Pharmacokinetics, safety, and tolerability of sodium phenylacetate and sodium benzoate in healthy Japanese volunteers: A phase I, single-center, open-label study.

TAK-123, a combination of sodium phenylacetate (NaPA) and sodium benzoate (NaBZ), is an intravenously administered drug developed for the treatment of acute hyperammonemia in infants, children, and adults with urea cycle enzyme deficiencies. The aim of the current study was to evaluate the pharmacokinetics, safety, and tolerability after intravenous infusion of TAK-123 in Japanese healthy adult volunteers. Ten volunteers received a 3.75 g/m2 loading dose of TAK-123 over a period of 1.5 h followed by a maintenance infusion of the same dose over 24 h. Phenylacetate (PA) and benzoate (BZ) and their respective metabolites, phenylacetylglutamine (PAG) and hippurate (HIP) were measured over a 24-h period using a high-performance liquid chromatography/tandem mass spectrometry method. Non-compartmental analysis was performed using WinNonlin® Professional. During the loading dose, plasma levels of both PA and BZ peaked at 1.5 h. Plasma PA levels plateaued and were maintained up to 6.5 h, whereas plasma BZ levels declined rapidly after switching to maintenance infusion. Urinary excretion ratios of PAG and HIP at 48 h after the administration were 99.3% and 104%, respectively, suggesting that almost all NaPA and NaBZ were metabolized and excreted into urine. Overall, TAK-123 was well-tolerated in healthy Japanese adults.

Longitudinal metabolic and gut bacterial profiling of pregnant women with previous bariatric surgery.

OBJECTIVE: Due to the global increase in obesity rates and success of bariatric surgery in weight reduction, an increasing number of women now present pregnant with a previous bariatric procedure. This study investigates the extent of bariatric-associated metabolic and gut microbial alterations during pregnancy and their impact on fetal development. DESIGN: A parallel metabonomic (molecular phenotyping based on proton nuclear magnetic resonance spectroscopy) and gut bacterial (16S ribosomal RNA gene amplicon sequencing) profiling approach was used to determine maternal longitudinal phenotypes associated with malabsorptive/mixed (n=25) or restrictive (n=16) procedures, compared with women with similar early pregnancy body mass index but without bariatric surgery (n=70). Metabolic profiles of offspring at birth were also analysed. RESULTS: Previous malabsorptive, but not restrictive, procedures induced significant changes in maternal metabolic pathways involving branched-chain and aromatic amino acids with decreased circulation of leucine, isoleucine and isobutyrate, increased excretion of microbial-associated metabolites of protein putrefaction (phenylacetlyglutamine, p-cresol sulfate, indoxyl sulfate and p-hydroxyphenylacetate), and a shift in the gut microbiota. The urinary concentration of phenylacetylglutamine was significantly elevated in malabsorptive patients relative to controls (p=0.001) and was also elevated in urine of neonates born from these mothers (p=0.021). Furthermore, the maternal metabolic changes induced by malabsorptive surgery were associated with reduced maternal insulin resistance and fetal/birth weight. CONCLUSION: Metabolism is altered in pregnant women with a previous malabsorptive bariatric surgery. These alterations may be beneficial for maternal outcomes, but the effect of elevated levels of phenolic and indolic compounds on fetal and infant health should be investigated further.

Amperometric Detection of the Urinary Disease Biomarker p-HPA by Allosteric Modulation of a Redox Polymer-Embedded Bacterial Reductase.

We report an amperometric biosensor for the urinary disease biomarker para-hydroxyphenylacetate ( p-HPA) in which the allosteric reductase component of a bacterial hydroxylase, C1-hpah, is electrically wired to glassy carbon electrodes through incorporation into a low-potential Os-complex modified redox polymer. The proposed biosensing strategy depends on allosteric modulation of C1-hpah by the binding of the enzyme activator and analyte p-HPA, stimulating oxidation of the cofactor NADH. The pronounced concentration-dependence of allosteric C1-hpah modulation in the presence of a constant concentration of NADH allowed sensitive quantification of the target, p-HPA. The specific design of the immobilizing redox polymer with suitably low working potential allowed biosensor operation without the risk of co-oxidation of potentially interfering substances, such as uric acid or ascorbic acid. Optimized sensors were successfully applied for p-HPA determination in artificial urine, with good recovery rates and reproducibility and sub-micromolar detection limits. The proposed application of the allosteric enzyme C1-hpah for p-HPA trace electroanalysis is the first successful example of simple amperometric redox enzyme/redox polymer biosensing in which the analyte acts as an effector, modulating the activity of an immobilized biocatalyst. A general advantage of the concept of allosterically modulated biosensing is its ability to broaden the range of approachable analytes, through the move from substrate to effector detection.

Metabolic Response in Rabbit Urine to Occurrence and Relief of Unilateral Ureteral Obstruction.

Ureteral obstruction will lead clinically to hydronephrosis, which may further develop into partial or complete loss of kidney function and even cause permanent histological damage. However, there is little knowledge of metabolic responses during the obstructed process and its recoverability. In this study, a complete unilateral ureteral obstruction (CUUO) model was established in the rabbit, and 1H NMR-based metabolomic analysis of urine was used to reveal the metabolic perturbations in rabbits caused by CUUO and the metabolic recovery after the CUUO was relieved. Univariate and multivariate statistical analyses were used to identify metabolic characteristics. The gradually decreased levels of 3-hydroxykynurenine, 3-methylhistidine, creatinine, guanidoacetate, meta- and para-hydroxyphenylacetate, and phenylacetylglycine and the gradually increased levels of acetate, alanine, citrate, glycine, lactate, and methionine in urine could be regarded as potential biomarkers for the occurrence and severity of ureteral obstruction. And the reduced levels of 3-methylhistidine, creatinine, guanidoacetate, hippurate, meta-hydroxyphenylacetate, and methylguanidine and the elevated levels of 2-aminoisobutyrate, acetylcholine, citrate, lactate, lysine, valine, and α-ketoglutarate in urine compared with the obstructed level could characterize the metabolic recovery of ureteral obstruction. Our results depicted the disturbed biochemical pathways involved in ureteral obstruction and demonstrated the practicability of recovering renal functions for the patients with severe hydronephrosis in clinical practice by removing causes for obstruction.

