Urinary metabolomic analysis to identify potential markers for the diagnosis of tuberculosis and latent tuberculosis.

Tuberculosis (TB) is a serious infectious disease with high infection and mortality rates. 5%-10% of the latent tuberculosis infections (LTBI) are likely to develop into active TB, and there are currently no clinical biomarkers that can distinguish between LTBI, active TB and other non-tuberculosis populations. Therefore, it is necessary to develop rapid diagnostic methods for active TB and LTBI. In this study, urinary metabolome of 30 active TB samples and the same number of LTBI and non-TB control samples were identified and analyzed by UPLC-Q Exactive MS. In total, 3744 metabolite components were obtained in ESI- mode and 4086 in ESI + mode. Orthogonal partial least square discriminant analysis (OPLS-DA) and hierarchical cluster analysis (HCA) showed that there were significant differences among LTBI, active TB and non-TB. Six differential metabolites were screened in positive and negative mode, 3-hexenoic acid, glutathione (GSH), glycochenodeoxycholate-3-sulfate, N-[4'-hydroxy-(E)-cinnamoyl]-l-aspartic acid, deoxyribose 5-phosphate and histamine. The overlapping pathways differential metabolites involved were mainly related to immune regulation and urea cycle. The results showed that the urine metabolism of TB patients was disordered and many metabolic pathways changed. Multivariate statistical analysis revealed that GSH and histamine were selected as potential molecular markers, with area under curve of receiver operating characteristic curve over 0.75. Among the multiple differential metabolites, GSH and histamine changed to varying degrees in active TB, LTBI and the non-TB control group. The levels of GSH and histamine in 48 urinary samples were measured by ELISA in validation phase, and the result in our study provided the potential for non-invasive biomarkers of TB.

Urinary leukotrienes and histamine in patients with varying severity of acute dengue.

BACKGROUND: Vascular leak is a hallmark of severe dengue, and high leukotriene levels have been observed in dengue mouse models, suggesting a role in disease pathogenesis. We sought to explore their role in acute dengue, by assessing levels of urinary LTE4 and urinary histamine in patients with varying severity of acute dengue. METHODS: Urinary LTE4, histamine and creatinine were measured by a quantitative ELISA, in healthy individuals (n = 19), patients with dengue fever (DF = 72) and dengue haemorrhagic fever DHF (n = 48). The kinetics of LTE4 and histamine and diurnal variations were assessed in a subset of patients. RESULTS: Urinary LTE4 levels were significantly higher (p = 0.004) in patients who proceed to develop DHF when compared to patients with DF during early illness (≤ 4 days) and during the critical phase (p = 0.02), which continued to rise in patients who developed DHF during the course of illness. However, LTE4 is unlikely to be a good biomarker as ROCs gave an AUC value of 0.67 (95% CI 0.57 and 0.76), which was nevertheless significant (p = 0.002). Urinary LTE4 levels did not associate with the degree of viraemia, infecting virus serotype and was not different in those with primary vs secondary dengue. Urinary histamine levels were significantly high in patients with acute dengue although no difference was observed between patients with DF and DHF and again did not associate with the viraemia. Interestingly, LTE4, histamine and the viral loads showed a marked diurnal variation in both patients with DF and DHF. CONCLUSIONS: Our data suggest that LTE4 could play a role in disease pathogenesis and since there are safe and effective cysteinyl leukotriene receptor blockers, it would be important to assess their efficacy in reducing dengue disease severity.

Development of a HILIC-MS/MS method for the quantification of histamine and its main metabolites in human urine samples.

