New metabolites of riboflavin appear in human urine

By surveying compounds having isoalloxazine derived from flavins on a high performance liquid chromatogram with fluorescence detection, two new flavin derivatives were found in human urine. These two compounds were purified by partition chromatography on a cellulose column and by paper chromatography with several solvent systems, and their structures were determined to be 7 alpha-hydroxyriboflavin and 8 alpha-hydroxyriboflavin. The relative distributions, measured by high performance liquid chromatography, of 7 alpha- and 8 alpha-hydroxyriboflavin, riboflavin, and hydroxyethylflavin and its derivative were calculated to be 31.1, 5.0, 25.6, 4.9, and 21.9%, respectively, to total flavins in normal human urine obtained in early morning. The excretion of 7 alpha- and 8 alpha-hydroxyriboflavin in human urine indicates the occurrence of a metabolic pathway of the isoalloxazine ring of flavin at its 7 alpha and 8 alpha positions.     

New lignan metabolites in rat urine

Ten potential lignan metabolites were quantified in rat urine extracts using liquid chromatography-tandem mass spectrometry. The rats were orally administered with the plant lignans 7-hydroxymatairesinol, matairesinol, lariciresinol or secoisolariciresinol, or with the mammalian lignan enterolactone. The samples were enzymatically hydrolysed and solid-phase extracted before analysis. Of the analysed compounds, only trace amounts of 7-oxoenterolactone could be detected in the urine extracts before administration, but after administration of any of the lignans, the excretion of 7-oxoenterolactone increased and monodemethylated matairesinol and 4,4'-dihydroxyenterolactone could be detected. In addition, other novel lignan metabolites were detected, i.e., 7-oxomatairesinol, alpha-conidendrin, and alpha- and beta-conidendric acid.     

Characterization and quantification of endogenous fatty acid nitroalkene metabolites in human urine

The oxidation and nitration of unsaturated fatty acids transforms cell membrane and lipoprotein constituents into mediators that regulate signal transduction. The formation of 9-NO₂-octadeca-9,11-dienoic acid and 12-NO₂-octadeca-9,11-dienoic acid stems from peroxynitrite- and myeloperoxidase-derived nitrogen dioxide reactions as well as secondary to nitrite disproportionation under the acidic conditions of digestion. Broad anti-inflammatory and tissue-protective responses are mediated by nitro-fatty acids. It is now shown that electrophilic fatty acid nitroalkenes are present in the urine of healthy human volunteers (9.9 ± 4.0 pmol/mg creatinine); along with electrophilic 16- and 14-carbon nitroalkenyl β-oxidation metabolites. High resolution mass determinations and coelution with isotopically-labeled metabolites support renal excretion of cysteine-nitroalkene conjugates. These products of Michael addition are in equilibrium with the free nitroalkene pool in urine and are displaced by thiol reaction with mercury chloride. This reaction increases the level of free nitroalkene fraction >10-fold and displays a K_D of 7.5 × 10⁻⁶ M. In aggregate, the data indicates that formation of Michael adducts by electrophilic fatty acids is favored under biological conditions and that reversal of these addition reactions is critical for detecting both parent nitroalkenes and their metabolites. The measurement of this class of mediators can constitute a sensitive noninvasive index of metabolic and inflammatory status.     

Variation of metabolites in normal human urine

Urine is often sampled from patients participating in clinical and metabolomic studies. Biological homeostasis occurs in humans, but little is known about the variability of metabolites found in urine. It is important to define the inter- and intra-individual metabolite variance within a normal population before scientific or clinical conclusions are made regarding different pathophysiologies. This study investigates the variability of selected urine metabolites in a group of 60 healthy men and women over a period of 30 days. To monitor individual variation, 6 women from the normal population were randomly selected and followed for 30 days. To determine the influence of extraneous environmental factors urine was collected from 25 guinea pigs with similar genetics, diet, and living environment. For both studies, 24 metabolites were identified and quantified using high-resolution ¹H nuclear magnetic resonance spectroscopy (NMR). The data demonstrated large inter and intra-individual variation in metabolite concentrations in both normal human and control animal populations. A defined normal baseline is essential before any conclusions may be drawn regarding changes in urine metabolite concentrations.     

