Determination of the polyamine content of rat heart mitochondria by the use of heparin-sepharose

Heparin-sepharose has been utilized to remove polyamines adsorbed to the cytoplasmic surface of rat heart mitochondria. The results obtained can be summarized as follows: Heparin-sepharose removes 90% of the spermine, 98% of the spermidine, and 98% of the putrescine adsorbed. Polyamine contents of chromatographed mitochondria amount to 2.66 and 0.36 nmol spermine and spermidine, respectively, per mg of mitochondrial protein.     

Heparin-sepharose as a tool in the subcellular fractionation of a polyamine-rich organ (rat ventral prostate)

Heparin-sepharose forms complexes with putrescine, spermidine, and spermine and indirect measurements of its affinity for polyamines gives values similar to those obtained with free heparin. A direct measurement of the binding of heparin-sepharose to spermine gives an apparent dissociation constant (K _d) of 1.5×10⁻⁶ M spermine. Unlike free heparin, heparin-sepharose does not cause either disruption of the nuclei or more sutble modifications able to modify their sedimentation behavior. The heparin-sepharose polyamine complex formed by the addition of heparin-sepharose to the homogenate can easily be removed and the homogenate can be processed according to normal schedules. Heparin-sepharose is able to sequester 85% of the exchangeable spermine present in the homogenate of rat ventral prostate. The distribution of the marker enzyme galactosyltransferase (Golgi apparatus) on a sucrose density gradient was followed to assess the usefulness of heparin-sepharose in minimizing the aggregation of cellular organelles brought about by polyamines.     

Determination of polyamines by flow injection analysis with a chemiluminescence detector based on their complexation with copper(II).

Through the flow injection analysis experiments, we discovered that an unsaturated complex of Cu(II) and polyamines (spermine, spermidine, putrescine) had a strongly catalytic effect on luminol-H(2)O(2) chemiluminescence (CL) reaction, and that the CL intensity is proportional to the concentrations of polyamines. Based on the automatic formation of an unsaturated complex of polyamines and Cu(II) when the solution containing polyamines passed through a column packed with solid Cu(OH)(2), a new flow injection chemiluminescence analysis method was proposed for the determination of polyamines. The effects of pH, buffer concentration, the concentration of chemiluminescence reagent, and the influence of mixing coil length were examined. Under optimal conditions, the linear range was from 1.0 x 10(-7) mol L(-1) to 1.0 x 10(-5) mol L(-1), and the detection limits were 0.17, 0.38, 0.44 pmol for spermine, spermidine, and putrescine, respectively. Compared with other methods, the advantages of this method include convenience, time-saving and low cost.     

High performance liquid chromatography of biological polyamines using immobilized enzyme as post-column reactor followed by electrochemical detection

A novel analytical method for biological polyamines (putrescine, spermidine and spermine) was developed. Polyamines were separated by ion-pair reversed phase chromatography using a polymer-based octadecyl bonded column. A polyamine oxidase immobilized column worked effectively as a post-column reactor to convert polyamines to hydrogen peroxide which was eventually detected by electrochemical oxidation on platinum electrode. This method required neither tedious derivatization nor gradient elution, permitting us to perform simple and rapid analysis of polyamines. The detection limits were 0.3, 0.6, and 4 pmol injected for putrescine, spermidine, and spermine, respectively with a linear range of two to three orders of magnitude. Chromatograms obtained with samples from human urine and rat brain homogenates demonstrated the high sensitivity and selectivity of the method.     

Separation of polyamines, conjugated to DNA, by reversed-phase high-performance liquid chromatography

Genomic DNA was isolated from the lichen Evernia prunastri in order to analyze by high-performance liquid chromatography the occurrence of polyamines conjugated to the macromolecule. The acid-insoluble (PH) fraction of this DNA contained mainly conjugated spermidine, although small amounts of free putrescine and spermidine were also present. The PH fraction of DNA also contained conjugated evernic acid, the main phenol produced by this lichen species. Conjugation of polyamines to calf thymus DNA was carried out under in vitro conditions. Conjugation was to spermidine and mainly to spermine and produced DNA compactation. Evernic acid enhanced the action of polyamines in order to produce DNA aggregation.     