An LC-MS/MS method was developed enabling the separation and quantification of histamine and its main metabolites (imidazole acetaldehyde, imidazole acetic acid, methyl imidazole acetic acid, methyl histamine, acetyl histamine) in urine samples. A fast separation was achieved in 10 min on two HILIC columns connected in series by adopting a linear gradient followed by an isocratic hold. The sample preparation consisted of a simple dilution step wherein 10 µL of urine was diluted with acetonitrile (ACN) to a final volume comprising 95% ACN. For methyl imidazole acetic acid, an additional dilution step was incorporated due to its high natural levels. Hereafter, the samples were stored at -20 °C and centrifuged prior to injection. Matrix matched calibrators were unavailable due to the endogenous occurrence of the compounds of interest. The occurrence of matrix effects and the lack of labeled internal standards prompted the use of the standard addition method as a viable alternative to solvent calibration. The validation of the method entailed matrix effects, accuracy and precision and was performed in compliance with the recent guidelines on endogenous compounds issued by the International Conference of Harmonization (ICH). The method was then adopted for the quantification of histamine and its metabolites in human urine samples collected from healthy volunteers and patients suffering from gastrointestinal discomfort.

Urinary metabolomics analysis reveals the anti-diabetic effect of stachyose in high-fat diet/streptozotocin-induced type 2 diabetic rats.

As a new platform of systems biology, metabolomics provides a powerful approach to discover therapeutic biomarkers and mechanism of metabolic disease. Type 2 diabetes mellitus (T2DM) is a global metabolic disease, thus, a urinary metabolomics profiling was analyzed to study the anti-diabetic effects and mechanism of stachyose (ST) on high-fat diet- and low dose streptozotocinc-induced T2DM rats. The results showed that ST treatment regulated the level of insulin, low-density lipoprotein cholesterol, and triglycerides, which demonstrates improvement in T2DM on ST treatment. Urinary samples from the ST and T2DM group were enrolled in metabolomics study, 21 differential metabolites were identified from urinary metabolomics analysis, indicating that the ST treatment partly exerted the anti-diabetes activity by regulating energy metabolism, gut microbiota changes and inflammation. A metabolomics strategy is both suitable and reliable for exploring the anti-diabetes effects and understanding the mechanisms of ST treatment against T2DM.

A highly selective "turn-on" electroanalysis strategy with reduced copper metal-organic frameworks for sensing histamine and histidine.

A highly selective and sensitive electroanalysis strategy has been developed for sensing histamine (HTA) and histidine (His) with "turn-on" signal outputs using copper nanocomposites (Cu NCs) of reduced copper metal-organic frameworks (Cu MOFs). It was discovered that the Cu NC-modified electrodes could display the sharp and stable oxidation peaks of solid-state CuCl electrochemistry at a low potential (about -0.10 V). More interestingly, once HTA or His was introduced, the peaking currents of the electrodes would increase due to the specific interaction between Cu2+ and imidazole groups of HTA or His. A highly selective electroanalysis method was thereby developed for the detection of both HTA and His in the concentration range of 0.010-100 µM. Besides, the application feasibility of the developed electroanalysis strategy was demonstrated for the evaluation of HTA and His separately in red wine and urine samples. Such an electroanalysis candidate for HTA and His holds great potential for wide applications in the fields of food analysis and clinical disease diagnosis.

High Affinity Aptamer for the Detection of the Biogenic Amine Histamine.

The importance of histamine in various physiological functions and its involvement in allergenic responses make this small molecule one of the most studied biogenic amines. Even though a variety of chromatography-based methods have been described for its analytical determination, the disadvantages they present in terms of cost, analysis time, and low portability limit their suitability for in situ routine testing. In this work, we sought to identify histamine-binding aptamers that could then be exploited for the development of rapid, facile, and sensitive assays for histamine detection suitable for point-of-need analysis. A classic SELEX process was designed employing magnetic beads for target immobilization and the selection was completed after ten rounds. Following Next Generation Sequencing of the last selection rounds from both positive and counter selection magnetic beads, several sequences were identified and initially screened using an apta-PCR affinity assay (APAA). Structural and functional characterization of the candidates resulted in the identification of the H2 aptamer. The high binding affinity of the H2 aptamer to histamine was validated using four independent assays ( KD of 3-34 nM). Finally, the H2 aptamer was used for the development of a magnetic beads-based competitive assay for the detection of histamine in both buffer and synthetic urine, achieving very low limits of detection of 18 pM and 76 pM, respectively, while no matrix effects were observed. These results highlight the suitability of the strategy followed for identifying small molecule-binding aptamers and the compatibility of the selected H2 aptamer with the analysis of biological samples, thus facilitating the development of point-of-care devices for routine testing. Ongoing work is focused on extending the application of the H2 aptamer to the detection of spoilage in meat, fish, and beverages, as well as evaluating the affinity of truncated forms of the aptamer.