Identification of fendiline metabolites in human urine

After oral application of 14C labelled fendiline, 13 metabolites of this drug could be identified in human urine. Only traces of parent fendiline were excreted in the urine. The main pathway of metabolism is hydroxylation of phenyl groups with subsequent glucuronidation and sulphation. On the other hand, oxidative dealkylation occurs with the amino group remaining at the 3,3-diphenylpropyl moiety and p-hydroxyacetophenone being formed almost entirely from the 1-phenylethyl group.     

New metabolites of riboflavin appeared in rat urine

By using paper and silica gel thin layer chromatography with various solvent systems, flavin compounds appeared in rat urine after administration of radioactive riboflavin were analyzed in detail. Two new metabolites having radioactivity were separated and their structures were determined to be 7-carboxy lumichrome and 8-carboxy lumichrome. The sum of radioactivities of these two compounds was about 46% of total radioactivity excreted in the urine during 24 h.     

Psychological stress increases bilirubin metabolites in human urine

Some authors have suggested that psychological stress induces the production of reactive oxygen species (ROS). Some studies have supported that bilirubin exerts anti-oxidative effects in vivo. However, it is not known whether ROS induced by psychological stress provoke bilirubin oxidation in vivo. We investigated if the concentration of bilirubin oxidative metabolite (BOM), a bilirubin oxidative metabolite, increased in urine from subjects exposed to psychological stress. Sixty healthy male volunteers working in a pharmaceutical company were divided into a Group I which did not attend a conference, a Group II which attended a conference but did not deliver a speech, and a Group III which attended a conference and delivered speeches in the presence of the company executives. Subjective stress was scored (self-rating score) after subjects in Group III delivered their speeches at the conference. Urine was collected on the next day. The BOM concentrations, as measured by enzyme-linked immunosorbent assay, were normalized to the urinary concentration of creatinine. The concentration of BOM in Group III was significantly higher compared to that in Groups I and II (p<0.01 and p<0.05, respectively). Furthermore, in Group III, the concentration of BOM correlated with the self-rating stress score (r=0.53, p<0.01). These findings suggest that emotional stimuli are associated with an increase in the oxidative metabolites of bilirubin in human urine, and that BOMs could be useful markers of psychological stress.     

Quantification of 22 phthalate metabolites in human urine

Phthalates are ubiquitous industrial chemicals with high potential for human exposure. Validated analytical methods to measure trace concentrations of phthalate metabolites in humans are essential for assessing exposure to phthalates. Previously, we developed a sensitive and accurate automated analytical method for measuring up to 16 phthalate metabolites in human urine by using on-line solid phase extraction coupled with isotope dilution-high performance liquid chromatography (HPLC)-electrospray ionization-tandem mass spectrometry. To include the measurement of seven additional analytes, including oxidative metabolites of diisononyl and diisodecyl phthalates, two chemicals used extensively in numerous consumer products, we used a novel nontraditional HPLC solvent gradient program. With this approach, we achieved adequate resolution and sensitivity for all 22 analytes with limits of detection in the low ng/mL range, without increasing the analytical run time. The method also has high accuracy with automatic recovery correction, high precision, and excellent sample throughput with minimal matrix effects. Although it is possible to measure these 22 phthalate metabolites with adequate precision and accuracy at sub-parts-per-billion levels, additional information, including toxicokinetic data, is needed to demonstrate the usefulness of these phthalate metabolites for exposure assessment purposes.     

