in very good keeping with that reported by other authors [8, 9]. Although increased sensitivity is theoretically possible using o-phthalaldehyde (OPA) derivatives, the shorter half-lifes of the OPA derivatives (between 6 and 60 min) can cause problems during quantitative determination [15]. The chromatograms of Dansyl derivatives of polyamines in cell-free extracts after continuous perifusion in the presence of 0.5 mM ornithine and different polyamines closely matched the profiles of the standard polyamine mixture (Fig. 2). It is noteworthy that in all cases the natural biogenic polyamines putrescine, cadaverine, spermidine, spermine, as welt as histamine, and the amino acid ornithine are clearly separated. This method allows the simultaneous determination of histamine, serotonin, polyamines and other related amines in a rapid, easy and reliable manner; the method described by Endo [16-] is based on the separation of amines by selective elution from small phosphorylated cellulose columns.
Agents Actions 36 (1992)
& ~S 11
F_, w
I I0
L _t
10
i
i
20
30
|
0
I
10
i
20
r
--
30 min
Figure 2 Cellular polyamine content after 3.5 h of continuous perifusion in the presence of 0.5 m M ornithine and (A) 55 pM histamine, (B) 55 pM 1,3-diaminopropane. Number code as in Fig. 1.
[5] [6]
[7]
[8]
Acknowledgements [9] This paper was supported by the Grant PB 88 0445 of the DGICYT, and funds from University of Mfilaga (1991). Thanks are due to R. Cameselle for technical assistance. [10] Received 14 June 1991; accepted by W. Lorenz, 7 November 1991 [11]
References [i] J. Bartholeyns and M. Bouclier, Involvement of histamine in growth of mouse and rat tumours. Antitumoral properties of monofluoromethylhistidine, an enzyme-activated irreversible inhibitor of histidine decarboxylase. Cancer Res. 44, 639-645 (1984). [2] A. J. Scolnik, M. C. Rubio and R. A. Caro, Histamine and Cancer. Trends Pharmacol. Sci. 6, 356-357 (1985). [3] J. Bartholeyns and J. R. Fozard, Role of histamine in tumour development. Trends Pharmacol. Sci. 6, 123-125 (1985). [4] J. M. Mat6s, F. M. S~inchez-Jim6nez, M. Garcia-Caballero
I
0
[12]
and I. Nfifiez de Castro, Histamine and serotonin inhibit induction of ornithine decarboxylase by ornithine in perifused Ehrlich ascites tumour cells. FEBS Lett. 250, 257-261 (1989). D. M. L. Morgan, Polyamines and cellular regulation: perspectives. Biochem. Soc. Trans. 18, 1080-1084 (1990). N. Seller, Liquid chromatographic methods for assaying polyamines usin# prechromatographic derivatization. In Methods in Enzymology, Vol. 94. (Eds. H. Tabor and C. W. Tabor) pp. 10-25, Academic Press, New York 1983. C. W. Tabor and H. Tabor, Quantitative determination of naturally occurring aliphatic diamines and polyamines by an automated liquid chromatosraphy procedure. In Methods in Enzymology, Vol. 94. (Eds. H. Tabor and C. W. Tabor) pp. 39-42, Academic Press, New York 1983. M.A. Smith and P. J. Davies, Separation and quantification of polyamhws in Plant tissue by high performance liquid chromatography of their dansyl-derivatives. Plant Physiol. 78, 89-91 (1985). C. Stefanelli, D. Carati and C. Rossoni, Separation of N 1 and NS-acetylspermidine isomers' by reversed-phase column liquid chromatography after derivatization with Dansylchloride. J. Chromatogr. Biomed. Applic. 375, 49-55 (1986). W. Lorenz, E. Neugebauer, B. Uvn~is, M. Ennis, G. Granerus, J. P. Green, J. J. Keyzer, P. T. McBride, P. F. Mannaioni, F. L. Pearce and J. Watkins, K. Munich Consensus Development Conference on Histamine Determination. Handb. Exp. Pharm. 97, 81-92 (1991). J. Mfirquez, A. R. Quesada, F. S~mchez-Jim6nez and L Nfifiez de Castro, Determination of 27 dansyl-amino acid derivatives in biological fluids by reversed-phase high performance liquid chromatography. J. Chromatogr. Biomed. Applic. 380, 275 283 (1986). A. R. Quesada, M. A. Medina, J. Mfirquez, F. SfinchezJim+nez and I. Nflfiez de Castro, Contribution by host tissues to circulating glutamine in mice inoculated with Ehrlich ascites tumor cells. Cancer Res. 44, 1551 1553 (1988).
