|Abstract: ||Abstract: The polyamine profiles of the toxic dinoflagellate Alexandrium minutum during different growth stages were measured by high-performance liquid chromatography. Both free and conjugated polyamines were found, including putrescine, cadaverine, spermine, spermidine and norspermidine. During the growth cycle of A. minutum T1, the levels of norspermidine and putrescine in the free polyamines were the highest after 3 days culture. Putrescine and cadaverine were the major components in the conjugated polyamines. The amount of conjugated amines was higher than that of free amines during the exponential phase of A. minutum T1.
Paralytic shellfish poisons (PSP) associated with food poisoning incidents have caused two human fatalities and many illnesses in Taiwan (Hwang et al., 1987, 1992, 1995). The source of PSP in Taiwan was thought to be the toxic dinoflagellate Alexandrium minutum (Hwang et al., 1999). The growth and toxicity of A. minutum are affected by some biologically active organic compounds and environmental factors (Hwang and Lu, 2000). Recently, polyamines, biologically active organic compounds widely distributed in all living cells, are also considered to stimulate and regulate the growth of bloom-forming phytoplankton (Iwasaki, 1984; Nishibori and Nishio, 1997; Nishibori et al., 2001). Polyamines are linked mainly to the cell cycle and growth in algae as well as plants (Maestrini et al., 1999). Numerous investigations have correlated an increase in polyamine levels with developmental processes and morphogenesis in plants (Slocum and Galston, 1985; Davies, 1995). Besides, polyamine is responsive to such physiological controls as light, hormones, injury and stress in plants (Galston and Kaur-Sawhney, 1995). More recently, there seems to be a relationship among cell growth, the development of harmful algal blooms and polyamine levels (Nishibori et al., 2001), but studies on endogenous polyamines in harmful algae have so far been few. In this study, the toxic alga A. minutum T1 was used for the determination of endogenous free and conjugated polyamines by high-performance liquid chromatography (HPLC). The polyamine profiles of A. minutum T1 in different growth stages were further compared.
A single clonal cell of A. minutum was isolated from an aquaculture pond at Tunkang, Pingtung Prefecture, Taiwan and cultured axenically in modified K medium (Hwang and Lu, 2000) at 25°C under a 14 : 10 light : dark cycle. The cells in 750 ml culture solution were harvested by centrifugation every 3–7 days. A 0.5 ml sample of the culture solution was preserved in 1% Lugol's iodine solution (Chang et al., 1995) and the cells were counted by microscopy. The cell pellet was washed with axenically distilled water to remove extracellular polyamines and sonicated in 2 ml of cold 6% perchloric acid (PCA) and then centrifuged. One millilitre of the supernatant was used for free polyamine extraction. The others reactions were performed in sealed glass vials with 11 N HCl at 110°C for 16 h. Hydrolysates were dried under vacuum in a water bath at 60°C. The residue was dissolved in 1 ml 6% PCA and polyamines levels were determined. The amount of each polyamine was subtracted from that of free polyamines to gain the amount of conjugated polyamines. The polyamines were measured as benzoyl derivates as reported by Hwang et al. (Hwang et al., 1997). Briefly, 2 ml polyamine extractions were mixed sequentially with 1 ml of 2 M sodium hydroxide and 10 μl of benzoyl chloride, and allowed to stand at 30°C for 40 min. The resulting solutions were then treated with 2 ml saturated sodium chloride solution, and extractions were performed with 3 ml diethyl ether. The ether layers were evaporated to dryness under a stream of nitrogen. The residues were dissolved with 1 ml methanol, and analysed by HPLC.
The HPLC system was performed according to Hwang et al. (Hwang et al., 1997), and the gradient programme was slightly modified. The effect of the maximum methanol concentration in the solvent of gradient elution on the separation of five polyamines derivatized with benzoyl chloride is shown in Figure 1. It was found that the optimum gradient elution programme for separating these five polyamines was carried out at a flow rate of 0.8 ml min−1, starting with a methanol–water mixture (50 : 50, v : v) for 0.5 min. Then the programme proceeded linearly with different mixtures of methanol–water (50 : 50 to 75 : 25) over 6.5 min, and then methanol–water (75 : 25) for 5 min (Figures 1 and 2A). All five polyamines were well separated in a total duration of 10 min, with good peak resolution, sharpness and symmetry (Figure 2A). Standard curves of five polyamines including putrescine (Put), cadaverine (Cad), spermine (Spm), spermidine (Spd) and norspermidine (Norspd) were separately prepared in the range of 0.02–3 μg, and the data were subsequentlysubjected to linear regression analysis. The correlation coefficients for each polyamine were 0.99 (Table I). The detection limit was 0.01 μg for each polyamine. The recoveries of these five polyamines from the toxic alga A. minutum T1 were higher than 80%, except for Cad (Table II). In the preliminary study, the HPLC patterns for Spm and Spd were overlapped when the standard solution contained the above-mentioned five authentic polyamines. A very small adjustment of the maximum concentration of methanol in the elution gradient programme may result from the polarity of the mobile phase and the interaction between the mobile and stationary phases. Judging from the above data, the extraction, derivation and elute systems were suitable for analysing the polyamines from algal cells.
