Chronic stress decreases ornithine decarboxylase expression and protects against 1,2-dimethylhydrazine-induced colon carcinogenesis.

Biological response to stress depends on the type, timing, and severity of the stressor. Acute stressful environments may positively activate molecular and cellular mechanisms to favor adaptation; however, chronic stress is often associated with detrimental health effects. Colon cancer (CC) is one of the leading causes of death associated with cancer and has been mentioned as a stress-related disease. In the present work, the effect of chronic stress on the initial phase of CC was evaluated, and special emphasis was placed on ornithine decarboxylase (ODC) expression and polyamines for their role in hyperproliferative diseases. BALB/c mice (n = 5/group) were administered the pro-carcinogen 1,2-dimethylhydrazine (DMH) for 8 weeks (20 mg/kg body weight/week) to induce colon carcinogenesis, and then exposed for 4 weeks to two physical stressors: restraint and forced-swimming. Distal colon inflammatory lesions and histomorphological changes were evaluated by hematoxylin-eosin staining; plasma corticosterone levels, colon ODC expression, and urinary polyamines were determined by competitive ELISA, RT-qPCR, Western Blot, and HPLC, respectively. The short-term exposure to DMH triggered colon inflammation, initiated colon carcinogenesis and increased ODC expression; meanwhile, the exposure to chronic stress activated the hypothalamic-pituitary-adrenal (HPA) axis, elicited the production of plasmatic corticosterone, and decreased ODC expression. The exposure of DMH-treated mice to chronic stress counteracted the inflammatory effect of DMH and maintained ODC homeostasis. In early phase of carcinogenesis, the exposure of DMH-treated mice to chronic stress had a positive effect against colon inflammation and maintained ODC homeostasis. The cross-talk between corticosterone, ODC expression, and inflammation in a tumor environment is discussed.

Sex-related differences in urinary immune-related metabolic profiling of alopecia areata patients.

INTRODUCTION: Alopecia areata is a well-known autoimmune disease affecting humans. Polyamines are closely associated with proliferation and inflammation, and steroid hormones are involved in immune responses. Additionally, bile acids play roles in immune homeostasis by activating various signaling pathways; however, the roles of these substances and their metabolites in alopecia areata remain unclear. OBJECTIVES: In this study, we aimed to identify differences in metabolite levels in urine samples from patients with alopecia areata and healthy controls. METHODS: To assess polyamine, androgen, and bile acid concentrations, we performed high-performance liquid chromatography-tandem mass spectrometry. RESULTS: Our results showed that spermine and dehydroepiandrosterone levels differed significantly between male patients and controls, whereas ursodeoxycholic acid levels were significantly higher in female patients with alopecia areata than in controls. CONCLUSION: Our findings suggested different urinary polyamine, androgen, and bile acid concentrations between alopecia areata patients and normal controls. Additionally, levels of endogenous substances varied according to sex, and this should be considered when developing appropriate treatments and diagnostic techniques. Our findings improve our understanding of polyamine, androgen, and bile acid profiles in patients with alopecia areata and highlight the need to consider sex-related differences.

Gender-Related Differences on Polyamine Metabolome in Liquid Biopsies by a Simple and Sensitive Two-Step Liquid-Liquid Extraction and LC-MS/MS.

Polyamines are involved in the regulation of many cellular functions and are promising biomarkers of numerous physiological conditions. Since the concentrations of these compounds in biological fluids are low, sample extraction is one of the most critical steps of their analysis. Here, we developed a comprehensive, sensitive, robust, and high-throughput LC-MS/MS stable-isotope dilution method for the simultaneous determination of 19 metabolites related to polyamine metabolism, including polyamines, acetylated and diacetylated polyamines, precursors, and catabolites from liquid biopsies. The sample extraction was optimized to remove interfering compounds and to reduce matrix effects, thus being useful for large clinical studies. The method consists of two-step liquid-liquid extraction with a Folch extraction and ethyl acetate partitioning combined with dansyl chloride derivatization. The developed method was applied to a small gender-related trial concerning human serum and urine samples from 40 obese subjects. Sex differences were found for cadaverine, putrescine, 1,3-diaminopropane, γ-aminobutyric acid, N8-acetylspermidine, and N-acetylcadaverine in urine; N1-acetylspermine in serum; and spermine in both serum and urine. The results demonstrate that the developed method can be used to analyze biological samples for the study of polyamine metabolism and its association with human diseases.