[13] J. A. Segura, M. A. Medina, F. J. Alonso, F. SfinchezJim~nez and I. Nfifiez de Castro, Glycolysis andglutaminolysis in perifused Ehrlich ascites tumour cells. Cell. Biochem. Funct. 7, 7-10 (1989).
Agents Actions 36 (1992) [14] J. M/trquez, F. Sfinchez-Jim~nez, M. A. Medina, A. R. Qaesada and I. Nfifiez de Castro, Nitrogen metabolism in tumor bearing mice. Arch. Biochem. Biophys. 268, 667-675 (1989). [15] H. Engelhardt, HPLC of amino acid and proteins. In Practice of High Performance Liquid Chromatography. (Ed. H. Engelhardt) pp. 410-425, Springer, Berlin, Heidelberg 1986.
21 [16] Y. Endo A simple method for determination of polyamines and histamine and its application to assay of ornithine and histidine decarboxylase activities. In Methods in Enzymology, Vol. 94. (Eds. H. Tabor and C. W. Tabor) pp. 40-47, Academic Press, New York 1983.
0065-4299/92/02001%05 $1.50+0.20/0 9 1992 Birkh/iuser Verlag,Basel
Simultaneous fluorometric determination of intracellular polyamines separated by reversed-phase high-performance liquid chromatography J. M. Mat6s 1, j. M/trquez t, M. Garcia-Caballero z, I. Nfifiez de Castro 1 and F. Sfinchez-Jim6nez 1,3 1Departamentode Bioquimica y Biologia Molecular, Facultad de Ciencias, 2 Cfitedra de Cirugia, Facultad de Medicina, Universidad de M/tlaga, 29071 Mfilaga,Spain
Abstract
A reversed-phase H P L C technique in combination with fluorescent detection is described for simultaneous quantification of the precolumn Dansyl derivatives of intracellular amines. The derivatives were stable for at least one week, kept protected from the light at - 20 ~ The detection limit was between 1 and 5 pmol for all tested polyamines. Serotonin coeluted with tryptamine. The method has a very good reproducibility for both, retention times and chromatographic peak areas. The average recovery of standard amine solutions added to cellular extracts was estimated to be higher than 90%. The described method enables a rapid, reliable and reproducible quantification of biogenic and related polyamines in biological fluids and tissues. Introduction
and simultaneous determination of them. Several methods have been proposed to analyze polyamines in biological tissues. High-performance liquid chromatography procedures have been used to separate fluorescent derivatives of polyamines [6-9]. As the K. Munich Consensus Development Conference of Histamine Determination advises, any histamine assay must be validated for each particular situation [113]. The present work describes a reversed-phase H P L C method in combination with fluorescent detection, useful to separate and quantify intracellular histamine, serotonin and other polyamines in Ehrlich ascites tumor ceils continuously perifused in the presence of different amines.
There is evidence supporting a relationship between metabolism of histamine and other polyamines, Bartholeyns and Bouclier [1] postulate that histamine could play a similar role to the biogenic polyamines in rapidly growing tissues. Histamine may interact with DNA like polyamines do. Scolnik et al. [2] report an increased uptake (3H) histaniine by tumor nuclear fraction, somehow modulating genomic expression of DNA [3]. Recently, Mat6s et al. [4] have reported the suppression by histamine of the ornithine decarboxylase activity induced by ornithine in perifused Ehrlich ascites cells; different concentrations of histamine produce a clear dose-response effect. Since the intracellular concentrations of histamine and natural polyamines are usually low [5], a sensible and rapid method is needed for the routine
Material and methods
3Author for correspondence.
Chemicals. Histamine dihydrochloride was from Fluka Biochemika (Switzerland). Other chemicals
18 used were purchased from Sigma Chemical Co. (USA). HPLC reagents were used as described by Mfirquez et al. [11].