Environmental cadmium exposure induces alterations in the urinary metabolic profile of pregnant women.

Cadmium (Cd) is a well-recognized, hazardous toxic heavy metal, and the adverse effects of high-level Cd exposure on human health have been well documented. However, little is known about the health effects of low-level environmental Cd exposure on pregnant women. The objective of this study was to assess urinary metabolic alterations in pregnant women exposed to environmental Cd, and to identify informative biomarkers. Urine samples from 246 pregnant women in the first trimester of pregnancy were collected, and urinary Cd concentrations were quantified using inductively coupled plasma mass spectrometry (ICP-MS). Urinary metabolomics was analyzed by ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). Cd-related metabolic biomarkers were examined by comparing the samples of the first and third tertiles of Cd exposure classifications, using a partial least-squares discriminant (PLS-DA) model. Five putative biomarkers were identified, including L-cystine, L-tyrosine, dityrosine, histamine, and uric acid, all of which were related to oxidative stress and nephrotoxic effects induced by Cd exposure. The results show that low-level environmental Cd exposure could induce metabolite profile alterations in pregnant women, which might be associated with adverse health effects. Our findings provide new insights into the early molecular events following Cd exposure, and may be valuable for the health risk assessment of Cd exposure during pregnancy.

Metabonomics delineates allergic reactions induced by Shuang-huang-lian injection in rats using ultra performance liquid chromatography-mass spectrometry.

Shuang-huang-lian Injection (SHLI) is the first successfully developed drug from traditional Chinese medicine (TCM) powder for injection, since its use for the treatment of acute respiratory tract infection, pneumonia, influenza, etc. At the same time, its allergic reactions have also emerged, which limits clinical applications. However, few scholars pay attention to the mechanism of allergic reactions. In this present study, metabonomics technology was used to explore the changes in endogenous metabolites in urine of the rat model of SHLI induced allergic reaction; we and analyzed the metabolites, metabolic pathway, and the mechanism which were closely related to the allergic reactions. The levels of serum histamine and tryptase were examined and changes in histomorphology were also observed. Based on the UPLC-Q-TOF/MS metabonomics, we carried out the pattern recognition analysis, selected potential biomarkers associated with allergic reactions, and explored the pathological mechanism for SHLI induced allergic reaction, which laid the foundation for the safety research of SHLI. Our results showed that SHLI increased the levels of serum histamine and tryptase in rats with allergic reaction; we determined 15 biomarkers in rat allergic reaction model induced by SHLI and found multiple metabolic pathways involved, such as metabolism of linolenic acid, phenylalanine, amino acid, 2-oxo acid, and purine and other metabolic pathways.

New approach for the diagnosis of histamine intolerance based on the determination of histamine and methylhistamine in urine.

Histamine intolerance is a disorder in the homeostasis of histamine due to a reduced intestinal degradation of this amine, mainly caused by diamine oxidase (DAO) enzyme deficiency, which provokes its accumulation in plasma and the appearance of adverse health affects. A new approach for the diagnosis of this intolerance could be the determination of histamine and its metabolites in urine. The aim of this work was to develop and validate a rapid method to determine histamine and methylhistamine in human urine by Ultra High Performance Liquid Chromatography and Fluorimetric detection (UHPLC-FL). The proposed method is a consistent procedure to determine histamine and methylhistamine in less than 11min with adequate linearity and sensitivity. Relative standard deviation was always lower than 5.5%, ensuring method precision; and mean recovery was greater than 99% for both analytes. The structure of histamine and methylhistamine conjugated with OPA were confirmed by UHPLC-ITD-FTMS which enabled to unequivocally identify both analytes in standards and also in urine samples. The analysis of histamine and methylhistamine in urine samples could be a potential new approach for the routine diagnosis of histamine intolerance, more patient-friendly and with clear advantages in terms of equipment and personnel demand for sample collection in comparison with current plasmatic DAO activity determination.