Metabolomic study of polyamines in rat urine following intraperitoneal injection of γ-hydroxybutyric acid.

INTRODUCTION: Recently, illegal abuse of γ-hydroxybutyric acid (GHB) has increased in drug-facilitated crimes, but the determination of GHB exposure and intoxication is difficult due to rapid metabolism of GHB. Its biochemical mechanism has not been completely investigated. And a metabolomic study by polyamine profile and pattern analyses was not performed in rat urine following intraperitoneal injection with GHB. OBJECTIVES: Urinary polyamine (PA) profiling by gas chromatography-tandem mass spectrometry was performed to monitor an altered PA according to GHB administration. METHODS: Polyamine profiling analysis by gas chromatography-mass spectrometry combined with star pattern recognition analysis was performed in this study. The multivariate statistical analysis was used to evaluate discrimination among control and GHB administration groups. RESULTS: Six polyamines were determined in control, single and multiple GHB administration groups. Star pattern showed distorted hexagonal shapes with characteristic and readily distinguishable patterns for each group. N1-Acetylspermine (p < 0.001), putrescine (p < 0.006), N1-acetylspermidine (p < 0.009), and spermine (p < 0.027) were significantly increased in single administration group but were significantly lower in the multiple administration group than in the control group. N1-Acetylspermine was the main polyamine for discrimination among control, single and multiple administration groups. Spermine showed similar levels in single and multiple administration groups. CONCLUSIONS: The polyamine metabolic pattern was monitored in GHB administration groups. N1-Acetylspermine and spermine were evaluated as potential biomarkers of GHB exposure and addiction.

Altered Polyamine Profiles in Colorectal Cancer.

BACKGROUND: The declining mortality rate of patients with colorectal cancer (CRC) can be explained, at least partially, with early diagnosis. Simple diagnostic methods are needed to achieve a maximal patient participation rate in screening. MATERIALS AND METHODS: Liquid chromatography electrospray tandem mass spectrometry (LC-MS/MS) was used to determine urinary polyamine (PA) profiles. In a prospective setting, 116 patients were included in the study: 57 with CRC, 13 with inflammatory bowel disease (IBD), 12 with adenoma, and 34 controls. RESULTS: N1,N12-diacetylspermine (DiAcSPM) level was significantly higher in patients with CRC than controls (sensitivity=78.0%, specificity=70.6%; p=0.00049). The level of diacetylated cadaverine (p=0.0068) was lower and that of diacetylated putrescine (p=0.0078) was higher in patients with CRC than in those with IBD. Cadaverine (p=0.00010) and spermine (p=0.042) levels were lower and that of DiAcSPM (p=0.018) higher in patients with CRC than in those with adenoma. CONCLUSION: The simultaneous determination of urinary PAs by means of LC-MS/MS can be used to discriminate CRC from controls and patients with benign colorectal diseases.

Urinary Polyamine Biomarker Panels with Machine-Learning Differentiated Colorectal Cancers, Benign Disease, and Healthy Controls.

Colorectal cancer (CRC) is one of the most daunting diseases due to its increasing worldwide prevalence, which requires imperative development of minimally or non-invasive screening tests. Urinary polyamines have been reported as potential markers to detect CRC, and an accurate pattern recognition to differentiate CRC with early stage cases from healthy controls are needed. Here, we utilized liquid chromatography triple quadrupole mass spectrometry to profile seven kinds of polyamines, such as spermine and spermidine with their acetylated forms. Urinary samples from 201 CRCs and 31 non-CRCs revealed the N1,N12-diacetylspermine showing the highest area under the receiver operating characteristic curve (AUC), 0.794 (the 95% confidence interval (CI): 0.704-0.885, p < 0.0001), to differentiate CRC from the benign and healthy controls. Overall, 59 samples were analyzed to evaluate the reproducibility of quantified concentrations, acquired by collecting three times on three days each from each healthy control. We confirmed the stability of the observed quantified values. A machine learning method using combinations of polyamines showed a higher AUC value of 0.961 (95% CI: 0.937-0.984, p < 0.0001). Computational validations confirmed the generalization ability of the models. Taken together, polyamines and a machine-learning method showed potential as a screening tool of CRC.

Development of a fast and simple gas chromatographic protocol based on the combined use of alkyl chloroformate and solid phase microextraction for the assay of polyamines in human urine.