The typical profiles of free and mixed (free and conjugated) polyamines in A. minutum T1 cells cultured for 22 days are shown in Figure 2B,C. Both free and conjugated polyamines contained Put, Cad, Spm, Spd and Norspd. The growth curve of A. minutum T1 was as follows: 0–5 days belonging to lag growth phase, 6–20 days belonging to exponential growth phase, 21–30 days belonging tostationary growth phase, and after 30 days belonging to death growth phase (Hwang and Lu, 2000). The variations of free and conjugated amines during the growth cycle are shown in Figure 3. With the exception of during the lag growth phase, the amount of total conjugated polyamines was found to be several-fold higher than that of total free polyamines during the life cycle of A. minutum T1. Moreover, except for Cad and Norspd, the level of free polyamines in A. minutum T1 cells was higher during the lag growth phase and decreased soon after the beginning of the exponential growth phase. Among these polyamines, the amount of Norspd and Put was higher than that of other polyamines after 3 days' culture. Cad and Norspd increased slightly between the period of the lag and the exponential growth phase and then decreased by the mid-exponential growth phase. However, Put and Cad were the major components in the conjugated polyamines. Both polyamines quickly reached their maximal levels at day 10 of culture, significantly decreased by mid-exponential growth phase, and then slightly increased again at the beginning of the death growth phase. The variation of ratio of the amount of each free or conjugated polyamine to the amount of total free and conjugated polyamines during the growth cycle of A. minutum T1 is shown in Figure 4. Although variations of the levels of conjugated Spd, Spm and Norspd were not found during the growth cycle of A. minutum T1, the ratio of conjugated Put and Cad reached their maximal levels during the exponential growth phase.
Hamana and Matsuzaki (1985) pointed out that the pattern of polyamines distributed in eukaryotic algae can be divided into four types and may be useful as a chemotaxonomic marker. It was interesting that A. tamarense should belong to type II (algal type) (Nishibori and Nishio, 1997), but A. minutum could be categorized into the type III (mixed type) because it contained Put, Cad, Spm, Spd and Norspd in the whole growth cycle. Because the amount of conjugated polyamine was found to be several-fold higher than that of free polyamine except for the lag phase, it is speculated that the cellular levels of free polyamine are regulated through reversible conjugated formation (Slocum and Galston, 1985). However, Pfosser (1993) assumed that the synthesis of free and conjugated polyamine is regulated by different mechanisms and only a limited exchange occurs between free and conjugated polyamines. There has been some evidence that an increase in Put levels or a high ratio of Put : Spd are closely associated with elongation in higher plants or phytoplankton (Shen and Galston, 1985; Maestrini et al., 1999). Put and Spd were assumed to be significant compounds in coastal sea water during phytoplankton blooms (Nishibori et al., 2001). In connection with this, conjugated Put may take part in the regulation of cell growth in A. minutum T1, because they are abundant over the whole cell life cycle. Hamana et al. (Hamana et al., 1990) have suggested that the increase of Norspd and Norspm was favourable to heat tolerance in alga. The suggestion might explain how Norspd exists at a high level in A. minutum T1 during the lag growth phase, when adapting to environmental stresses. On the other hand, the aging of A. minutum T1 was supposed to be related to Spm due to an increase of Spm during the death phase. Similarly, Spm and Spd inhibited cell growth and mitotic activity and increased lignification in chickpea seeds (Bueno et al., 1993). The plant growth inhibited by Spm is due to interrupting the biosynthesis of chorophyll (Beigbeder and Kotzabasis, 1994). As a whole, free polyamines could stimulate the cell division from a quiescent state, while conjugated polyamines might play an important role in morphogenic processes and the formation of secondary metabolites (Flores and Martin-Tanguy, 1991; Sawhney and Applewhite, 1993).
HPLC is a powerful tool with which to analyse the polyamine pattern of the toxic alga A. minutum. Results presented here have indicated that the requirement of free polyamines may be higher for the lag growth phase and that conjugated polyamines accumulated by the exponential growth phase. Furthermore, the free polyamines Norspd and Put might provide the evidence for elucidating the physiological significance of A. minutum T1 in the lag phase, while the conjugated polyamines Put and Cad might contribute to some physiological functions in the exponential phase. Besides, Spm significantly increased when the cells were in culture during the death phase.