Determination of polyamines in human tissues by precolumn derivatization with 9-fluorenylmethyl chloroformate and high-performance liquid chromatography

A high-performance liquid chromatography (HPLC) assay with fluorescence detection was developed for the determination of the polyamines putrescine, spermidine, spermine in samples of human spinal cord, cerebellum, cerebrospinal fluid (CSF), skeletal muscle, and muscle microdialysates without an extensive sample preparation. The precolumn derivatization was performed with 9-fluorenylmethyl chloroformate (FMOC), and the derivatizated polyamines were stable for at least 14 h at 4 degrees C. All polyamines were separated within 35 min. The method was checked for linearity, and mean correlation coefficient values of 0.995, 0.999, and 0.991 were achieved for putrescine, spermidine, and spermine, respectively. The within- and between-assay coefficient of variation percentages evaluated in standard solutions varied between 1.0 and 4.9% and between 1.3 and 6.9%, respectively. The corresponding values obtained in samples of human spinal cord were between 1.0 and 5.0% and between 0.6 and 5.8%. The values of the recovery, evaluated in spinal cord tissue, varied between 83.7 and 93.5%.     

毛细管电泳-激光诱导荧光分析血清多胺的研究

为进一步探讨多胺的生物学作用,建立了毛细管电泳-激光诱导荧光(λ_ex=488 nm,λ_em=513 nm)分析血清多胺方法.在碱性介质中,多胺与荧光素异硫氰酸酯进行衍生化反应,硼酸盐(pH 8.6)作为运行缓冲液,运行电压20 kV,腐胺、精胺、精脒和1,6-己二胺（内标）在8 min内达到基线分离.考察了方法的线性范围、稳定性、检测限和方法的回收率等,方法具有样品处理简单,灵敏度高,速度快等特点.用于正常对照大鼠和肿瘤大鼠血清多胺的测定.结果提示：实验组肿瘤大鼠血清精胺和精脒水平显著高于正常对照大鼠和实验组未出现肿瘤大鼠血清精胺和精脒水平（P＜0.05）,正常对照组大鼠和实验组未出现肿瘤大鼠血清精胺和精脒水平间无显著性差异（P＞0.05）,各组间血清腐胺水平均无显著性差异（P＞0.05）.     

Investigation of polyamine metabolism by high-performance liquid chromatographic and gas chromatographic profiling methods

Measurements of polyamines, polyamine conjugates and their metabolites in tissues, cells and extracellular fluids are used in biochemistry, (micro)biology, oncology and parasitology. Decarboxylation of ornithine yields putrescine. Aminopropylation of putrescine yields spermidine, and aminopropylation of spermidine yields spermine. Spermidine and spermine are retroconverted to putrescine and spermidine, respectively, by initial N-acetylation and subsequent polyamine oxidation. The intermediate N-acetylputrescine, N¹-acetylspermidine and N⁸-acetylspermidine are the major urinary N-acetylpolyamines. Polyamines and N-acetylpolyamines are terminally degraded to non-α-amino acid metabolites by oxidative deamination and aldehyde dehydrogenation. Chromatography with on-line detection is the most commonly applied profiling method for polyamines, N-acetylpolyamines and their non-α-amino acid metabolites. Cation-exchange and reversed-phase high-performance liquid chromatography require pre- or post-column derivatisation, followed by UV-Vis spectrophotometric or fluorimetric detection. Isolation and derivatisation precedes gas chromatography with flame-ionisation, nitrogen-phosphorus, electron-capture or mass spectrometric detection. High-performance liquid chromatography and gas chromatography of polyamines are not competitive techniques, but rather supplementary.     