Ehrlich ascites cells. A hyperdiploid Lettr6 strain was maintained as previously reported [12]. The cells were washed once with 0.9% NaC1, and twice with phosphate saline buffer (6.16 mM KC1, 154 m M NaC1, 1.65 m M NaH/PO4, 9.35 mM Na2HPO 4 pH 7.4) and centrifuged at 500x 9 for 5rain. The final cell suspension was 4.2 x 108 cells/ml. Determination of free intracellular polyamines. Continuous incubations were performed as previously described by Segura et al. [13] and Mat6s et al. [4]. Immediately after perifusion, cells were collected, centrifuged at 2000 x 9 for 5 rain, and suspended in 1 ml of phosphate saline buffer, and counted; the whole cells were centrifuged at 2000 x 9 for 90 s through 1 ml of silicone oil mixture (AR 200 and AR 20, 2:4:1, w/w, Wacker-Chemie, Munich, FRG) in a 3 ml polypropylene tube, loaded with 0.5 ml of 0.25 M HC104 as the bottom layer. The calculated total volume of the cells was 0.60 + 0.03 gl/106 cells [14]. The Dansyl derivatives of the diamines were obtained as described by Smith and Davies [8] with minor modifications. Perchloric acid suspension (400 ~1) was supplemented with 100 ~tl of 1,8-diaminooctane (1 ~mol/3 x 108 cells) as internal standard. Then, the mixture was homogenized and centrifuged at 3000 x 9 for 20 min at 4 ~ The supernatant was mixed with 200 tll of saturated Na2COs (final pH 10.3) and 200 lal of acetone DnsC1 solution (10 mg/ml). The mixture was incubated overnight, protected from the light at room temperature. Afterwards, 10 ~tl of proline solution (100 mg/ml) was added to the mixture to remove the unreacted Dns-C1. After 30 min, the polyamine derivatives were extracted with 1.5 ml of toluene with vigorous vortexing, centrifuged at 2000 x 9 for 5 rain, and 1 m] of the organic phase was dried in a Speedvac Concentrator (Savant Instrument, Inc., New York, USA). The dried Dansyl derivatives were dissolved in 0.5 ml acetonitrile:water (70: 30), filtered through HVLP 013 Millipore membranes (0.45 Jam pore size; Bedford, MA, USA) and immediately assayed or stored at - 2 0 ~ A Beckman HPLC system (San Ram6n, CA, USA) was used, equipped with a 421A Controller and a 110-B Solvent Delivery Module doted with a 20 ~1
Agents Actions 36 (1992)
sample loop. A 420-AC Waters Millipore (Milford, MA, USA) fluorescent detector (excitation filter at 338 nm and emission cutoff filter at 425 nm) was used. Continuous on-line quantification of chromatographic peaks was carried out by a Spectra Physics Model SP 4290 computing integrator (San Jos~, CA, USA). The separation of Dansyl derivatives was performed on a 250 x 4 mm LiChrosorb | RP18 (5 g) reversed-phase column (Merck, FRG) at constant room temperature 22+ 1 ~ Two mobile phases were used: (A) water: acetonitrile: methanol (5: 3: 2) and (B) acetonitrile: methanol (3 : 2). A sigmoidal-like gradient described in the Results sections was used. Results and discussion
The aim of the present work was to devise a rapid, routine and reliable method to quantify free biogenic and related polyamines in tumor cells.
t~ Z no t/~ W rr" cr o c) W ~_1 a
3 1
2
10 11 4 5
8 9 6
Z W W 0s ~/
...3 ta_ 0 Figure1
I
10
J
|
20
30
min
Chromatogram of a standard mixture containing 11 Dansyl polyamine derivatives. The elution program is described in Table 1. Injection volume 20~tl; for experimental details see the Material and methods section. (1) N-Acetylputrescine, (2) tryptamine, (3) 1,4-benzenediamine, (4) 1,3-diaminopropane, (5) putrescine, (6) cadaverine, (7) histamine, (8) ornithine, (9) 1,8-diaminooctane, (i0) spermidine and (11) spermine.
