Determination of polyamines in human prostate by high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method for the determination of polyamines in human prostate has been developed. This method is based on pre-column derivatization with dansyl chloride (Dns-Cl). The derivatives were separated on a μBondapak C₁₈ column (250×4.6 mm I.D.; 10 μm), and eluted with methanol and distilled water using a one-step linear gradient. The column eluate was monitored by fluorescence detection (excitation, 370 nm; emission, 506 nm). The within-assay precision of the study (C.V.) was as follows: putrescine (PUT) 2.88%, spermidine (SPD) 2.94% and spermine (SP) 1.17%. The between-assay precision (C.V.) was: PUT 2.66%, SPD 3.06%, SP 2.79%. The recovery was greater than 97%. The detection limit for PUT, SPD and SP were 0.05, 0.08 and 0.06 nmol/ml, respectively. In contrast to other studies, sample or polyamine derivatives did not require extraction with an organic solvent such as ethanol, evaporation under vacuum or other condensation procedures. This is a simple, rapid and sensitive method that can be applied to the determination of polyamines in nearly all biological tissues and body fluids, such as urine and serum.     

Changes in Polyamine Concentrations in Amygdaloid-Kindled Rats

Concentrations of the polyamines putrescine, spermidine, and spermine were investigated in the left and right amygdala and in the remaining cerebrum, in which kindling was induced by repeated application of electrical stimulation of the left amygdala of rats. In kindled rats, the concentrations of spermidine and spermine increased slightly, but elevations did not reach significant levels in any brain regions. The most profound increase was detected in the putrescine concentration in all parts of the cerebrum 1-8 h after the final stimulation. These results suggest that the increases in concentrations of polyamines, particularly of putrescine, are involved in the pathogenesis of amygdaloid kindling.     

Urinary polyamine evaluation for effective diagnosis of various cancers

With a newly modified analytical method, the concentrations of free and acetylated urinary polyamines were simultaneously determined in a control group (32 cases) and patients with various types of cancers (104 cases, 20 males and 84 females) by gas chromatography-nitrogen-phosphorus detection. Significant concentration differences between normal subjects and various cancer patients were found. The various types of cancers (advanced gastric carcinoma, ovarian cancer, acute myelocyte leukemia, non-Hodgkin's lymphoma) gave unique patterns of urinary polyamine profile as well as significant differences of concentration. To indirectly evaluate the possible involvement of enzymes, precursor-to-product concentration ratios were compared between controls and patients with various types of cancers.     

Polyamines as biomarkers for plant regeneration capacity: improvement of regeneration by modulation of polyamine metabolism in different genotypes of indica rice

The importance of cellular polyamine (PA) levels and the ratio of putrescine (Put) to spermidine (Spd) for plant regeneration ability via somatic embryogenesis in several commercially grown indica rice varieties is reported here. The genotypes namely NDR-624, IR-20, IR-36, BJ-1 (having Put:Spd ratio2.3) showed superior plant regeneration while KL, PB-1 and TN-1 (having Put:Spd ratio3.8) showed moderate plant regeneration ability. The genotypes namely HS, Bindli, DV-85, ACB-72, IR-64 and IR-72 (having Put:Spd ratio5.0) showed poor plant regeneration ability. In contrast KH-7 (Put:Spd ratio10.0) showed no response at all. Favorable modification of cellular PA titers and their Put:Spd ratio by the addition of exogenous PAs (Put, Spd) or their biosynthesis inhibitor, difluoromethylarginine (DFMA) led to the induction/promotion of plant regeneration in poorly responding genotypes. These results showed a close relationship between cellular PA levels and their Put:Spd ratio with in vitro morphogenetic capacity in indica rice and suggest that the cellular PAs and Put:Spd ratios are important determinants (biomarkers) of plant regeneration ability in indica rice, and the improvement/induction of plant regeneration in morphogenetically poor and recalcitrant species could be achieved by modulating PA metabolism.     

Determination of dansylated polyamines in red blood cells by liquid chromatography-tandem mass spectrometry

The concentration of polyamines in red blood cells (RBCs) is considered to be an index of cell proliferation. This index has been demonstrated to be of clinical importance for the follow-up and treatment of some cancer patients. The concentration of polyamines in RBCs is usually determined by high-performance liquid chromatography (HPLC) with fluorescence detection. In the current work, we present a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of putrescine, spermidine, and spermine, the three major polyamines in RBCs. The polyamines were dansylated and analyzed by an LC gradient of 20-min duration on a C18 column on-line with a tandem mass spectrometer. An internal standard (1,8-diaminooctane) was used for quantification. This method exhibited excellent linearity for the three polyamines with regression coefficients higher than 0.99. The limits of detection for putrescine, spermidine, and spermine were 0.10, 0.75, and 0.50 pmol/ml, respectively. The intrarun precision values for putrescine, spermidine, and spermine all were better than 10%, and the interrun precision values were 13%, 9%, and 20%, respectively. The LC-MS/MS method is sufficiently simple and reliable enough to replace the currently used HPLC method with fluorescence detection in which putrescine is not always detectable.     