Dual-Mode Optical Sensing of Histamine at Nanomolar Concentrations in Complex Biological Fluids and Living Cells.

An easily synthesized fluorescein-based luminescent dye has been utilized for the dual-mode detection of histamine at nanomolar concentrations at pH 7.0 in water. The specific response to histamine was achieved by imidazole-catalyzed 'imine formation' reaction. The protocol was subsequently applied for the estimation of histamine in complex biological milieu such as human blood serum and urine samples. Furthermore, the dose-dependent cellular uptake of histamine and de novo synthesis (by thapsigargin treatment) was visualized in RAW 264.7, a mouse macrophage cell line. We have also developed portable paper strips for rapid, on-site detection of histamine without involving costly instruments.

FODMAPs alter symptoms and the metabolome of patients with IBS: a randomised controlled trial.

OBJECTIVE: To gain mechanistic insights, we compared effects of low fermentable oligosaccharides, disaccharides and monosaccharides and polyols (FODMAP) and high FODMAP diets on symptoms, the metabolome and the microbiome of patients with IBS. DESIGN: We performed a controlled, single blind study of patients with IBS (Rome III criteria) randomised to a low (n=20) or high (n=20) FODMAP diet for 3 weeks. Symptoms were assessed using the IBS symptom severity scoring (IBS-SSS). The metabolome was evaluated using the lactulose breath test (LBT) and metabolic profiling in urine using mass spectrometry. Stool microbiota composition was analysed by 16S rRNA gene profiling. RESULTS: Thirty-seven patients (19 low FODMAP; 18 high FODMAP) completed the 3-week diet. The IBS-SSS was reduced in the low FODMAP diet group (p<0.001) but not the high FODMAP group. LBTs showed a minor decrease in H2 production in the low FODMAP compared with the high FODMAP group. Metabolic profiling of urine showed groups of patients with IBS differed significantly after the diet (p<0.01), with three metabolites (histamine, p-hydroxybenzoic acid, azelaic acid) being primarily responsible for discrimination between the two groups. Histamine, a measure of immune activation, was reduced eightfold in the low FODMAP group (p<0.05). Low FODMAP diet increased Actinobacteria richness and diversity, and high FODMAP diet decreased the relative abundance of bacteria involved in gas consumption. CONCLUSIONS: IBS symptoms are linked to FODMAP content and associated with alterations in the metabolome. In subsets of patients, FODMAPs modulate histamine levels and the microbiota, both of which could alter symptoms. TRIAL REGISTRATION NUMBER: NCT01829932.

Towards a specific marker for acute bradykinin-mediated angioedema attacks: a literature review.

BACKGROUND: Bradykinin-mediated angioedema (AE) is a rare disease characterised by recurrent angioedema linked to acquired (e.g. angiotensin converting enzyme inhibitor induced AE) or hereditary disorders (e.g. AE type I or II). As the clinical picture can be misleading, diagnosis of this disease is sometimes difficult. A bradykinin-mediated AE attack may be a therapeutic emergency which requires access to effective, but expensive, treatments. Their prescription must therefore be justified. No specific marker of acute bradykinin-mediated AE attacks has yet been identified to facilitate the therapeutic decision but it has been sought in many studies. PURPOSE: This article reviews the literature on this type of biomarker, comparing candidate bradykinin-mediated AE markers to candidate markers of mast cell activation. CONCLUSION: The most interesting biomarkers are those linked to endothelial stress (VE cadherin, E-selectin, endothelin-1, von Willebrand factor and its activity) which is significantly increased during an AE attack. All these markers must now be validated by prospective studies to determine their specificity and utility in diagnosis.

