高效液相色谱法测定人胎盘中的多胺

本文研究了一种简单而又快速的处理人胎盘的方法。用苯甲酰氯进行柱前衍生，衍生的多胺用反相高效液相色谱进行分离分析，最佳的衍生试剂的用量是７μｌ，衍生温度为３６．５℃。分析表明，老化胎盘中的精脒、精胺的含量比正常胎盘的要高得多，它们之间的差异非常显著；而正常胎盘中三种多胺含量和钙化胎盘的相比确无明显差异。这一结论可为临床检验胎盘老化与否提供科学依据。     

Determination of polyamine metabolome in plasma and urine by ultrahigh performance liquid chromatography–tandem mass spectrometry method: Application to identify potential markers for human hepatic cancer

To evaluate the potential relationship between cancer and polyamine metabolome, a UHPLC–MS/MS method has been developed and validated for simultaneous determination of polyamine precursors, polyamines, polyamine catabolite in human plasma and urine. Polyamine precursors including l-ornithine, lysine, l-arginine and S-adenosyl-l-methionine; polyamines including 1,3-diaminopropane, putrescine, cadaverine, spermidine, spermine, agmatine, N-acetylputrescine, N-acetylspermine and N-acetylspermidine; polyamine catabolite including γ-aminobutyric acid had been determined. The analytes were extracted from plasma and urine samples by protein precipitation procedure, and then separated on a Shim-pack XR-ODS column with 0.05% heptafluorobutyric acid (HFBA) in methanol and 0.05% HFBA in water. The detection was performed on UHPLC–MS/MS system with turbo ion spray source in the positive ion and multiple reaction-monitoring mode. The limits of quantitation for all analytes were within 0.125–31.25 ng mL⁻¹ in plasma and urine. The absolute recoveries of analytes from plasma and urine were all more than 50%. By means of the method developed, the plasma and urine samples from hepatic cancer patients and healthy age-matched volunteers had been successfully determined. Results showed that putrescine and spermidine in hepatic cancerous plasma were significant higher than those in healthy ones, while spermidine, spermine and N-acetylspermidine in hepatic cancerous urine were significant higher than those in healthy ones. The methods demonstrated the changes of polyamine metabolome occurring in plasma and urine from human subjects with hepatic cancer. It could be a powerful manner to indicate and treat hepatic cancer in its earliest indicative stages.     

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.     

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.     

Monitoring of biologically active amines in cereals and cereal based food products by HPLC

Summary Biologically active amines (putreanine sulphate, N-acetyl putrescine, putrescine, cadaverine, histamine, agmatine, N-acetyl spermidine, spermidine, spermine) were separated and quantified in cereal flour and cereal products by a liquid chromatographic method. The method consists of the separation of ion pairs formed between biologically active amines and octanesulphonic acid on a reversed-phase column, postcolumn derivatization with o-phtalaldehyde-2-mercapthoethanol and spectrofluorometric detection. Results of the reliability study were satisfactory. The method was linear for each amine at 1–10 mg L⁻¹. Putrescine and spermidine were the only amines always detected in cereal flour and cereal products, ranging from 2.45 to 47.83 mg kg⁻¹ for putrescine and 3.27 to 37.14 mg kg⁻¹ for spermidine. The most important differences among types of samples were found in polyamine derivatives. Presented at: Balaton Symposium on High-Performance Separation Methods, Siófok, Hungary, September 3–5, 1997.     

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.     

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.     

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.     

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.     

Determination of polyamines in human plasma by high‐performance liquid chromatography coupled with Q‐TOF mass spectrometry

A high‐performance liquid chromatography coupled with Q‐time of flight mass spectrometry (HPLC/Q‐TOF MS) method was developed and validated for the determination of 1, 3‐diaminopropane, putrescine, cadaverine, spermidine and spermine in human plasma. The plasma samples were first pretreated by 10% HClO₄ and then derived by benzoyl chloride with 1, 6‐diaminohexane as internal standard. The derived polyamines were separated on a C₁₈ column using a gradient program. The detection was performed on a Q‐TOF MS by positive ionization mode. Calibration curve for each polyamine was obtained in the concentration range of 0.4 ~ 200.0 ng ? ml^?1, with limit of detection of 0.02 ~ 0.1 ng ? ml^?1. The intra‐ and inter‐day RSD for all polyamines were 2.5–14.0% and 2.9 ~ 13.4%, respectively. The method was applied to determine the polyamines in human plasma from cancer patients and healthy volunteers. Results showed that the mean levels of polyamines in the plasma of cancer patients were higher than that of healthy volunteers, which suggested that the plasma polyamines could be employed as cancer diagnostic indicators in clinical testing. Copyright © 2012 John Wiley & Sons, Ltd.     