Reversed-Phase Ion-Pairing Liquid Chromatographic Separation and Fluorimetric Detection of Polyamines

Abstract

A rapid and specific reversed-phase ion-pairing high performance liquid chromatographic procedure for putrescine, spermidine and spermine is reported. The ion-pairing reagent, heptanesulfonate, was employed and o-phthalaldehyde and 2-mercaptoethanol were used for on-line post-column derivatization and subsequent fluorescence detection. Experiments were carried out to determine the effects of several variables such as pH, concentration of the aqueous buffer, counter-ion concentration, and the percentage of organic modifier in the moving phase. The minimum detection limits for the polyamines ranged from 120 pmoles for spermine to 12 pmoles for putrescine. The method includes a gradient program which provides complete separation from amino acids and specificity for the three polyamines. The procedure was applied successfully to urine and serum samples.     

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 15 pmole for spermine and the others in 0.5 ml of serum, respectively.     

Determination of polyamines in urine of normal human and cancer patients by capillary gas chromatography

A capillary gas chromatographic method is described for the determination of polyamines (putrescine, spermidine and spermine) in the urine of normal human and cancer patients. Morning urine after acid hydrolysis is cleaned up on a silica gel column and derivatized with trifluoroacetic-anhydride. Creatinine in human urine is used as internal standard. Recoveries of polyamines are 96.7% putrescine, 102.6% spermidine (Spd), and 98.7% spermine. SD of the method for Spd is 1.949 +/- 0.041 (micrograms/mg creatinine, mean +/- SD, n = 5). The results show that the mean level of polyamines in cancer patients urine is much higher than that in normal human urine. The mean of total polyamines in the normal human and the cancer patients is 2.01 and 44.74, respectively (g/mg creatinine).     

Selective fluorometric detection of polyamines using micellar electrokinetic chromatography with laser-induced fluorescence detection

The polyamines putrescine, cadaverine, spermine and spermidine were separated and quantified by micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence detection. The derivatization reagent, 1-pyrenebutanoic acid succinimidyl ester (PSE), allowed for the selective detection of the polyamines at 490 nm. Multiple labeling of the polyamines with PSE allows the formation of intramolecular excimers that emit at longer wavelengths (450-520 nm) than mono-labeled analytes (360-420 nm). Optimal separation of the labeled polyamines was achieved using a separation buffer consisting of 10 mM phosphate pH 7.2, 30 mM cholate, and 30% acetonitrile. Using these conditions, the four polyamines were separated in under 10 min. Limits of detection for putrescine, cadaverine, spermine and spermidine were 6, 5, 15 and 13 nM, respectively. These are superior or comparable to those previously reported in the literature using fluorescence detection.     

HPLC of 9-fluorenylmethylchloroformate-polyamine derivatives with fluorescence detection

Summary There are a number of reagents available for fluorescent labelling of primary amines. These include dansyl chloride, o-phthalaldehyde, fluorescamine, and a new reagent, 9-fluorenylmethylchloroformate (FMOC), reported recently. This paper describes a reversed-phase HPLC procedure for the separation and fluorescence detection of polyamines following pre-column derivatization with FMOC. The polyamines studied by this method include putrescine, cadaverine, spermidine, and spermine. Experiments were carried out to determine maximum fluorescence excitation and emission wavelengths, optimum reaction pH, linear ranges, and minimum detection limits for each of the polyamines. The HPLC method includes a gradient program which provides complete separation from serum hydrolysate components and specificity for the four polyamines with detection limits ranging from 2 to 9 pg. This procedure was applied to hydrolyzed serum samples.     

The polyamines of Xanthium strumarium and their response to photoperiod

Flowering plants of Xanthium strumarium L., grown in 8 h photoperiods, were analysed for polyamines. Putrescine, spermidine and spermine were found throughout the plant in three forms: (a) as free polyamines; (b) conjugates soluble in 5% trichloracetic acid (TCA); and (c) bound to the TCA-insoluble precipitate. On a fresh weight basis, total polyamines are most abundant in young leaves and buds, especially flower buds. Spermidine predominates in the free polyamine fractions, while spermine is dominant in the conjugated fraction. Transfer of vegetative plants from 16 h photoperiods to 1, 2, 3, or 4 inductive cycles (8 h light + 16 h uninterrupted dark) caused rapid and marked changes in the polyamine titer of the leaves and ultimately, floral initiation. The titer of free putrescine per mg protein declined progressively with induction in all leaf sizes, while the titers of free spermidine and spermine rose during days 2 and 3 in small and expanding leaves. Conjugated putrescine, spermidine and spermine rose sharply after only 1 inductive cycle, especially in small and expanding leaves, and maintained the higher level for at least several cycles. In plants given 4 inductive cycles, buds harvested after 4 additional days had sharply elevated levels of conjugated polyamines, especially spermine, on a protein basis.     