19
Agents Actions 36 (1992)
Figure 1 shows a typical chromatogram of a mixture of 11 standard Dansyl polyamine derivatives separated by a Cls reversed-phase H P L C column under the chromatographic conditions described in Table 1. There was a good separation for all assayed polyamines. Because of the short retention time of Dns-C1, Dns-OH and Dns-NH 2, they did not interfere with the polyamine derivatives which left the column from the 9th rain onward. The total separation time was 40 rain. Under the experimental conditions serotonin coeluted with tryptamine. The reproducibility of the retention times of the Dansyl derivatives of tested polyamines was calculated from at least 20 analyses: except for the internal standard 1,8-diaminooctane and spermi-
dine, the variation coefficient (C.V.) was less than 5%. The reproducibility of the areas was also studied: all tested polyamine derivatives showed a linear response in the concentration range 10-3000 pmol in the sample. The Dansyl derivatives were stable for at least one week; this was confirmed by the reproducibility of the peak areas when samples were protected from the light and kept at - 20 ~ Dansyl histamine is less stable than other Dansyl polyamines; consequently, the recovery of histamine added was lower (Table 2); to get the best results in the histamine analyses the chromatographic run must be performed directly after Dansyl derivatization. The average recovery of standard polyamines added to different cellularfree extracts was 97%, showing the accuracy of the
Table 1 Chromatographic conditions for HPLC analysis of Dansyl polyamines.
Duration (rain)
Flow-rate (ml/min)
From ratio A: B
To ratio A: B
Gradient
0.0-0.2 0.2-3,0 3.0-13 13-15 15-17 17 18 18-20 20-24 24 29 29-35 35-40
0.2 1 1 1 1 1 1 1 1 1 1
72:28 72:28 72:28 60:40 47:53 40:60 28:72 20:80 15:85 10:90 5:95
72:28 72:28 60:40 47:53 40:60 28:72 20:80 15:85 10:90 5:95 0:100
Linear Linear Linear Linear Linear Linear Linear Linear Linear
Solvents: (A) water: acetonitrile: methanol (5 : 3 : 2); (B) acetonitrile: methanol (3 : 2).
Table 2 Recovery of standard polyamine added to cellular-free extracts.
Polyamine
1,3-Diaminopropane" Putrescine Cadaverine Spermidine Spermine Histamine Serotonin a 1,4-Benzenediamine a Acetylputrescine Ornithine
Polyamine in the sample (nmol) Cells (alone)
Cells + 3.0 nmol (actual)
Cells + 3.0 nmol (theoretical)
(%)
18.72 3.41 3.02 1.09 2.39 1.06 37.82 19.61 0.93 1.64
21.94 6.22 5.78 3.81 5.17 3.74 40.41 23.29 3.69 4.50
21.72 6.41 6.02 4.09 5.39 4.06 40.82 22.61 3.93 4.46
101 97 96 93 96 92 99 103 94 97
Cells perifused in the presence of 1,3-diaminopropane, serotonin or 1,4-benzenediamine.
Recovery
20
method. The detection limit obtained by this method was about 1 pmol for serotonin, 1,4benzenediamine, 1,3-diaminopropane, 1,6-diaminohexane, 1,8-diaminooctane, ornithine, putrescine and spermine, and 5 pmol for histamine, acetylputrescine, 1,5-diaminopentane and spermidine, although higher amounts are found in tumor cells (Fig. 2). The sensitivity of the determination of polyamines is in very good keeping with that reported by other authors [8, 9]. Although increased sensitivity is theoretically possible using o-phthalaldehyde (OPA) derivatives, the shorter half-lifes of the OPA derivatives (between 6 and 60 min) can cause problems during quantitative determination [15]. The chromatograms of Dansyl derivatives of polyamines in cell-free extracts after continuous perifusion in the presence of 0.5 mM ornithine and different polyamines closely matched the profiles of the standard polyamine mixture (Fig. 2). It is noteworthy that in all cases the natural biogenic polyamines putrescine, cadaverine, spermidine, spermine, as welt as histamine, and the amino acid ornithine are clearly separated. This method allows the simultaneous determination of histamine, serotonin, polyamines and other related amines in a rapid, easy and reliable manner; the method described by Endo [16-] is based on the separation of amines by selective elution from small phosphorylated cellulose columns.
Agents Actions 36 (1992)
& ~S 11
F_, w
I I0
L _t
10
i
i
20
30
|
0
I
10
i
20
r
--
30 min
Figure 2 Cellular polyamine content after 3.5 h of continuous perifusion in the presence of 0.5 m M ornithine and (A) 55 pM histamine, (B) 55 pM 1,3-diaminopropane. Number code as in Fig. 1.