Quantification of six herbicide metabolites in human urine

We developed a sensitive, selective and precise method for measuring herbicide metabolites in human urine. Our method uses automated liquid delivery of internal standards and acetate buffer and a mixed polarity polymeric phase solid phase extraction of a 2 mL urine sample. The concentrated eluate is analyzed using high-performance liquid chromatography-tandem mass spectrometry. Isotope dilution calibration is used for quantification of all analytes. The limits of detection of our method range from 0.036 to 0.075 ng/mL. The within- and between-day variation in pooled quality control samples range from 2.5 to 9.0% and from 3.2 to 16%, respectively, for all analytes at concentrations ranging from 0.6 to 12 ng/mL. Precision was similar with samples fortified with 0.1 and 0.25 ng/mL that were analyzed in each run. We validated our selective method against a less selective method used previously in our laboratory by analyzing human specimens using both methods. The methods produced results that were in agreement, with no significant bias observed.     

Coffee aroma constituents and odorant metabolites in human urine

Excretion of odorants and their metabolites into human urine was investigated in urine samples obtained from 14 volunteers after consumption of common dosages of coffee with respect to blank urine samples prior to coffee consumption. Analyses were targeted both on common potent odor constituents of the coffee beverage, as characterized by aroma extract dilution analysis, as well as on volatile constituents of coffee occurring at elevated concentrations and thereby representing quantitatively dominating compounds in the coffee volatile fraction. Identification and quantification of the target substances was accomplished by two-dimensional high resolution gas chromatography mass spectrometry (2D-HR-GC-MS; heart-cut technique) in conjunction with stable isotope dilution assays. Quantification was made on free volatile or odoros marker substances in the urine and their glucuronidated derivatives. Data revealed that some odorants were present at considerably elevated concentrations after the ingestion of coffee. This was confirmed by the odor profiles of the respective urine samples during sensory analysis.     

alpha-Hydroxy-beta-keto-gamma-aminobutyric acid in human urine.

A new amino acid has been isolated from the normal human urine. The chemical structure of the amino acid was determined to be alpha-hydroxy-beta-keto-gamma-aminobutyric acid based on its physical properties involving NMR, infrared and mass spectra, as well as chemical degradation and synthesis. In six healthy adults the urinary contents of the new amino acid were 3.2--4.5 mumol/24 h.     

PhIP metabolites in human urine after consumption of well-cooked chicken

We devised an assay to quantify the metabolites of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in human urine following a single exposure to well-cooked meat. Our method uses LC/MS/MS to detect four metabolites and four deuterated internal standard peaks in a single chromatographic run. N2-OH-PhIP-N2-glucuronide was the most abundant urinary metabolite excreted by the 12 individuals who participated in our study. N2-PhIP glucuronide was the second most abundant metabolite for 8 of the 12 volunteers. The stability of PhIP metabolism over time was studied in three of the volunteers who repeated the assay eight times over a 2.5 year-period. PhIP metabolite excretion varied in each subject over time, although the rate of excretion was more constant. Our results suggest that quantifying PhIP metabolites should make future studies of individual susceptibility and dietary interventions possible.     

High-performance liquid chromatographic determination of the polar metabolites of nifedipine in plasma, blood and urine

A relatively simple reversed-phase high-performance liquid chromatographic method for the determination of the polar metabolites of nifedipine in biological fluids is described. After conversion of 2-hydroxymethyl-6-methyl-4-(2-nitrophenyl)pyridine-3,5-dicarboxylic acid 5-methyl ester (IV) into 5,7-dihydro-2-methyl-4-(2-nitrophenyl)-5-oxofuro[3,4-b]pyridine-3-carboxylic acid methyl ester (V) by heating under acidic conditions, V was extracted with n-pentane—dichloromethane (7:3) and analysed on a C₁₈ column with ultraviolet detection. Subsequently, 2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylic acid monomethyl ester (III) was extracted with chloroform and analysed on the same system. Limits of determination in blood were 0.1 μg/ml for III and 0.05 μg/ml for IV and V; these limits were two to ten times higher for urine. This inter-assay variability was always less than 7.5%.