Metabolism and function of polyamines in plants: recent development (new approaches)

A review is presented of the recent developments in the metabolism andfunction of polyamines in plants. Polyamines appear to be involved in a widerange of plant processes so their exact role is not completely understood. Inthis review, the metabolic pathways involved in polyamine biosynthesis anddegradation are explained, along with the transport and conjugation of thesecompounds. The methodologies involved in the analysis of polyamine functionusing metabolic inhibitors and genetic and molecular approaches are described.The occurrence and distribution of polyamine-derived alkaloids are also dealtwith. The direction of future research in the study of plant polyamines isindicated.     

Polyamine uptake in cultured astrocytes: characterization and modulation by protein kinases

The properties and regulation of the polyamine transport system in brain are still poorly understood. The present study shows, for the first time, the existence of a polyamine transport system in cerebellar astrocytes and suggests that polyamine uptake is mediated by a single and saturable high-affinity transport system for putrescine, spermine, and spermidine (K:(m) = 3.2, 1.2, and 1.8 microM:, respectively). Although substitution of NaCl by choline chloride produced a decrease in the putrescine, spermine, and spermidine uptake, it seems that polyamine transport in cerebellar astrocytes is not mediated by an Na(+) cotransport as in the presence of Na(+) and cholinium, polyamine uptake was much lower than when measured in a sucrose-based medium. On the other hand, ouabain, gramicidin (a Na(+) ionophore), and ionomycin (a Ca(2+) ionophore) produced a strong inhibition of polyamine uptake, suggesting that membrane potential could have an important role in the functioning of the astroglial polyamine uptake system. Moreover, protein kinase C inhibition produced an enhancement of polyamine uptake, whereas stimulation of protein kinase C with phorbol esters inhibited polyamine uptake. Alternatively, the tyrosine kinase inhibitor genistein caused a marked reduction in the uptake. No effects on polyamine uptake were observed with inhibitors and activators of cyclic AMP-dependent protein kinase or when Ca(2+)/calmodulin-dependent protein kinase II was inhibited with KN-62. These results suggest that the polyamine uptake system in cerebellar astrocytes could be modulated by protein kinase C and tyrosine kinase activities.     

Changes in free polyamine levels induced by salt stress in leaves of cultivated and wild tomato species

The effects of N_aCl on endogenous free levels of the poluamines putrescine, spermi dine and spermine, and the relationships between polyamines, K⁺ levels and Na⁺ accumulation were determined in leaves of the cultivated tomato ( Lycopersicon esculentum Mill.) and its wild, salt-tolerant relative L. pennellii (Correll) D' Arcy at different exposure times during a 32-day period. Both stress treatments (100 and 200 m M NaCl) decreased the levels of putrescine and spermidine, although to a different degree for the cultivated and wild tomato species. The spermine levels did not decrease with salinity in L. pennellii over the salinization period, whereas they decreased in L. esculentum , except at the first application of the 100m M NaCl treatment. In both species, the changes induced by salinity in total polyamines and K⁺ were very similar, with the accumulation of Na⁺ in the leaf being concomitant with a decrease in both total polyamines and K⁺. This suggests that the main role of the polyamines in the leaf tissues. In this sense, a direct relationship between total polyamines and K⁺, and inverse relationship between polyamines and Na⁺ and between K⁺ and Na⁺ were found for both species. In the short term (up to 4 days) a peculiar physiological behavior was found in L. pennellii , as the total polyamine and K⁺ levels decreased at 100 m M but not at 200 m M NaCl, while after this time the latter plants had values lower than those of the 100 m M NaCl-treated plants at day 11.     

