Detection of free polyamine in coastal seawater using ion exchange chromatography
ICES Journal of Marine Science, 58: 1201–1207. 2001
doi:10.1006/jmsc.2001.1115, available online at http://www.idealibrary.com on
Detection of free polyamine in coastal seawater using ion
exchange chromatography
Naoyoshi Nishibori, Akinori Nishii, and
Haruyoshi Takayama
Nishibori, N., Nishii, A., and Takayama, H. 2001. Detection of free polyamine in
coastal seawater using ion exchange chromatography. – ICES Journal of Marine
Science, 58: 1201–1207.
Polyamines are widely distributed in living organisms and are known to be essential
elements for normal growth and development. Although they may affect phytoplankton growth their existence and concentration in seawater is unclear. In this paper the
polyamine concentrations in coastal seawater are measured by high performance liquid
chromatography (HPLC) using cation exchange resin and OPA reagent. This method
seems sufficient for their detection in seawater. Putrescine and spermidine were found
to be the major polyamines in the coastal seawater, though their respective concentrations varied widely. In addition to them cadaverine, norspermidine,
norspermine, and spermine were also detected.
2001 International Council for the Exploration of the Sea
Keywords: polyamine, seawater, HPLC, cation exchange resin.
Received 19 September 2000; accepted 25 May 2001; published electronically
2 October 2001.
N. Nishibori: Shikoku University Junior College, Ojin, Tokushima 771-1192, Japan. A.
Nishii and H. Takayama. Hiroshima Fisheries Experimental Station, Ondo, Hiroshima
732-1207, Japan. Correspondence to N. Nishibori: e-mail:
Introduction
Among the plant-growth regulators polyamines are
widely distributed in living organisms and detectable in
seaweeds (Badini et al., 1994), phytoplanktons (Hamana
and Matsuzaki, 1985; Nishibori and Nishio, 1997), and
plants (Galston and Kaur-Sawhney, 1990), as well as in
bacteria (Hamana and Matsuzaki, 1992) and vertebrates
(Tabor and Tabor, 1984). The endogenous polyamines
are known to be essential for normal growth and
development and to play several important roles in
growth and differentiation through their binding to
DNA, RNA, and proteins due to their polycationic
nature (Tabor and Tabor, 1984; Kaminska et al., 1990;
Pfosser et al., 1990; Kotzabasis and Senger, 1994;
Scoccianti et al., 1995). In addition, exogenous
polyamines have been shown to stimulate growth and
cell division in a wide range of plants (Smith, 1985; Wu
and Kuniyuki, 1985).
Palenik and Morel (1991) reported that some marine
phytoplankton species are able to take up ammonium
produced by cell surface deaminases from primary
amines and amino acids. Pantoja et al. (1993) reported
on the oxidation of fluorescent-labelled cadaverine
and lysine by phytoplankton culture and suggested the
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possible importance of this nitrogen-uptake pathway in
the marine nitrogen cycle. Lee and Jøgensen (1995)
reported that the putrescine concentration profiles in a
coastal salt pond appeared to follow the pattern of
primary production. Recently the increase of in vivo
fluorescence by the addition of polyamine to natural
phytoplankton assemblages and a cyanobacterial culture
was reported (Maestrini et al., 1999), and these
polyamines were probably used as one of the nitrogen
sources in their study.
Although these biologically active amines may well
affect phytoplankton growth the existence and concentration of polyamines in seawater remain relatively
unknown. Here we describe and apply a detection
method for polyamines in seawater by high performance
liquid chromatography (HPLC) using cation-exchange
resin.
Materials and methods
HPLC conditions
The samples containing polyamines were separated with
a column (2.6 mm id.50 mm) of cation-exchange resin
(Hitachi No. 2619F) kept at 75C, using two buffers: A,
2001 International Council for the Exploration of the Sea
1202
N. Nishibori et al.
12
3
1 2
4
3
5 6
4
7
5
0
30
Retention time (min)
60
0
30
Retention time (min)
60
Figure 1. HPLC profiles of seawater sample (upper) and standard (lower) obtained from 80% B (left) and 65% B (right).
1, putrescine; 2, cadaverine; 3, norspermidine; 4, spermidine; 5, diaminoheptane; 6, norspermine; 7, spermine.
0.045 M sodium citrate, 0.061 M citric acid, 0.064 M
NaCl (pH 4); and B, 0.20 M sodium citrate, 2.0 M NaCl
(pH 7) (Hamana et al., 1994). The isocratic elution
flow rate was 0.25 ml/min. After the reaction with
o-phthalaldehyde (OPA) reagent (0.8 g OPA in 10 ml
ethanol, 15.0 g boric acid, 8.0 g sodium hydroxide,
2.0 ml of 2-mercaptoethanol in one litre of OPA reagent), polyamines were detected on a fluorescence
spectrometer at the excitation and emission wavelength
of 340 and 435 nm, respectively, according to Hitachi
Technical Data Sheet No. 72. To determine the suitable
buffer condition a sample containing 200 nM of each
standard polyamine (putrescine, cadaverine, norspermidine, spermidine, norspermine, and spermine) and
diaminoheptane was eluted with various concentrations
of B buffer.
Standard sample analysis
The standard polyamines were dissolved in the artificial
seawater to the concentrations of 0, 2, 10, 50, and
100 nM. Six millilitres of the artificial seawater samples
were evaporated to dryness and re-dissolved in 0.6 ml of
2% perchrolic acid (PCA). The supernatant obtained by
centrifugation was analysed using HPLC after ultra-free
treatment. Then the linearity of polyamine concentrations vs. the peak areas under the analytical
conditions was examined and the detection limit and
coefficient of variation were determined.
Seawater sample preparation
Seawater was collected from two stations (Stn Kure and
Stn Kaita) in Hiroshima Bay of Japan using a Kitahara
(1-l) water sampler at 0, 5, and B1 m between 19
March and 20 May 1999 (eight samples at each depth of
both sampling sites). These sampling areas are noted for
the incessant and extensive shellfish poisoning caused
by the harmful and toxic algal blooms that occur. After
collection the samples were filtered through a precombusted GF/C (Whatmann) filter under a gentle
vacuum and stored at 20C until polyamine analysis.
To each of the 6 ml of seawater samples, 0.2 ml of 30%
PCA and 120 pmol of diaminoheptane were added as an
internal standard, and then evaporated to dryness. The
samples were treated as described above and analysed
using HPLC.
Results
In the seawater samples analysed without the internal
standard (diaminoheptane) no peak was detected at the
retention time of diaminoheptane: this confirmed the
absence of diaminoheptane in the seawater. The peak of
Detection of free polyamine
5.0
5.0
3.0
2.0
1.0
0
Norspermidine
4.0
Relative peak area
4.0
5.0
Cadaverine
Relative peak area
3.0
2.0
1.0
0
0
20 40 60 80 100 120
Concentration (nM)
5.0
3.0
2.0
1.0
0
20 40 60 80 100 120
Concentration (nM)
1.0
0
20 40 60 80 100 120
Concentration (nM)
5.0
4.0
3.0
2.0
1.0
0
0
2.0
Norspermine
Relative peak area
4.0
3.0
20 40 60 80 100 120
Concentra (...truncated)