Excretion of urinary histamine and N-tele methylhistamine in patients with gastrointestinal food allergy compared to non-allergic controls during an unrestricted diet and a hypoallergenic diet.

BACKGROUND: Patients with gastrointestinal food allergy are characterised by increased production of mast cell derived mediators upon allergen contact and present often with unspecific symptoms. The aim of this study was to evaluate urinary histamine and methylhistamine excretion in patients with food allergy and to compare their values with food-tolerant controls. METHODS: In a retrospective case control study the urinary excretion parameters were analysed from 56 patients (40.9, 19 - 58 years) in whom later food challenge tests confirmed food allergy. During their diagnostic work-up urine was collected during a 12-h period under an unrestricted diet with staple foods and a hypoallergenic potato-rice-diet (each 2 days). Healthy controls underwent the same diet types to define normal excretion parameters. Urinary histamine and n-methylhistamine were determined by ELISA or tandem mass spectrometry, respectively, and were expressed as median (25 - 75% range, µg/mmol creatinine x m(2)BSA). RESULTS: During unrestricted diet urinary histamine was significantly higher in gastrointestinal food allergy than healthy controls (1.42, 0.9 - 2.7 vs 0.87, 0.4 - 1.3; p < 0.0001), while the difference between both groups became marginal during potato-rice diet (1.30, 0.7 - 2.1 vs 1.05, 0.5 - 1.5; p = 0.02). N-methylhistamine was found to be significantly elevated in gastrointestinal food allergy both during unrestricted diet (7.1, 5.0 - 11.2) and potato-rice diet (5.7, 3.7 - 8.7) compared to controls (p < 0.0001). Interestingly, urinary methylhistamine excretion (p < 0.004) and clinical symptom score (p < 0.02) fell significantly when the diet was switched from unrestricted to hypoallergenic food, but was not correlated with symptom scores. CONCLUSIONS: In gastrointestinal food allergy significantly higher levels of urine histamine and methylhistamine excretion were found under unrestricted diet, reflecting an increased secretion of histamine due to offending foods. Measurement of urinary n-methylhistamine levels may help to find out patients with increased histamine production and/or food-allergen induced clinical symptoms, respectively.

[Determination and correlation analysis of contents of putrescine, cadaverine, and histamine in necrotic tissue, blood, and urine of patients with diabetic foot].