Biosynthesis, oxidation and conjugation of aliphatic polyamines in higher plants

Summary. This chapter will focus on polyamine biosynthesis, oxidation, conjugation processes, mainly to hydroxycinnamic acids, and compartmentation of enzymes, substrates and products, giving an overview about recent results especially in higher plants. New research advances regarding the cloning of the main cDNA encoding for polyamine biosynthetic and oxidative enzymes, will be taken into consideration. Received January 4, 2000 Accepted March 7, 2000     

Arabidopsis stress-inducible gene for arginine decarboxylase AtADC2 is required for accumulation of putrescine in salt tolerance

Arginine decarboxylase (ADC) catalyzes the first step of polyamine (PA) biosynthesis to produce putrescine (Put) from arginine (Arg). One of the 2 Arabidopsis ADC genes, AtADC2, is induced in response to salt stress causing the accumulation of free Put. To analyze the roles of stress-inducible AtADC2 gene and endogenous Put in stress tolerance, we isolated a Ds insertion mutant of AtADC2 gene (adc2-1) and characterized its phenotypes under salt stress. In the adc2-1 mutant, free Put content was reduced to about 25% of that in the control plants and did not increase under salt stress. Furthermore, the adc2-1 mutant was more sensitive to salt stress than the control plants. The stress sensitivity of adc2-1 was recovered by the addition of exogenous Put. These results indicate that endogenous Put plays an important role in salt tolerance in Arabidopsis. AtADC2 is a key gene for the production of Put under not only salinity conditions, but also normal conditions.     

Atmospheric pressure chemical ionization-mass spectrometry method to improve the determination of dansylated polyamines

Determination of polyamine pools is still a step impossible to circumvent in studies aimed at determining the pathophysiological role of natural polyamines. In addition, polyamine measurement in biological fluids and tissues may have clinical relevance, especially in cancer patients. Among the wide panel of analytical methods developed for the quantification of polyamines, high-performance liquid chromatographic (HPLC) separation of polyamines after derivatization with dansyl chloride remains the most commonly used method. In this work, we show that atmospheric pressure chemical ionization-mass spectrometry (MS) can be used to detect and quantify biologically relevant polyamines after dansylation, without chromatographic separation. Positive-ion mass spectra for each dansylated polyamine were generated after optimization by flow injection analysis (FIA). FIA coupled with MS detection by selected ion monitoring greatly increased the sensitivity of the polyamine detection. The method is linear over a wide range of polyamine concentrations and allows detection of quantities as low as 5 fmol. The FIA/MS method is about 50-fold more sensitive than the conventional HPLC/fluorimetry procedure. A good correlation (r>0.98) between these two methods was observed. The FIA/MS method notably reduces the time of analysis per sample to 1.5 min and turns out to be rapid, efficient, cost saving, reproducible, and sufficiently simple to allow its routine application.     

Perturbations in polyamines and related enzymes following chlordecone-potentiated bromotrichloromethane hepatotoxicity

The mechanism by which chlordecone (CD) amplifies the hepatotoxicity of halomethanes such as CCl₄, CHCl₃, and BrCCl₃ has been a subject of intense study. Recent work has shown that suppression of hepatocellular regeneration leads to accelerated progression of liver injury leading to complete hepatic failure due to an unusual interaction between individually nontoxic low-dose combination of CD and CCl₄. Since polyamines are involved in cell division, their levels reflect the extent to which there is suppression of hepatocellular regeneration during CD and CCl₄ interaction. The present studies were designed to investigate the polyamine levels and associated enzymes in livers of rats treated with BrCCl₃ alone or CD and BrCCl₃ low-dose combination in order to confirm whether the sequence of events of hepatotoxicity is similar to that seen in CCl₄ toxicity or that seen during CD and CCl₄ interaction. The extent of liver toxicity in rats fed 10 ppm chlordecone (CD) for 15 days prior to the injection of a single low dose of BrCCl₃ (15 μL/kg body weight) or after exposure to a high dose of BrCCl₃ (80 μL/kg body weight) without CD pretreatment, was similar 6 and 24 hr later as assessed by plasma transaminase levels. There was also an increase in transaminase levels, in rats exposed to a single low dose of BrCCl₃ alone (15 μL/kg body weight) but this increase was far below the high-dose exposure alone or the combination treatment. Hepatic levels of ornithine decarboxylase, S-adeno-sylmethionine decarboxylase, N¹-acetylputrescine, N¹-acetylspermidine, putrescine, spermidine, and spermine at the end of 24 hr increased after exposure to a low dose of BrCCl₃ alone as compared to exposure to a high dose alone or the low-dose combination of CD and BrCCl₃. Liver spermidine N¹-acetyltrans-ferase was elevated at 2, 6, and 24 hr after exposure to a high dose of BrCCl₃ alone as compared to treatment with a low-dose combination of CD and BrCCl₃ suggesting decreased synthesis of this enzyme, in spite of a greater need as seen from liver transaminase levels. In general, it was observed that there is significant elevation in some polyamines and related enzymes during toxicity of a low dose of BrCCl₃ which seemed to stabilize within 24 hr. This was not observed with the other two groups of rats exposed either to BrCCl₃ high dose alone or the low-dose combination of CD and BrCCl₃. Results indicate that CD and BrCCl₃ low-dose combination treatment causes increased liver toxicity resulting in compromised polyamine metabolism which is coincidental with suppressed hepatocellular regeneration leading to accelerated progressive phase of liver injury culminating in complete hepatic failure. These findings point to the possibility that the mechanism of potenti-ation of BrCCl₃ hepatotoxicity by CD is similar to that seen for CD and CCl₄ interaction.     