Analysis of polyamines as carbamoyl derivatives in urine and serum by liquid chromatography-tandem mass spectrometry

A quantitative analysis of polyamines in urine and serum by liquid chromatography-tandem mass spectrometry (LC-MS/MS) is described. The polyamines were carbamylated with isobutyl chloroformate, extracted with diethyl ether under pH 9.0, and analyzed by LC-MS/MS with single reaction monitoring mode. The limit of quantification was 1 ng/mL based on a signal-to-noise ratio>3, and the correlation coefficient (r2) for the calibration curves was >0.99 for both urine and serum samples. The present method was applied to urine and serum samples from 30 breast cancer patients and 30 normal female controls. There was no significant difference in the urinary polyamine levels between breast cancer patients and controls. However, 1,3-diaminopropane, putrescine, spermine and N-acetylspermidine levels in serum increased in breast cancer patients. These four serum polyamines may be a good index to study both production and metabolism of polyamines, and a useful tool in assessment of the polyamine status of breast cancer patients.     

Reversed-phase liquid chromatographic separation and simultaneous fluorimetric detection of polyamines and their monoacetyl derivatives in human and animal urine, serum and tissue samples: an improved, rapid and sensitive method for routine application

A highly sensitive and precise method for the determination of the polyamines putrescine, cadaverine, spermidine and spermine and all their monoacetyl derivatives in a single analysis in human and animal urine, serum and tissue samples is described. For polyamine separation, an ion-pairing reversed-phase high-performance liquid chromatographic (HPLC) method is used, followed by post-column derivatization with o-phthalaldehyde and consecutive fluorescence detection. Urine and serum samples are purified with a Bond Elut silica cartridge. The detection limit for polyamines is 0.5-1.0 pmol and excellent linearity is achieved in the range from 3 pmol up to more than 10 nmol. The influence of some modifications of different analytical steps such as the temperature of the HPLC column and the derivatization reaction coil and the o-phthalaldehyde flow-rate is described. Quality control data and measurements of the reproducibility of the method are presented. In order to establish a rapid analytical method for easy routine use, all steps for preparation and quantitative analysis are minimized. This method was applied to the determination of total polyamines in human urine and serum hydrolysate and of free and acetylated polyamines in human urine and pancreatic tissue of the rat. Values for normal polyamine concentrations in the urine and serum of fifteen male and fifteen female healthy volunteers and in the pancreas of ten normal rats are presented.     

Analysis of dansyl derivatives of di- and polyamines in mouse brain, human serum and duodenal biopsy specimens by high-performance liquid chromatography on a standard reversed-phase column

The concentrations of putrescine, spermine and spermidine were measured in human serum, children's duodenal biopsy specimens and mouse brain homogenates by high-performance liquid chromatography. The chromatographic analysis was performed on dansyl derivatives of the polyamines using a reverse-phase system with an ion-pairing retention mechanism (heptane sulphonate). Capacity factors were determined at different concentrations of acetonitrile. Simple linear gradients were set up for fast (15 min) or routine (25 min) analysis. Three fluorescence detectors were compared for these determinations and their detection limits determined. The minimum detectable amount of polyamines was 25 fmol compared to 500 fmol with standard detectors. While samples prepared from tissues did not require a high sensitivity, a detector of better performance was needed to assay the polyamines in human serum.     

Sensitive fluorimetric method for the determination of putrescine, spermidine and spermine by high-performance liquid chromatography and its application to human blood

A fast and sensitive method for the determination of putrescine, spermidine and spermine by high-performance liquid chromatography is described. These compounds are converted to their fluorescent dansyl derivatives and are separated by a reversed-phase chromatographic system (Micropak CH-10) with water and acetonitrile as mobile phase. The sensitivity of the method is 30 pmoles. The application of the method to the determination of polyamines in blood is described. It was found that most of the polyamines circulating in blood are localized in the erythrocytes, their content in normal human blood being spermidine 14.1 +/- 3.1, and spermine 8.4 +/- 2.8 nmoles/ml packed erythrocytes. The polyamine level in serum is less than 0.1 nmole/ml. The polyamine content of the erythrocytes from patients with malignant neoplasms was significantly elevated.     