OBJECTIVE: To determine and perform a correlation analysis of the contents of putrescine, cadaverine, and histamine in necrotic tissue, blood, and urine of patients with diabetic foot (DF). METHODS: Ten patients with severe wet necrotizing DF hospitalized from January 2011 to January 2012 were assigned as group DF, and 10 orthopedic patients with scar but without diabetes or skin ulcer hospitalized in the same period were assigned as control group. Samples of necrotic tissue from feet of patients in group DF and normal tissue from extremities of patients in control group, and samples of blood and 24-hour urine of patients in both groups were collected, and the amount of each sample was 10 mL. Contents of putrescine, cadaverine, and histamine were determined with high performance liquid chromatography-mass spectrometry. The data got from the determination of blood and urine were processed with t test, and those from necrotic or normal tissue with Wilcoxon rank sum test. The correlation of contents of polyamines between necrotic tissue and blood, blood and urine were processed with simple linear regression analysis. RESULTS: (1) Contents of putrescine, cadaverine, and histamine in the necrotic tissue of group DF were (186.1 ± 26.8), (78.553 ± 12.441), (33 ± 10) mg/kg, which were significantly higher than those in normal tissue of control group [(2.2 ± 1.2), (1.168 ± 0.014), 0 mg/kg, with Z values respectively -3.780, -3.781, -4.038, P values all below 0.01]. The content of putrescine in necrotic tissue of group DF was significantly higher than those of cadaverine and histamine (with Z values respectively -3.780, -3.630, P values all below 0.01). (2) Contents of putrescine, cadaverine, and histamine in the blood of group DF were (0.075 ± 0.013), (0.022 ± 0.003), (0.052 ± 0.014) mg/L, and they were significantly higher than those in the blood of control group [(0.014 ± 0.009), (0.013 ± 0.003), (0.016 ± 0.008) mg/L, with t values respectively 6.591, 2.207, 3.568, P < 0.05 or P<0.01]. The content of putrescine in the blood of group DF was significantly higher than those of cadaverine and histamine (with t values respectively 13.204, 3.096, P values all below 0.01). (3) Contents of putrescine, cadaverine, and histamine in the urine of group DF were (0.735 ± 0.088), (0.450 ± 0.012), (0.1623 ± 0.0091) mg/L, and only the contents of putrescine and cadaverine were significantly higher than those in the urine of control group [(0.050 ± 0.014), (0.035 ± 0.007) mg/L, with t values respectively 3.270, 4.705, P<0.05 or P<0.01]. The content of putrescine in the urine of group DF was significantly higher than that of cadaverine (t = 6.686, P < 0.01). (4) There were significant and positive correlations in contents of putrescine, cadaverine, and histamine between necrotic tissue and blood in patients of group DF (with r values respectively 0.981, 0.994, 0.821, P values all below 0.01). There were no significant correlations in contents of putrescine, cadaverine, and histamine between blood and urine in patients of group DF (with r values respectively 0.150, 0.239, 0.177, P values all above 0.05). CONCLUSIONS: Putrescine, cadaverine, and histamine exist in the necrotic tissue of patients with DF in high concentrations, among which putrescine predominates. These polyamines can be absorbed into the blood through wound and excreted through the urine.

Histamine determination in human urine using sub-2 µm C18 column with fluorescence and mass spectrometric detection.

A fast, sensitive, and selective method for the determination of histamine in human urine samples by ultrahigh pressure liquid chromatography (LC) with fluorescence and mass spectrometry (MS) detection is investigated. A fluorescent reagent, 4-(1-pyrene) butyric acid N-hydroxysuccinimide ester was conjugated to the primary and secondary amino moieties of histamine. The structure of dipyrene-labeled histamine in human urine was determined by quadrupole time-of-flight MS with electospray ionization interface. The determination of the dipyrene derivative of histamine in urine samples was achieved within 3.9 min on an ultrahigh pressure LC Eclipse Zorbax XDB-C(18) column with 1.8 µm particle diameter. In this work, histamine separation was achieved significantly faster (3.9 min) with improved detection limit (signal-to-noise = 3) of 0.04 nM than 19.5 min with a detection limit of 0.183 nM as reported in a previous method.

Urinary excretion of histamine and methylhistamine after burns.

BACKGROUND: The increased vascular permeability seen after burn contribute to morbidity and mortality as it interferes with organ function and the healing process. Large efforts have been made to explore underlying pathophysiological mechanisms that generate increased vascular permeability after burns. Many different substances have been proposed as mediators of which histamine, serotonin and oxygen radicals are claimed most important. However, no specific blocker has convincingly been shown to be clinically effective. Early work has claimed increased histamine plasma-concentrations in humans after burn and data from animal models pointed at histamine as an important mediator. Modern human clinical studies investigating the role of histamine as a mediator of the generalized post burn increase in vascular permeability are lacking. METHOD: We examined histamine turnover by measuring the urinary excretion of histamine and methyl histamine for 48 h after burns in 8 patients (mean total burn surface area 24%). RESULTS: Over time, in this time frame and compared to healthy controls we found a small increase in the excretion of histamine, but no increase of its metabolite methylhistamine. CONCLUSION: Our findings do not support that histamine is an important mediator of the increased systemic vascular permeability seen after burn.