The use of selected ion flow tube mass spectrometry to detect and quantify polyamines in headspace gas and oral air

Polyamines are a class of aliphatic compounds which include putrescine, cadaverine, spermine and spermidine. They are involved in a variety of cellular processes and have been implicated in a number of different pathophysiological mechanisms. Polyamines are volatile compounds having a distinctive odour normally perceived as being unpleasant. The measurement of their abundance has, however, been restricted to compounds present in the aqueous phase. Using selected ion flow tube mass spectrometry (SIFT‐MS) we have shown that the polyamines react with the ions H₃O⁺, NO⁺ and O to form distinctive product ions allowing their levels to be quantified in the vapour phase. The low volatility of spermine did not allow extensive analysis of this compound by SIFT‐MS while the adherent properties of cadaverine and putrescine required the use of PTFE transfer lines and couplers. Our data suggested the presence of cadaverine and putrescine in both oral air and the headspace of putrefying bovine muscle, while product ions corresponding to putrescine and spermidine were found in the headspace of human semen. SIFT‐MS therefore appears to be a practical means of measuring vapour‐phase polyamine levels, having applications in biology, medicine and dentistry, and food science. Copyright © 2009 John Wiley & Sons, Ltd.     

Liquid chromatographic determination of polyamines in human urine based on intramolecular excimer-forming fluorescence derivatization using 4-(1-pyrene)butanoyl chloride

A liquid chromatographic method for highly sensitive and selective fluorometric determination of polyamines (putrescine, cadaverine, spermidine and spermine) in human urine is described. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(1-pyrene)butanoyl chloride (PBC), followed by reversed-phase liquid chromatography. The method offers higher sensitivity for determination of spermidine and spermine than previously reported method utilizing 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester as a derivatization reagent. Samples containing free polyamines in diluted human urine were directly derivatized with PBC and separated on an octyl column. The derivatives were detected at excitation 345 and emission 475 nm wavelengths. For determination of total polyamine content, the conjugated polyamines were first hydrolyzed in 4 M HCl. The detection limits (signal-to-noise ratio = 3) for polyamines in urine were 1.1-3.4 pmol/mL. At optimized derivatization and chromatographic conditions, interferences such as biogenic monoamines gave no peaks or the peaks did not interfere with the peaks of polyamine derivatives. In conclusion, the present derivatization method allows direct determination of polyamines in human urine samples without the need for sample clean-up procedures.     

Simplified micro-method for the quantitative analysis of putrescine, spermidine and spermine in urine

A simplified micro-method for the quantitative analysis of urinary polyamines is described. After acid hydrolysis of urine, the polyamines are converted to fluorescent 1-dimethylaminonaphthalene-5-sulfonyl (Dns; dansyl) derivatives and separated by means of thin-layer chromatography. Dns-NH2, which has been reported to interfere with the determination of putrescine, is well separated from di-Dns-putrescine. Putrescine, spermidine and spermine are quantitated by in situ scanning of their fluorescent spots on the chromatogram. The present method is both sensitive and reproducible. It eliminates a number of time-consuming steps and thus reduces preparative losses. Yet an adequate chromatographic resolution is obtained. Representative polyamine analyses of urine from normal volunteers and from cancer patients are reported. Elevated levels occur in the urines of pregnant women and of patients with various types of cancer.     

LC Analysis of Histamine and Other Biogenic Amines in Digesta Cultures

In the intestine, microbial protein degradation to histamine and other biologically active amines is believed to be relevant to the health of species. An LC method with fluorescence detection has been developed for analysis of histamine, phenylethylamine, isoamylamine, putrescine, cadaverine, tyramine, and spermidine. After pre-column derivatisation with 9-fluorenylmethyl chloroformate, amines were separated on a reversed-phase C₁₈ polyamine column with a mobile phase gradient. Validation of the method included calibration experiments, determination of amine recovery, and repeatability tests. The method proved especially useful for detection of histamine, phenylethylamine, putrescine, and cadaverine at relevant concentrations in caecum cultures from horses. The limits of quantification for these four amines ranged between 80 nM (histamine) and 400 nM (phenylethylamine).     