Polyamine distribution in the rat intestinal mucosa

As the first step in a study of mucosal polyamine metabolism during intestinal adaptation, we have measured mucosal polyamine concentrations at different sites along the normal rat intestine. Putrescine, spermidine, spermine and cadaverine were measured by spectrofluorometric analysis after thin-layer chromatography of their dansylated derivatives. Spermidine was present in the largest amounts at each of the sampling sites. The ratio of the concentration of spermidine to that of spermine paralleled the established pattern of cellular proliferation in the normal intestine as did the putrescine concentration (nmol per 10 cm) which decreased from duodenum to colon. These results provide the essential background to an assessment of the role of polyamines in the intestinal adaptive response.     

HPLC analysis of polyamines and their acetylated derivatives in the picomole range using benzoyl chloride and 3,5-dinitrobenzoyl chloride as derivatizing agent

Analytical methods are described for the quantitative determination of putrescine, cadaverine, spermidine, spermine and the acetylated derivatives of spermidine and spermine in biological fluids using pre-column derivatization with either benzoyl chloride or 3,5-dinitrobenzoyl chloride, which were added to each sample as solutions in diethyl ether. Putrescine, spermidine and spermine can be analysed in seminal plasma at nanogram levels when benzoyl chloride is used as derivatizing agent. In the analysis of putrescine, cadaverine, spermidine and acetyl derivatives of spermidine and spermine, higher sensitivity is obtained with 3,5-dinitrobenzoyl-chloride. This method can readily be used in the determination of acetylated polyamines in urine samples.     

An improved and easy technique for polyamine determination in biological samples. Application to cell-free system from hypertrophied rat heart.

An accurate, improved cation-exchange chromatographic method using o-phthalaldehyde and ultraviolet detection at 280 nm for the determination of free polyamines (putrescine, spermidine, spermine) has been developed. Different samples, such as the 105,000 g supernatant of reticulocyte or heart muscle, and KCl ribosomal wash containing initiation factors, can be analysed. The minor modification of reagents results in a good precision and sensitivity, which is demonstrated by a relative standard deviation of 5--9% and recoveries of 98%. This technique is of particular interest because it allows polyamine determination in biological samples with high concentrations of salt.     

Determination of polyamines in urine via electrospun nanofibers–based solid-phase extraction coupled with GC-MS and application to gastric cancer patients

The simultaneous determination of polyamines and their metabolites in urine samples was achieved by gas chromatography–mass spectrometry in the selected ion monitoring mode. After conjugating with the ion-pair reagent bis-2-ethylhexylphosphate in the aqueous phase, the polyamines in the samples were extracted with polystyrene nanofiber-based packed-fiber solid-phase extraction followed by a derivatization step using pentafluoropropionyl anhydride. With optimal conditions, all analytes were separated well. For analytes of putrescine, cadaverine, N-acetylputrescine, and N-acetylcadaverine, the linearity was good in the range of 0.05–500 μmol/L (R² ≥ 0.993). While for spermidine, spermine, acetylspermidine, N⁸-acetylspermidine, and N-acetylspermine, the linearity was good in the range of 0.5–500 μmol/L (R² ≥ 0.990). The recoveries of three spiked concentrations (0.5, 5, 300 μmol/L) were 85.6%–108.4%, and relative standard deviations for intra- and interday were in the range of 2.9%–13.4% and 4.5%–15.1%, respectively. The method was successfully applied to the analysis of urine samples of gastric cancer patients. The results showed that the levels of most polyamines and N-acetylated polyamines from the patient group were significantly higher than those from the control group. The altered concentrations of the above-mentioned metabolites suggest their role in the pathogenesis of gastric cancer, and they should be further evaluated as potential markers of gastric cancer.     

Rapid and Simple Technique for the Quantitation of Polyamines in Biological Samples

Abstract

A rapid and simple technique has been developed to quantify putrescine, spermidine, and spermine in biological tissue. The method, based upon several published procedures, involves protein precipitation with perchloric acid followed by dansylation with 5-dimethylamino-1-naphthalenesulfonyl chloride (dansyl chloride). After extraction on a Waters C₁₈ Sep-Pak cartridge, the samples are analyzed by high pressure liquid chromotography using a step solvent change and a 3μ C₁₈ reverse phase column. The chromotographic conditions allowed complete analysis of the three polyamines within 10 min with a total run time of 13 min (sample injection and re-equilibrium of column). Standard curves were linear up to 1 μg polyamine and the coefficient of variation for the assay ranged from 4% at l μg polyamine per sample to 11% at 50 ng polyamine per sample. The assay is therefore both rapid and simple. Moreover, unlike other available methods, the present technique does not require duel pumps, ion pairing agents, solvent extraction or a gradient control system. The concentrations of putrescine, spermidine and spermine in rat lung, liver and kidney are reported.