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

A high-performance liquid chromatographic method for the determination of polyamines (spermine, spermidine and putrescine) in human saliva was developed. This method is based on pre-column derivatization with o-phthaldialdehyde (OPA). The derivatives were separated on a Nucleosil ODS column (250×4.6 mm I.D.; 5 μm). The gradient elution was performed with two mobile phases A (water) and B (methanol) at a flow rate of 0.8 ml/min. The column eluate was monitored by fluorescence detection (excitation, 360 nm; emission, 510 nm). The within- and between-assay coefficients of variation for all the compounds were below 5%. The detection limits for spermine, spermidine and putrescine were 0.04, 0.05 and 0.06 nmol/ml, respectively. The recovery was greater than 90%. Our analytical technique requires neither preliminary extraction with an organic solvent, nor long multi-step procedures. For saliva samples, this is a simple, rapid and highly reproducible method that can be easily applied to the routine determination of salivary polyamines, whose levels increase early in several pathological conditions.     

Separation and quantitation of polyamines in plant tissue by high performance liquid chromatography of their dansyl derivatives

High performance liquid chromatography in combination with fluorescence spectrophotometry can be used to separate and quantitate polyamines (putrescine, cadaverine, spermidine, spermine), prepared as their dansyl derivatives, from plant tissue. The procedure gives sensitive and consistent results for polyamine determinations in plant tissue. In a standard mixture, the minimal detection level was less than 1 picomole of polyamines.     

High performance liquid chromatography of the dansyl derivatives of putrescine, spermidine, and spermine

A high performance liquid chromatographic (HPLC) method, based on dansylation and fluorescence detection, is described for the estimation of putrescine, spermidine, and spermine in lichen (Evernia prunastri [L.]) samples. Because of the high concentrations of phenols and salts, dansylation was followed by a pre-HPLC purification step. Both flow rate and mobile phase (methanol:water) followed a gradient for optimum resolution on a reverse-phase column. Amounts as small as 0.3 picomole of standard polyamines could be detected. In applying the method to lichens, it was found that 5.45% (w/w) of the exogenous putrescine taken up by the thallus was unbound in the algal partner and that 60% (w/w) was conjugated in the thallus, perhaps to lichen phenolics.     

Lack of detectable polyamines in an extremely halophilic bacterium

Polyamines (putrescine, spermidine, spermine and other analogs) were not detectable by the dansylation procedure coupled with HPLC analysis in an extremely halophilic bacterium, Halobacterium halobium. Based on the detection limit of this analytical method, we estimated that the polyamine content in H. halobium, if present, was less than 0.06% of that of E. coli. Putrescine uptake and the metabolic conversion of ornithine or arginine to polyamines were negligible in this bacterium. In a H. halobium cell-free extract, a saturated amount of KC1 was needed for poly(U) directed polyphenylalanine synthesis; neither putrescine nor spermidine could replace KC1. These results suggest that polyamines may play an insignificant role in the growth of this halophilic bacterium.     

Polyamines in the photosynthetic apparatus

The three main polyamines putrescine (Put), spermidine (Spd) and spermine (Spm) were characterized by HPLC in intact spinach leaf cells, intact chloroplasts, thylakoid membranes, Photosystem II membranes, the light-harvesting complex and the PS II complex. All contain the three polyamines in various ratios; the HPLC polyamine profiles of highly resolved PS II species (a Photosystem II core and the rection center) suggest an enrichment in the polyamine Spm.Abbreviations Chl chlorophyll - HPLC high performance liquid chromatography - LHC light-harvesting complex - PS II Photosystem II - PS II-RC Photosystem II reaction center - Put putrescine - Spd spermidine - Spm spermine - 10%S-core D₁-D₂-Cyt b559-47 kD-43 kD complex     

Simultaneous determination of histamine and polyamines by capillary zone electrophoresis with 4-fluor-7-nitro-2,1,3-benzoxadiazole derivatization and fluorescence detection

Capillary zone electrophoresis (CZE) with fluorescence detection was applied to the simultaneous determination of histamine and polyamines including spermine, spermidine, diaminopropane, putrescine, cadaverine, diaminohexane with 4-fluor-7-nitro-2,1,3-benzoxadiazole (NBD-F) as the fluorescent derivatization reagent. The seven NBD-F labeled amines was separated within 200 s using 85 mM phosphate running buffer at pH 3.0. The concentration limits of these amines ranged from 5.1 x 10(-8) M for spermine to 2.1 x 10(-8) M for histamine. The relative standard deviations for migration time and peak height were less than 1.5% and 6.0%, respectively. The method was successfully applied to the analysis of biogenic amines in the lysate of tobacco mesophyll protoplasts, and spermidine and putrescine were detected in the lysate with satisfying recovery.     