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.     

Simultaneous determination of 10 kinds of biogenic amines in rat plasma using high‐performance liquid chromatography coupled with fluorescence detection

We describe a simple, rapid, selective and sensitive HPLC method coupled with fluorescence detection for simultaneous determination of 10 kinds of biogenic amines (BAs: tryptamine, 2‐phenethylamine, putrescine, cadaverine, histamine, 5‐hydroxytryptamine, tyramine, spermidine, dopamine and spermine). BAs and IS were derivated with dansyl chloride. Fluorescence detection (λ_ex/λ_em = 340/510 nm) was used. A satisfactory result for method validation was obtained. The assay was shown to be linear over the ranges 0.005–1.0 μg/mL for tryptamine, 2‐phenethylamine and spermidine, 0.025–1.0 μg/mL for putrescine, 0.001–1.0 μg/mL for cadaverine, 0.25–20 μg/mL for histamine, 0.25–10 μg/mL for 5–hydroxytryptamine and dopamine, and 0.01–1.0 μg/mL for tyramine and spermine. The limits of detection and the limits of quantification were 0.3–75.0 ng/mL and 1.0–250.0 ng/mL, respectively. Relative standard deviations were ≤5.14% for intra‐day and ≤6.58% for inter‐day precision. The recoveries of BAs ranged from 79.11 to 114.26% after spiking standard solutions of BAs into a sample at three levels. Seven kinds of BAs were found in rat plasma, and the mean values of tryptamine, 2‐phenethylamine, putrescine, cadaverine, histamine, spermidine and spermine determined were 52.72 ± 7.34, 11.45 ± 1.56, 162.56 ± 6.26, 312.75 ± 18.11, 1306.50 ± 116.16, 273.89 ± 26.41 and 41.51 ± 2.07 ng/mL, respectively.     

Automated determination of polyamines by high-performance liquid chromatography with simple sample preparation

Recently, a new fully endcapped reversed-phase packing material, Inertsil, was introduced, especially suitable for the determination of basic compounds. We used this packing material to separate o-phthaldialdehyde (OPA) derivatives of amino acid derivatives completely from the OPA derivatives of spermine (SPM), spermidine (SPD), putrescine (PUT) and cadaverine (CAD). The obtained separation made the commonly used off-line extraction procedure redundant and thus an on-line sample clean-up was introduced. This enabled automation of the procedure resulting in a better reproducibility and a more efficient use of equipment. Furthermore, no studies are required to determine the extraction recovery.

The present method has a cycle time of 30 min. A linear response for each polyamine was found up to 250 pmol, with an R² ranging from 0.9981 (SPM) to 0.9998 (CAD). The limit of detection, calculated at a signal-to-noise ratio of 3, was 0.1 pmol, corresponding to a plasma concentration of 0.1 μmol/l. The coefficient of variation (CV) for the peak area was below 3% and for retention times below 0.5% (n=15).

In order to evaluate the applicability of the method, three different types of sample were chromatographed, e.g. urine (obtained from healthy human volunteers), pig plasma and sulfosalicylic acid homogenates of pig intestine biopsies. Tissue homogenates and urine-specimen could easily be quantitated, while plasma concentrations were just above the limit of detection, resulting in a plasma CV ranging from 4.8% (SPM) to 13.6% (SPD) and a tissue CV ranging from 2.1% (SPM) to 8.5% (CAD), The urinary CVs were not determined.

In conclusion, the present method provides an easy way to measure polyamine concentrations for most applications.     