The role of free polyamines in the alternate-bearing of pistachio (<Emphasis Type="Italic">Pistacia vera</Emphasis> cv. Pontikis)

The present study was conducted in order to examine the physiological role of free polyamines in flower bud abscission. For this reason five 15-year old pistachio trees cv. "Pontikis" were selected and half of the main branches were manually defruited in early May. Polyamines were analyzed in three different organs (shoots, leaves and flower buds) from both fruiting and non-fruiting branches, during the period of kernel growing. Five samplings took place and the polyamines putrescine, spermidine and spermine were assayed. The flower bud abscission percentage was recorded every 5–10 days during kernel formation. Polyamine concentration declined during the period that coincides with that of kernel development, in both fruiting and non-fruiting branches, while significant bud abscission occurred from mid-July till late September in fruiting branches. Polyamine concentration in organs from fruiting branches was in most cases lower than that of non-fruiting ones. Most of the individual polyamines exhibited a high and significant negative correlation with bud abscission. By measuring the spermidine content of leaves and the spermine content of buds, it was possible to estimate the forthcoming bud abscission with significant accuracy (approximately 93%). On the other hand, the total polyamine content of the buds exhibited a significant strong negative relationship with bud abscission. Consequently, polyamines could have an important physiological function in the development of flower bud abscission of pistachio.     

Interaction of phospholipid bilayers with polyamines of different length

The tip-dip patch clamp method was used to study the effect of three polyamines, putrescine, spermidine and spermine, on bilayer lipid membrane (BLM) stability. Two kinds of mixed lipid-polyamine membranes were investigated. The poration voltage (V _p), and the closed (σ_cl) and open (σ_op) state conductances for pure and polyamine-treated lipid membranes were determined by the method of current-voltage surfaces. It was demonstrated that putrescine and spermidine destabilized lipid membranes under all circumstances. BLM stabilization by spermine was observed when it was added to preformed membranes. Received: 19 November 1999 / Revised version: 25 February 2000 / Accepted: 25 February 2000     

Methylthioadenosine and polyamine biosynthesis in a Saccharomyces cerevisiae meu1delta mutant

As part of our studies on polyamine biosynthesis in yeast, the metabolism of methylthioadenosine was studied in a mutant that lacks methylthioadenosine phosphorylase (meu1delta). The nucleoside accumulates in this mutant and is mainly excreted into the culture medium. Intracellular accumulation of the nucleoside is enough to account for the inhibition of spermidine synthase and thus to indirectly regulate the polyamine content of the meu1delta cells. By comparing the results with this mutant with a meu1delta spe2delta mutant that cannot synthesize spermidine or spermine, we showed that >98% of methylthioadenosine is produced as a byproduct of polyamine synthesis (i.e., from decarboxylated S-adenosylmethionine). In contrast, in MEU1+ SPE2+ cells methylthioadenosine does not accumulate and is metabolized through the methionine salvage pathway. Using a met15delta mutant we show that this pathway (i.e., involving polyamine biosynthesis and methylthioadenosine metabolism) is a significant factor in the metabolism of methionine, accounting for 15% of the added methionine.     

Effects of polyamine biosynthesis inhibitors on the progesterone-initiated increase in intracellular free Ca2+ and acrosome reactions in human sperm