Chromophoric determination of putrescine, spermidine and spermine with dabsyl chloride by high-performance liquid chromatography and thin-layer chromatography

A fast and sensitive method for the determination of putrescine, spermidine, spermine and ammonia by high-performance liquid chromatography (HPLC) with dabsyl chloride is described. These compounds are converted to their chromophoric dabsyl derivatives and are separated by a normal-phase chromatographic column (μPorasil, 10 μm) with 2% acetone in chloroform as isocratic mobile phase. The sensitivity of the method is 20 pmoles. The present method was shown to be a straightforward procedure for estimating polyamines in various rat tissues.The chromophoric derivatives of polyamines are also well separated by thin-layer chromatography (TLC) on silica gel, and the combination of the HPLC and TLC procedures provides a reliable method for qualitative and quantitative analysis of polyamines.     

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.     

Androgenic control of polyamine concentrations in rat epididymis.

Unilateral orchidectomy resulted in a significant decrease in tissue content of putrescine and polyamines. However, no differences were detected when the results were expressed in terms of ng g-1 tissue. At 48 h after bilateral orchidectomy, a significant decrease in putrescine content was observed, but spermidine and spermine content were unaffected. The observed decrease in putrescine was prevented by treatment with testosterone propionate, but neither spermidine nor spermine were affected. Bilateral orchidectomy resulted in a significant decrease in the tissue content of putrescine, spermidine and spermine after 7 days. Treatment with testosterone propionate increased the content of putrescine, spermidine and spermine in the epididymis by about 200%, 92% and 34%, respectively. When results were expressed as nmol g-1, a significant decrease after castration in putrescine and spermidine, but not in spermine, was observed. Treatment with testosterone propionate restored putrescine concentration, but had no effect on spermidine and spermine concentrations. In castrated rats treated with testosterone propionate, the anti-androgen flutamide abolished the effect of the androgen on putrescine and spermidine content, but there was no effect on spermine. Acetylputrescine was not detected in the epididymis, while acetylpolyamines were detected at much lower concentrations than polyamines. After bilateral orchidectomy there was a decrease in the tissue content of all acetylpolyamines and an increase in their tissue concentration. The effect of castration on acetylpolyamine content was reversed by testosterone propionate treatment. We conclude that an active synthesis of polyamines occurs in the rat epididymis, and that this process depends upon the androgen environment. Regulation of ornithine decarboxylase activity appears to be the main step that is controlled by androgens.     

Active transport and metabolic characteristics of polyamines in the rat lens

Putrescine, spermidine and spermine were transported into the rat lens against a concentration gradient. This process appeared to be energy-dependent and involved a carrier system different from those for amino acids. Competition experiments suggested that the three polyamines were transported by the same system or very similar systems. Incorporated spermine was converted to spermidine and putrescine, and spermidine was converted to putrescine. In contrast, the conversion of putrescine to spermidine and spermine, or the conversion of spermidine to spermine was not observed. Furthermore, ornithine was not utilized for the synthesis of putrescine. These metabolic characteristics of the polyamines in the rat lens were correlated with the extremely low activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase. Other enzymes of polyamine metabolisms, however, were relatively active. In conclusion, the lens has a very low ability for the de novo synthesis of polyamines. The polyamines in the lens are considered to be supplied form the surrounding intraocular fluid by an active transport system specific for polyamines.     