Polyamines are aliphatic amines with low molecular weight that are widely recognized as one of the most important cancer biomarkers for early diagnosis and treatment. The goal of the work herein presented is the development of a rapid and simple method for the quantification of free polyamines (i.e., putrescine, cadaverine, spermidine, spermine) and N-monoacetylated polyamines (i.e., N1-Acetylspermidine, N8-Acetylspermidine, and N1-Acetylspermine) in human urine. A preliminary derivatization with propyl chloroformate combined with the use of solid phase microextraction (SPME) allowed for an easy and automatable protocol involving minimal sample handling and no consumption of organic solvents. The affinity of the analytes toward five commercial SPME coatings was evaluated in univariate mode, and the best result in terms of analyte extraction was achieved using the divinylbenzene/carboxen/polydimethylsiloxane fiber. The variables affecting the performance of SPME analysis were optimized by the multivariate approach of experimental design and, in particular, using a central composite design (CCD). The optimal working conditions in terms of response values are the following: extraction temperature 40 °C, extraction time of 15 min and no addition of NaCl. Analyses were carried out by gas chromatography-triple quadrupole mass spectrometry (GC-QqQ-MS) in selected reaction monitoring (SRM) acquisition mode. The developed method was validated according to the guidelines issued by the Food and Drug Administration (FDA). The satisfactory performances reached in terms of linearity, sensitivity (LOQs between 0.01 and 0.1 µg/mL), matrix effect (68-121%), accuracy, and precision (inter-day values between -24% and +16% and in the range 3.3-28.4%, respectively) make the proposed protocol suitable to be adopted for quantification of these important biomarkers in urine samples.

Polyamine Metabolites Profiling for Characterization of Lung and Liver Cancer Using an LC-Tandem MS Method with Multiple Statistical Data Mining Strategies: Discovering Potential Cancer Biomarkers in Human Plasma and Urine.

Polyamines, one of the most important kind of biomarkers in cancer research, were investigated in order to characterize different cancer types. An integrative approach which combined ultra-high performance liquid chromatography-tandem mass spectrometry detection and multiple statistical data processing strategies including outlier elimination, binary logistic regression analysis and cluster analysis had been developed to discover the characteristic biomarkers of lung and liver cancer. The concentrations of 14 polyamine metabolites in biosamples from lung (n = 50) and liver cancer patients (n = 50) were detected by a validated UHPLC-MS/MS method. Then the concentrations were converted into independent variables to characterize patients of lung and liver cancer by binary logic regression analysis. Significant independent variables were regarded as the potential biomarkers. Cluster analysis was engaged for further verifying. As a result, two values was discovered to identify lung and liver cancer, which were the product of the plasma concentration of putrescine and spermidine; and the ratio of the urine concentration of S-adenosyl-l-methionine and N-acetylspermidine. Results indicated that the established advanced method could be successfully applied to characterize lung and liver cancer, and may also enable a new way of discovering cancer biomarkers and characterizing other types of cancer.

Use of microextraction by packed sorbents and gas chromatography-mass spectrometry for the determination of polyamines and related compounds in urine.

A novel methodology for the determination of ornithine, putrescine, cadaverine, spermidine and gamma-amino butyric acid in urine samples has been developed. The method uses in situ aqueous derivatization followed by automated microextraction by packed sorbent coupled to a gas chromatography-mass spectrometry system equipped with a programmed temperature vaporizer. This instrumental configuration minimizes sample manipulation due to from the mixing of the reagents, the process is completely automated. The analytes were derivatized using ethyl chloroformate as derivatization reagent. The reaction occurred in aqueous medium and was carried out in 1min in the vial of an autosampler used to perform microextraction by packed sorbent. The parameters affecting derivatization, extraction and separation were optimized in order to obtain maximum sensitivity. Calibration curves were obtained for five calibration levels in three different matrices. All the calibration models displayed good linearity, with R(2) values higher than 0.95. The validity of the models was checked using ANOVA, and it was observed that they did not exhibit any lack of fit. Repeatability and reproducibility was evaluated, with values below 15% in both cases. LOD and LOQ values were found to be in the low µg/L level. Influence of the matrix was confirmed, thus quantification was performed using the standard additions method and normalization to IS. The method developed was applied to the analysis of these compounds in urine samples from healthy individuals and cancer diagnosed patients (Internal Medicine Unit of the Virgen de la Vega Hospital, Salamanca, Spain). Significant differences (Mann-Whitney U test) were observed for putrescine and ornithine concentrations.