[5] [6]
[7]
[8]
Acknowledgements [9] This paper was supported by the Grant PB 88 0445 of the DGICYT, and funds from University of Mfilaga (1991). Thanks are due to R. Cameselle for technical assistance. [10] Received 14 June 1991; accepted by W. Lorenz, 7 November 1991 [11]
References [i] J. Bartholeyns and M. Bouclier, Involvement of histamine in growth of mouse and rat tumours. Antitumoral properties of monofluoromethylhistidine, an enzyme-activated irreversible inhibitor of histidine decarboxylase. Cancer Res. 44, 639-645 (1984). [2] A. J. Scolnik, M. C. Rubio and R. A. Caro, Histamine and Cancer. Trends Pharmacol. Sci. 6, 356-357 (1985). [3] J. Bartholeyns and J. R. Fozard, Role of histamine in tumour development. Trends Pharmacol. Sci. 6, 123-125 (1985). [4] J. M. Mat6s, F. M. S~inchez-Jim6nez, M. Garcia-Caballero
I
0
[12]
and I. Nfifiez de Castro, Histamine and serotonin inhibit induction of ornithine decarboxylase by ornithine in perifused Ehrlich ascites tumour cells. FEBS Lett. 250, 257-261 (1989). D. M. L. Morgan, Polyamines and cellular regulation: perspectives. Biochem. Soc. Trans. 18, 1080-1084 (1990). N. Seller, Liquid chromatographic methods for assaying polyamines usin# prechromatographic derivatization. In Methods in Enzymology, Vol. 94. (Eds. H. Tabor and C. W. Tabor) pp. 10-25, Academic Press, New York 1983. C. W. Tabor and H. Tabor, Quantitative determination of naturally occurring aliphatic diamines and polyamines by an automated liquid chromatosraphy procedure. In Methods in Enzymology, Vol. 94. (Eds. H. Tabor and C. W. Tabor) pp. 39-42, Academic Press, New York 1983. M.A. Smith and P. J. Davies, Separation and quantification of polyamhws in Plant tissue by high performance liquid chromatography of their dansyl-derivatives. Plant Physiol. 78, 89-91 (1985). C. Stefanelli, D. Carati and C. Rossoni, Separation of N 1 and NS-acetylspermidine isomers' by reversed-phase column liquid chromatography after derivatization with Dansylchloride. J. Chromatogr. Biomed. Applic. 375, 49-55 (1986). W. Lorenz, E. Neugebauer, B. Uvn~is, M. Ennis, G. Granerus, J. P. Green, J. J. Keyzer, P. T. McBride, P. F. Mannaioni, F. L. Pearce and J. Watkins, K. Munich Consensus Development Conference on Histamine Determination. Handb. Exp. Pharm. 97, 81-92 (1991). J. Mfirquez, A. R. Quesada, F. S~mchez-Jim6nez and L Nfifiez de Castro, Determination of 27 dansyl-amino acid derivatives in biological fluids by reversed-phase high performance liquid chromatography. J. Chromatogr. Biomed. Applic. 380, 275 283 (1986). A. R. Quesada, M. A. Medina, J. Mfirquez, F. SfinchezJim+nez and I. Nflfiez de Castro, Contribution by host tissues to circulating glutamine in mice inoculated with Ehrlich ascites tumor cells. Cancer Res. 44, 1551 1553 (1988).
[13] J. A. Segura, M. A. Medina, F. J. Alonso, F. SfinchezJim~nez and I. Nfifiez de Castro, Glycolysis andglutaminolysis in perifused Ehrlich ascites tumour cells. Cell. Biochem. Funct. 7, 7-10 (1989).
Agents Actions 36 (1992) [14] J. M/trquez, F. Sfinchez-Jim~nez, M. A. Medina, A. R. Qaesada and I. Nfifiez de Castro, Nitrogen metabolism in tumor bearing mice. Arch. Biochem. Biophys. 268, 667-675 (1989). [15] H. Engelhardt, HPLC of amino acid and proteins. In Practice of High Performance Liquid Chromatography. (Ed. H. Engelhardt) pp. 410-425, Springer, Berlin, Heidelberg 1986.
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