Polyamine regulation of ornithine decarboxylase and its antizyme in intestinal epithelial cells

Ornithine decarboxylase (ODC) is feedback regulated by polyamines. ODC antizyme mediates this process by forming a complex with ODC and enhancing its degradation. It has been reported that polyamines induce ODC antizyme and inhibit ODC activity. Since exogenous polyamines can be converted to each other after they are taken up into cells, we used an inhibitor of S-adenosylmethionine decarboxylase, diethylglyoxal bis(guanylhydrazone) (DEGBG), to block the synthesis of spermidine and spermine from putrescine and investigated the specific roles of individual polyamines in the regulation of ODC in intestinal epithelial crypt (IEC-6) cells. We found that putrescine, spermidine, and spermine inhibited ODC activity stimulated by serum to 85, 46, and 0% of control, respectively, in the presence of DEGBG. ODC activity increased in DEGBG-treated cells, despite high intracellular putrescine levels. Although exogenous spermidine and spermine reduced ODC activity of DEGBG-treated cells close to control levels, spermine was more effective than spermidine. Exogenous putrescine was much less effective in inducing antizyme than spermidine or spermine. High putrescine levels in DEGBG-treated cells did not induce ODC antizyme when intracellular spermidine and spermine levels were low. The decay of ODC activity and reduction of ODC protein levels were not accompanied by induction of antizyme in the presence of DEGBG. Our results indicate that spermine is the most, and putrescine the least, effective polyamine in regulating ODC activity, and upregulation of antizyme is not required for the degradation of ODC protein.     

Polyamine metabolism in an obligately alkalophilic Bacillus alcalophilus that grows at pH 11.0

Bacillus alcalophilus, an obligately alkalophilic bacterium that grows at pH 11.0, has an intracellular pH of 9.5 or less. Unlike all other living organisms, polyamines (putrescine, spermidine and spermine) in B. alcalophilus, if present, will be largely unprotonated. HPLC analysis indicated that spermidine is the major polyamine in B. alcalophilus, accounting for more than 90% of total polyamines, and the level of spermidine varies during growth. Ornithine decarboxylase activity was not detectable in B. alcalophilus under all conditions examined. When [3H]arginine was added to the culture medium, the radioactivity can be recovered from polyamine pool; the distribution is 3% for putrescine, 94% for spermidine, and 3% for spermine, suggesting the presence of arginine pathway for polyamine biosynthesis. The polyamine transport system in B. alcalphilus appears to be Na+-dependent and is highly sensitive to the inhibition of gramicidin S and valinomycin.     

Simultaneous liquid chromatography determination of polyamines and arylalkyl monoamines

The diamines putrescine (PUT) and diaminopropane (DAP), the polyamines spermidine (SPD) and spermine (SPM), and the arylalkyl amines phenethylamine (PEA), tyramine (TYR), dopamine (DA), and salsolinol (SAL) were dansylated and baseline separated by LC using a Waters ODS-2 column. The dansyl derivatives were detected by fluorescence (lambda(ex): 337 nm; lambda(em): 520 nm). Besides the amine function, the phenolic OH groups of TYR, DA, and SAL were also dansylated (LC-MS, formation of N,O-didansyl [TYR] and N,O,O'-tridansyl derivatives [DA and SAL]). Calibration curves revealed response factors being appreciably lower for (N,O-didansyl) aminophenol TYR and (N,O,O'-tridansyl) DA and SAL than for N-dansylamines. However, the method is suitable as a cheap alternative to LC-MS for the simultaneous determination of polyamines and arylalkyl amines of large quantities of samples.     

Localization and biosynthesis of polyamines in insulin-producing cells.