Metabolomic study of urinary polyamines in rat exposed to 915 MHz radiofrequency identification signal.

Metabolomic analysis of urinary polyamines (PAs) from rat exposed to 915 MHz radiofrequency identification (RFID) signal for 8 h/day for 2 weeks was performed by gas chromatography-mass spectrometry as N-ethoxycarbonyl/N-pentafluoropropionyl derivatives. Large alterations in nine PA levels including four aliphatic and five acetylated PAs were monitored in sham-exposed and RFID-exposed groups. Total PA and urinary levels of N (1)-acetylputrescine, N (1)-acetylcadaverine, putrescine, cadaverine, N (1)-acetylspermidine, N (8)-acetylspermidine, spermidine and spermine were reduced, whereas N (1)-acetylspermine was significantly increased after sham and RFID exposure compared with those before exposure. Their levels were normalized to the corresponding group means before exposure and then plotted into star symbol patterns. N (1)-Acetylspermine after RFID exposure was 54 % higher compared to the level before RFID exposure, while it was elevated by only 17 % in the sham group. The results suggest that 915 MHz RFID exposure may induce metabolic disturbance of PA. It may also elevate spermidine/spermine acetyltransferase (SSAT) activity. Thus, the present metabolic profiling combined with star pattern recognition method might be useful for understanding the complexity of biochemical events after exposure to RFID signal.

A Phase I Trial of DFMO Targeting Polyamine Addiction in Patients with Relapsed/Refractory Neuroblastoma.

BACKGROUND: Neuroblastoma (NB) is the most common cancer in infancy and most frequent cause of death from extracranial solid tumors in children. Ornithine decarboxylase (ODC) expression is an independent indicator of poor prognosis in NB patients. This study investigated safety, response, pharmacokinetics, genetic and metabolic factors associated with ODC in a clinical trial of the ODC inhibitor difluoromethylornithine (DFMO) ± etoposide for patients with relapsed or refractory NB. METHODS AND FINDINGS: Twenty-one patients participated in a phase I study of daily oral DFMO alone for three weeks, followed by additional three-week cycles of DFMO plus daily oral etoposide. No dose limiting toxicities (DLTs) were identified in patients taking doses of DFMO between 500-1500 mg/m2 orally twice a day. DFMO pharmacokinetics, single nucleotide polymorphisms (SNPs) in the ODC gene and urinary levels of substrates for the tissue polyamine exporter were measured. Urinary polyamine levels varied among patients at baseline. Patients with the minor T-allele at rs2302616 of the ODC gene had higher baseline levels (p=0.02) of, and larger decreases in, total urinary polyamines during the first cycle of DFMO therapy (p=0.003) and had median progression free survival (PFS) that was over three times longer, compared to patients with the major G allele at this locus although this last result was not statistically significant (p=0.07). Six of 18 evaluable patients were progression free during the trial period with three patients continuing progression free at 663, 1559 and 1573 days after initiating treatment. Median progression-free survival was less among patients having increased urinary polyamines, especially diacetylspermine, although this result was not statistically significant (p=0.056). CONCLUSIONS: DFMO doses of 500-1500 mg/m2/day are safe and well tolerated in children with relapsed NB. Children with the minor T allele at rs2302616 of the ODC gene with relapsed or refractory NB had higher levels of urinary polyamine markers and responded better to therapy containing DFMO, compared to those with the major G allele at this locus. These findings suggest that this patient subset may display dependence on polyamines and be uniquely susceptible to therapies targeting this pathway. TRIAL REGISTRATION: Clinicaltrials.gov NCT#01059071.

Dietary polyamine intake and polyamines measured in urine.