This laboratory has previously reported that progesterone can initiate a rapid transient increase in the concentration of intracellular free Ca²⁺([Ca²⁺]_i) and an increase in a Ca²⁺-requiring exocytotic event, the acrosome reaction (AR) in human sperm. Rapid increases in Ca²⁺ fluxes of some mammalian cells caused by another steroid, testosterone, require polyamine biosynthesis. Herein, we tested two polyamine biosynthesis suicide inhibitors for their effects on the progesterone-initiated increase in [Ca²⁺]_i and AR in capacitated human sperm in vitro: DL-α-(difluoromethyl)ornithine hydrochloride (DFMO), an inhibitor of putrescine synthesis by ornithine decarboxylase and (5′-{[(Z))-4-amino-2-butenyl]methylamino}-5′-deoxyadenosine (MDL 73811), an inhibitor of S-adenosylmethionine decarboxylase (required for spermidine and spermine synthesis). Sperm were capacitated in vitro and preincubated 10 min with 4.9 mM DFMO or 9.8 μM MDL 73811 with or without various polyamines (245 μM). Progesterone (3.09 μM final concentration) or progesterone solvent (ethanol, 0.1% final concentration) was then added, sperm fixed 1 min after additions and AR assayed by indirect immunofluorescence or with fluorescein-labeled Con A lectin. DFMO strongly inhibited the AR but putrescine (product of ornithine decarboxylase and precursor of spermidine and spermine) reversed that inhibition. Preincubation for 25 min with DMFO + spermidine also reversed DFMO inhibition. MDL 73811 inhibited the progesterone-initiated AR, and a 10 min preincubation with spermidine, but not putrescine or spermine, reversed that inhibition. Preincubations with putrescine alone or with spermidine alone followed by addition of the progesterone solvent did not initiate the AR, and such preincubations followed by progesterone addition did not increase the AR more than progesterone alone. MDL 73811 and DFMO partially inhibited the rapid progesterone-initiated increase in [Ca²⁺]_i (assayed with fura-2), and those inhibitions were partially reversed by putrescine and spermidine, respectively. Putrescine or spermidine alone did not increase [Ca²⁺]_i nor did preincubation with either polyamine followed by progesterone addition increase [Ca²⁺]_i more than progesterone alone. Neither inhibitor was able to inhibit the AR initiated by the calcium ionophore, ionomycin. Our results suggest that human sperm polyamine biosynthesis is necessary for the progesterone-initiated rapid increase in [Ca²⁺]_i and subsequent membrane events of the AR. © 1993 Wiley-Liss, Inc.     

Leishmania tropica major: effect of paromomycin and pentamidine on polyamine levels in the skin of normal and infected mice

The polyamine content of the skin of BALB/c and C3H mice was determined at intervals, after injecting Leishmania tropica major. In BALB/c mice, putrescine and spermidine levels increased three- to seven-fold; in C3H mice, spontaneous recovery occurred after 3 weeks, accompanied by a reduction in putrescine and spermidine levels. Ornithine decarboxylase activity was negligible in normal, uninfected skin of both BALB/c and C3H mice, but increased steadily during infection. Treatment with drugs that inhibit the growth of leishmanial amastigotes in the skin of mice also reduced polyamine levels and ornithine decarboxylase activity of previously infected skin. There was a close correlation between the therapeutic activity of the drugs and their effect on polyamine content and synthesis. The aminoglycoside paromomycin, which was chemotherapeutically more effective than pentamidine, also had a greater effect on polyamine levels. S-adenosyl-L-Methionine decarboxylase activity in the skin of BALB/c and C3H mice was only slightly affected by the parasites. Polyamine levels and ornithine decarboxylase activity could possibly serve as means for measuring the growth of leishmanial parasites in skin and other tissues and as a measure of the efficacy of anti-leishmanial chemotherapeutics.     

Tolerance to salt stress in maize callus lines with different polyamine content

Four callus lines from immature embryos of a self-crossed maize (Zea mays L.) hybrid cultivar were selected for “high” (two lines) and “low” (two lines) polyamine (PA) levels. Each selected line was exposed to culture media containing no (control) or 1% (0.171 m) NaCl and the relative growth rates were compared after subculture. Low-PA lines appeared to be tolerant to salt stress, while high-PA lines were sensitive. Analysis of PA at the end of the subculture showed that treated calli of sensitive lines had increased their putrescine content in comparison with their control, while putrescine remained constant in tolerant lines. Callus lines were analysed by RAPD (random amplification of polymorphic DNA) markers. One polymorphism (550-bp band) was found, demonstrating a genetic difference between the lines. Received: 18 January 1997 / Revision received: 14 April 1997 / Accepted 15 July 1997     

Leishmania spp.: effect of inhibitors on growth and on polyamine and macromolecular syntheses

Ethidium bromide, pentamidine isethionate, and MGBG [methylglyoxal-bis (guanylhydrazone)] inhibited the uptake of radioactive putrescine by leishmanial (Leishmania spp.; Leishmania tropica major; Leishmania mexicana; Leishmania donovani) promastigotes and interfered with their polyamine synthesis. Inhibition was apparent as early as 1 hr after adding these drugs to the parasites at growth-inhibiting concentrations. Ethidium bromide also inhibited the incorporation of radioactive uracil into leishmanial RNA at growth-inhibiting concentrations, while DNA synthesis was inhibited by ethidium bromide at high concentrations after a lag period. MGBG inhibited the synthesis of leishmanial DNA and RNA at growth-inhibiting concentrations.