Two recently developed fluorescence cytochemical methods, specific for spermidine and spermine, were used to localize polyamines in the endocrine pancreas. The polyamines were restricted to the insulin-producing beta-cells and were mainly associated with the secretory granules. Chemical polyamine determinations carried out on isolated rat and mouse pancreatic islets revealed large amounts of polyamines. Compared with extracts of whole pancreas, the islets contained very high concentrations of spermine relative to spermidine. Biosynthesis of polyamines from [3H]ornithine or from [3H]putrescine in isolated islets was significantly stimulated at high glucose concentrations. Moreover, significant incorporation of label from [3H]putrescine was also detected in gamma-aminobutyric acid. This incorporation, however, was not stimulated by high glucose. Possible roles for polyamines associated with the secretory granules in insulin-producing cells are discussed.     

High-performance liquid chromatographic determination of free and total polyamines in human serum as fluorescamine derivatives

A highly sensitive and simple fluorimetric method for the determination of free and total polyamines, spermidine, spermine, putrescine and cadaverine, in human serum by high-performance liquid chromatography is described. The polyamines, obtained after clean-up of deproteinized serum by Cellex P column chromatography, are converted to their fluorescamine derivatives in the presence of nickel ion which inhibits the reaction of interfering amines with fluorescamine, and the derivatives are separated simultaneously by reversed-phase chromatography (LiChrosorb RP-18) with a linear gradient elution. The lower limits of detection are 10 and 5 pmole for spermine and the others in 0.5 ml of serum, respectively.     

Regulation by polyamines of ornithine decarboxylase activity and cell division in the unicellular green alga Chlamydomonas reinhardtii

Polyamines are required for cell growth and cell division in eukaryotic and prokaryotic organisms. In the unicellular green alga Chlamydomonas reinhardtii, biosynthesis of the commonly occurring polyamines (putrescine, spermidine, and spermine) is dependent on the activity of ornithine decarboxylase (ODC, EC 4.1.1.17) catalyzing the formation of putrescine, which is the precursor of the other two polyamines. In synchronized C. reinhardtii cultures, transition to the cell division phase was preceded by a 4-fold increase in ODC activity and a 10- and a 20-fold increase, respectively, in the putrescine and spermidine levels. Spermine, however, could not be detected in C. reinhardtii cells. Exogenous polyamines caused a decrease in ODC activity. Addition of spermine, but not of spermidine or putrescine, abolished the transition to the cell division phase when applied 7 to 8 h after beginning of the light (growth) phase. Most of the cells had already doubled their cell mass after this growth period. The spermine-induced cell cycle arrest could be overcome by subsequent addition of spermidine or putrescine. The conclusion that spermine affects cell division via a decreased spermidine level was corroborated by the findings that spermine caused a decrease in the putrescine and spermidine levels and that cell divisions also could be prevented by inhibitors of S-adenosyl-methionine decarboxylase and spermidine synthase, respectively, added 8 h after beginning of the growth period. Because protein synthesis was not decreased by addition of spermine under our experimental conditions, we conclude that spermidine affects the transition to the cell division phase directly rather than via protein biosynthesis.     

Interconversion of putrescine,spermidine and spermine in goldfish and rat retina

Labelled putrescine is converted to spermidine and spermine in the retina of both the goldfish and of the rat, but the bulk remains as putrescine and spermidine in the goldfish retina whereas the bulk is present as spermine in the rat retina. Labelled spermidine is converted to spermine and to putrescine in the retina of both species, most remaining as spermidine in the goldfish retina whereas most is converted to spermine in the rat retina. Labelled spermine is converted to both spermidine and putrescine in the retina of both species with a greater conversion in the goldfish retina than in the rat retina. These results provide direct evidence of the interconversion of putrescine, spermidine and spermine in neural tissue from both fish and mammals and suggest that spermine should not be regarded solely as an end-product of putrescine metabolism but also as a source of spermidine and putrescine.The pattern of distribution of putrescine and the polyamines, spermidine and spermine, in goldfish retina is the reverse of that in rat retina: Putrescine is the most abundant in goldfish retina whereas spermine is most abundant in rat retina suggesting that the individual polyamines are of different importance in the two species.