Dietary polyamines have recently been associated with increased risk of pre-malignant colorectal lesions. Because polyamines are synthesized in cells and taken up from dietary sources, development of a biomarker of exposure is challenging. Excess polyamines are primarily excreted in the urine. This pilot study seeks to identify dietary correlates of excreted urinary polyamines as putative biomarkers of exposure. Dietary polyamines/other nutrients were estimated from a food frequency questionnaire (FFQ) and correlated with urinary levels of acetylated polyamines in 36 men using 24-h urine samples. Polyamines, abundant in cheese and citrus, were highly positively correlated with urinary N(8)-acetylspermidine (correlation coefficient; r = 0.37, P = 0.03), but this correlation was attenuated after adjustment for total energy intake (r = 0.07, P = 0.68). Dietary energy intake itself was positively correlated with urinary total acetylated polyamine output (r = .40, P = 0.02). In energy-adjusted analyses, folic acid and folate from food were associated with urinary N(1),N(12)-diacetylspermine (r = 0.34, P = 0.05 and r = -0.39, P = 0.02, respectively). Red meat negatively correlated with total urinary acetylated polyamines (r = -0.42, P = 0.01). Our findings suggest that energy, folate, folic acid, saturated fat, and red meat intake, as opposed to FFQ-estimated dietary polyamines, are correlated with urinary polyamines.

Simultaneous determination of polyamines and acetylpolyamines in human urine by capillary electrophoresis with fluorescence detection.

There has been evidence linking elevated polyamines (PAs) and acetylpolamines (AcPAs) level and cancer. So the simultaneous analysis of these compounds has become important task for cancer diagnosis and antitumor drug monitoring. A simple, fast and inexpensive CZE-LIF method has been developed for the determination of cadaverine (CAD), putrescine (PUT), spermine (SPM), spermidine (SPD), acetylspermine (ASPM), and acetylspermidine (ASPD) in human urine using 4-chloro-7-nitro-2,1,3-benzooxadiazole as a fluorescent reagent. Labeling reaction conditions were systematically investigated and were found to be 20 mM borate buffer at pH 7.4, labeling reaction time, and temperature were 10 min and 70°C, respectively. Under these optimized conditions the four PAs, two AcPAs and the internal standard were separated in 6 min. An Exactive-MS with an ESI source was used for identification of the bis-derivative of the ASPM. The method was validated in term of linearity, LODs, repeatability, intra- and interday assays, recovery, and selectivity. The LODs for CAD, PUT, SPM, SPD, ASPM, and ASPD were found to be 7.6, 10.0, 9.0, 8.8,7.8, and 3.3 nM, respectively. The method was successfully applied for the analysis of PAs and AcPAs in healthy human urine samples.

Analysis of free, mono- and diacetylated polyamines from human urine by LC-MS/MS.

Polyamines are promising biochemical markers of cancer and many other pathophysiological conditions, and thus their concentrations in biological fluids are a matter of interest. However, since the concentrations of these compounds are low, their quantitation is typically based on methods requiring laborious sample preparation. Here we developed and validated an LC-MS/MS method to analyze simultaneously free (DAP, PUT, CAD, SPD, SPM) monoacetylated (AcPUT, AcCAD, N(1)AcSPD, N(8)AcSPD, N(1)AcSPM) and diacetylated (DiAcPUT, DiAcCAD, DiAcSPD, DiAcSPM) polyamines from human urine without the need for derivatization. Deuterium labeled polyamines were the internal standards for each analyte. Diluted urine samples spiked with internal standards were filtered through a strong anion exchange resin prior to LC-MS/MS analysis. The chromatographic separation of 14 polyamines was achieved in 12min on C18 column with 0.1% HFBA (v/v) as the ion-pairing agent and a water-acetonitrile gradient. Ionization was performed with positive electrospray ionization (ESI) and detection was with a triple quadrupole mass spectrometer with selected reaction monitoring. Calibration curves ranged from up to 5 to 10,000nM. The accuracy and precision of the method were determined using urine based quality control samples, and matrix effects were examined by using standard addition methods. This novel method is suitable for elucidating differences in urinary polyamine excretion in cancer patients and healthy humans.

Analysis of chloroformate-derivatised amino acids, dipeptides and polyamines by LC-MS/MS.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed which, with sample preparation using a commercially available kit, allows rapid quantitation of 39 chloroformate-derivatised amino acids (AAs), polyamines (PAs) and dipeptides (DPs) in complex biological matrices. Lower limits of quantitation (LOQ) were 20-150nM for putrescine, spermine, spermidine, cadaverine, agmatine, and below 5µM for all analytes. Responses were linear for all analytes between 0.5 and 50µM. Quantitative measurements of all 39 metabolites were achieved within a 15min runtime. The method was evaluated with a Pseudomonas aeruginosa cell extract study (n=24) and a larger human urine study (n=308). Batch effects were observed in the urine study and an investigation of instrument and sample stability showed a wave-like pattern in the MS responses. Both the run order and inter-batch variation were successfully corrected by normalising to pooled urine quality control data. Thus, this method should be suitable for diverse biological matrices and for large as well as small sample sets.