An Electrochemical Flow Analysis System for Putrescine Using Immobilized Putrescine Oxidase and Horseradish Peroxidase

An electrochemical flow analysis system was optimized together with immobilized putrescine oxidase and horseradish peroxidase for putrescine measurement. Four coupling agents, suberic acid bis(N‐hydroxysuccinimide ester), γ‐maleimidobutyric N‐hydroxysuccinimide ester, 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide and glutaraldehyde, were used for immobilizing the two enzymes on porous aminopropyl glass beads to form a bienzymic detection column. Although the glutaraldehyde crosslinking procedure offered the highest response, the immobilized bienzyme system was responsive to putrescine, spermidine (123% of the putrescine response at 250 μM) and cadaverine (98% of the putrescine response). In contrast, the enzymes immobilized on the glass beads using suberic acid bis(N‐hydroxysuccinimide ester) offered significantly better selectivity towards putrescine at the same concentration. For comparison, cadaverine and spermidine only provoked a response of 4.7% and 27.5% of the putrescine signal. The response to cadaverine and spermidine was further suppressed by lowering the detection pH from 8 to 7. At 250 μM, the response obtained for cadaverine and spermidine was only 1.5% and 3.9%, respectively, of the signal obtained for putrescine. A linear response to putrescine was obtained from 5 to 75 μM (0.629 μAs μM^?1, R²=0.997) with a detection limit of 5 μM (S/N=3). The amperometric response retained 75% of its initial value after 600 repeated injections. The immobilized PUO/HRP (putrescine oxidase/horseradish peroxidase) was successfully demonstrated for measuring putrescine in fish extracts as an indicator of fish spoilage.     

Non-Covalent Interaction in Binary Thymidine/Polyamine Systems in Aqueous Solution

Results of equilibrium and NMR spectral studies have shown the formation of molecular complexes in the systems of thymidine with polyamines—ethylenediamine, 1,3-diaminopropane, putrescine, 3-aza-1,5-diaminopentane, 3-aza-1,6-diaminohexane, 4-aza-1,7-diaminoheptane, spermidine, 4,8-diaza-1,11-diaminoundecane or spermine. The overall stability constants of the adducts and the equilibrium constants of their formation have been determined. Relative to adenosine or cytidine, the pH range of complex formation is shifted towards higher values, which is a consequence of a significantly higher basicity of thymine and corresponds well with the assumed model of ion-ion or ion-dipole interactions. The pH range of adduct formation is found to coincide with that in which the polyamine is protonated and the thymidine deprotonated. The -NH₃⁺ groups from polyamine and the N(3) atom from thymidine have been identified as the centers of noncovalent interactions. The stability of the molecular complexes formed in the studied systems depends on the acid-base character of the substrates and on the structure of the reacting molecules.     

Metformin Inhibits the Urea Cycle and Reduces Putrescine Generation in Colorectal Cancer Cell Lines

The urea cycle (UC) removes the excess nitrogen and ammonia generated by nitrogen-containing compound composites or protein breakdown in the human body. Research has shown that changes in UC enzymes are not only related to tumorigenesis and tumor development but also associated with poor survival in hepatocellular, breast, and colorectal cancers (CRC), etc. Cytoplasmic ornithine, the intermediate product of the urea cycle, is a specific substrate for ornithine decarboxylase (ODC, also known as ODC1) for the production of putrescine and is required for tumor growth. Polyamines (spermidine, spermine, and their precursor putrescine) play central roles in more than half of the steps of colorectal tumorigenesis. Given the close connection between polyamines and cancer, the regulation of polyamine metabolic pathways has attracted attention regarding the mechanisms of action of chemical drugs used to prevent CRC, as the drug most widely used for treating type 2 diabetes (T2D), metformin (Met) exhibits antitumor activity against a variety of cancer cells, with a vaguely defined mechanism. In addition, the influence of metformin on the UC and putrescine generation in colorectal cancer has remained unclear. In our study, we investigated the effect of metformin on the UC and putrescine generation of CRC in vivo and in vitro and elucidated the underlying mechanisms. In nude mice bearing HCT116 tumor xenografts, the administration of metformin inhibited tumor growth without affecting body weight. In addition, metformin treatment increased the expression of monophosphate (AMP)-activated protein kinase (AMPK) and p53 in both HCT116 xenografts and colorectal cancer cell lines and decreased the expression of the urea cycle enzymes, including carbamoyl phosphate synthase 1 (CPS1), arginase 1 (ARG1), ornithine trans-carbamylase (OTC), and ODC. The putrescine levels in both HCT116 xenografts and HCT116 cells decreased after metformin treatment. These results demonstrate that metformin inhibited CRC cell proliferation via activating AMPK/p53 and that there was an association between metformin, urea cycle inhibition and a reduction in putrescine generation.