Clinical and laboratory parameters of mast cell activation as basis for the formulation of diagnostic criteria.

Mast cell (MC) activation occurs in a number of different pathologic conditions. Acute activation is commonly seen in patients with allergic reactions, with consecutive massive release of vasoactive and proinflammatory mediator substances from MCs, leading to the clinical signs and symptoms of anaphylaxis. In these patients, serum tryptase concentrations usually increase subtantially above baseline levels. Chronic MC activation is more difficult to diagnose, especially when symptoms are mild or atypical, and no underlying disease is found. In these patients, serum tryptase levels usually are normal. In a smaller group of patients, tryptase levels are constantly elevated and may point to an occult form of mastocytosis. These patients have to be examined for MC monoclonality, other criteria of a primary MC disease, non-MC hematopoietic neoplasms, and reactive disorders producing chronic MC activation or MC accumulation. In most patients in whom MC activation is found, histamine-induced symptoms can be documented and usually respond to treatment with histamine receptor antagonists or MC stabilizers. If this is not the case, alternative explanations for symptoms and differential diagnoses have to be considered.

A new spectrofluorimetric method for determination of trace amounts histamine in human urine and serum.

A new spectrofluorimetric method was developed for the determination of trace amounts of histamine in human urine and serum samples. In NaAc-HAc buffer solution of pH 4.0, histamine can react with the acetylacetone-formaldehyde system to produce a fluorescent derivative which emits yellow-green fluorescence at 476 nm, according to the Hantzsch reaction, and the enhanced fluorescence intensity is in proportion to the concentration of histamine. Optimum conditions for the determination of histamine were also investigated. The dynamic range and detection limit for the determination of histamine is 5.96 x 10(-8)-1.50 x 10(-5 )mol/L and 4.35 x 10(-8)mol/L, respectively. This method is practical and can be successfully applied to determination of histamine in human urine and serum samples. A proposal of the reaction pathway is suggested.

Identification of urinary modified nucleosides and ribosylated metabolites in humans via combined ESI-FTICR MS and ESI-IT MS analysis.

The physiological response of the human body to several diseases can be reflected by the metabolite pattern in biological fluids. Cancer, like other diseases accompanied by metabolic disorders, causes characteristic effects on cell turnover rate, activity of modifying enzymes, and RNA/DNA modifications. This results in an altered excretion of modified nucleosides and biochemically related compounds. In the course of our metabolic profiling project, we screened 24-h urine of patients suffering from lung, rectal, or head and neck cancer for previously unknown ribosylated metabolites. Therefore, we developed a sample preparation procedure based on boronate affinity chromatography followed by additional prepurification with preparative TLC. The isolated metabolites were analyzed by ion trap mass spectrometry (IT MS) and Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). IT MS was applied for LC-auto MS(3) screening runs and MS(n(n=4-6)) syringe pump infusion experiments, yielding characteristic fragmentation patterns. FTICR MS measurements enabled the calculation of corresponding molecular formulae based on accurate mass determination (mass accuracy: 1-5 ppm for external and sub-ppm values for internal calibration). We were able to identify 22 metabolites deriving from cellular RNA metabolism and related metabolic pathways like histidine metabolism, purine biosynthesis, methionine/polyamine cycle, and nicotinate/nicotinamide metabolism. The compounds 1-ribosyl-3-hydroxypyridinium, 1-ribosyl-pyridinium, and 3-ribosyl-1-methyl-l-histidinium as well as a series of ribosylated histamines, conjugated to carboxylic acids at the N(omega)-position were found as novel urinary constituents. The occurrence of the modified nucleosides 2-methylthio-N(6)-(cis-hydroxyisopentenyl)-adenosine, 5-methoxycarbonylmethyl-2-thiouridine, N(6)-methyl-N(6)-threonylcarbamoyladenosine, and 2-methylthio-N(6)-threonylcarbamoyladenosine in human urine is verified for the first time.