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.

Metabolomics reveals aging-associated attenuation of noninvasive radiation biomarkers in mice: potential role of polyamine catabolism and incoherent DNA damage-repair.

Development of methods for rapid screening and stratification of subjects after exposure is an integral part of countermeasures against radiation. The potential demographic and exposure history-related heterogeneity of exposed populations warrants robust biomarkers that withstand and reflect such differences. In this study, the effect of aging and repeated exposure on the metabolic response to sublethal irradiation was examined in mice using UPLC-ESI-QTOF mass spectrometry. Aging attenuated postexposure elevation in excretions of DNA damage biomarkers as well as N(1)-acetylspermidine. Although N(1)-acetylspermidine and 2'-deoxyuridine elevation was highly correlated in all age groups, xanthine and N(1)-acetylspermidine elevation was poorly correlated in older mice. These results may reflect the established decline in DNA damage-repair efficiency associated with aging and indicate a novel role for polyamine metabolism in the process. Although repeated irradiation at long intervals did not affect the elevation of N(1)-acetylspermidine, 2'-deoxyuridine, and xanthine, it did significantly attenuate the elevation of 2'-deoxycytidine and thymidine compared to a single exposure. However, these biomarkers were found to identify exposed subjects with accuracy ranging from 82% (xanthosine) to 98% (2'-deoxyuridine), irrespective of their age and exposure history. This indicates that metabolic biomarkers can act as robust noninvasive signatures of sublethal radiation exposure.

Determination of polyamines in human urine by precolumn derivatization with benzoyl chloride and high-performance liquid chromatography coupled with Q-time-of-flight mass spectrometry.

A simple and sensitive HPLC/Q-TOF MS method for simultaneous determination of 1,3-diaminopropane, putrescine, cadaverine, spermidine, spermine and acetyl-spermine in human urine was developed in electrospray-ionization source by positive ion mode. The samples were firstly pretreated by 10% HClO(4) and then derivatized by benzoyl chloride with 1,6-diaminohexane as internal standard. The derived polyamines were separated on a C(18) column by a gradient elution with methanol-water, and then sensitively detected with Q-TOF MS. The limits of detection for polyamines ranged from 0.02 to 1.0 ng ml(-1) with excellent linearity within the range from 1 to 1000 ng ml(-1) except acetyl-spermine from 5 to 1000 ng ml(-1). The intra- and inter-day R.S.D. for all polyamines were 2.0-14.7% and 3.9-12.9%, respectively. The method was applied to determine the polyamines in human urine from 10 cancer patients and 15 healthy volunteers. Results showed that the mean levels of polyamines in urine of patients were all higher than those in healthy volunteers. The cluster analysis was used to establish the distinction mode between cancer sufferers and healthy individuals.

Identification and determination of urinary acetylpolyamines in cancer patients by electrospray ionization and time-of-flight mass spectrometry.

A method for the quantification of acetylpolyamines, N(1),N(12)-diacetylspermine (DiAcSpm), monoacetylspermidine (AcSpd), and N(1),N(8)-diacetylspermidine (DiAcSpd), identifying each compound simultaneously, was developed with the goal of evaluating these acetylpolyamines as potential biomarkers of cancer. The method consists of prepurification of acetylpolyamines in urine with commercially available cartridges and derivatization with heptafluorobutyric (HFB) anhydride. HFB derivatives of acetylpolyamines were determined simultaneously using (15)N-labeled acetylpolyamines as internal standards by electrospray ionization and time-of-flight mass spectrometry (ESI-TOF MS). After the method was validated, the urinary acetylpolyamines of 38 cancer patients were quantified with this method. A comparison of the concentrations of DiAcSpm with those measured by a colloidal gold aggregation method demonstrated a correlation coefficient of 0.996, showing that the two methods were equally satisfactory. Analysis of the correlation between DiAcSpd or AcSpd and DiAcSpm, performed for the first time, indicated the usefulness of DiAcSpm as a urinary biomarker of cancer. During the course of this work, two simple methods for the preparation of alpha,omega-diacetylpolyamines were developed, and a possibility to separate and determine the concentrations of the two isomers, N(1)-acetylspermidine and N(8)-acetylspermidine in AcSpd, was shown by tandem mass spectrometry (MS/MS).