Assessing Exposome Effects on Pregnancy through Urine Metabolomics of a Portuguese (Estarreja) Cohort.

This nuclear magnetic resonance metabolomics study compared the influence of two different central Portugal exposomes, one of which comprised an important source of pollutants (the Estarreja Chemical Complex, ECC), on the urinary metabolic trajectory of a cohort of healthy pregnant women (total n = 107). An exposome-independent description of pregnancy metabolism was found to comprise a set of 18 metabolites reflecting expected changes in branched-chain amino acid catabolism and hormone and lipid metabolisms. In addition, a set of small changes in some metabolites was suggested to be exposome-dependent and characteristic of pregnant subjects from the Estarreja region. These results suggested that the Estarreja exposome may impact to a very low extent pregnancy metabolism, inducing slight changes in amino acid metabolism (alanine, glycine, and 3-hydroxyisobutyrate, possibly involved in valine metabolism), tricarboxylic acid (TCA) cycle (cis-aconitate), diet, or gut microflora (furoylglycine) as well as allantoin, 2-hydroxyisobutyrate, and an unassigned resonance at δ 8.45. Furthermore, the urine of Estarreja subjects was found to generally contain higher levels of 4-hydroxyphenylacetate and lower levels of citrate. However, out of the above metabolites, only glycine and citrate seemed to correlate with the proximity to the ECC, with slightly relative higher levels of these compounds found for subjects living closer to the ECC. This suggested possible small effects of local pollutants on energy metabolism, with the remaining exposome-dependent metabolite changes most probably originating from other aspects of the local exposome such as diet and lifestyle. Despite the limitation of this study regarding the unavailability of objective environmental parameters for the period under study, our results confirm the usefulness of metabolomics of human urine to gauge exposome effects on human health and, particularly, during pregnancy.

Quantitation of phenylbutyrate metabolites by UPLC-MS/MS demonstrates inverse correlation of phenylacetate:phenylacetylglutamine ratio with plasma glutamine levels.

Urea cycle disorders (UCDs) are genetic conditions characterized by nitrogen accumulation in the form of ammonia and caused by defects in the enzymes required to convert ammonia to urea for excretion. UCDs include a spectrum of enzyme deficiencies, namely n-acetylglutamate synthase deficiency (NAGS), carbamoyl phosphate synthetase I deficiency (CPS1), ornithine transcarbamylase deficiency (OTC), argininosuccinate lyase deficiency (ASL), citrullinemia type I (ASS1), and argininemia (ARG). Currently, sodium phenylbutyrate and glycerol phenylbutyrate are primary medications used to treat patients with UCDs, and long-term monitoring of these compounds is critical for preventing drug toxic levels. Therefore, a fast and simple ultra-performance liquid chromatography (UPLC-MS/MS) method was developed and validated for quantification of phenylbutyrate (PB), phenylacetate (PA), and phenylacetylglutamine (PAG) in plasma and urine. The separation of all three analytes was achieved in 2min, and the limits of detection were <0.04µg/ml. Intra-precision and inter-precision were <8.5% and 4% at two quality control concentrations, respectively. Average recoveries for all compounds ranged from 100% to 106%. With the developed assay, a strong correlation between PA and the PA/PAG ratio and an inverse correlation between PA/PAG ratio and plasma glutamine were observed in 35 patients with confirmed UCDs. Moreover, all individuals with a ratio ≥0.6 had plasma glutamine levels<1000µmol/l. Our data suggest that a PA/PAG ratio in the range of 0.6-1.5 will result in a plasma glutamine level<1000µmol/l without reaching toxic levels of PA.

Microbial metabolites are associated with a high adherence to a Mediterranean dietary pattern using a 1H-NMR-based untargeted metabolomics approach.

The study of biomarkers of dietary patterns including the Mediterranean diet (MedDiet) is scarce and could improve the assessment of these patterns. Moreover, it could provide a better understanding of health benefits of dietary patterns in nutritional epidemiology. We aimed to determine a robust and accurate biomarker associated with a high adherence to a MedDiet pattern that included dietary assessment and its biological effect. In this cross-sectional study, we included 56 and 63 individuals with high (H-MDA) and low (L-MDA) MedDiet adherence categories, respectively, all from the Prevención con Dieta Mediterránea trial. A 1H-NMR-based untargeted metabolomics approach was applied to urine samples. Multivariate statistical analyses were conducted to determine the metabolite differences between groups. A stepwise logistic regression and receiver operating characteristic curves were used to build and evaluate the prediction model for H-MDA. Thirty-four metabolites were identified as discriminant between H-MDA and L-MDA. The fingerprint associated with H-MDA included higher excretion of proline betaine and phenylacetylglutamine, among others, and decreased amounts of metabolites related to glucose metabolism. Three microbial metabolites - phenylacetylglutamine, p-cresol and 4-hydroxyphenylacetate - were included in the prediction model of H-MDA (95% specificity, 95% sensitivity and 97% area under the curve). The model composed of microbial metabolites was the biomarker that defined high adherence to a Mediterranean dietary pattern. The overall metabolite profiling identified reflects the metabolic modulation produced by H-MDA. The proposed biomarker may be a better tool for assessing and aiding nutritional epidemiology in future associations between H-MDA and the prevention or amelioration of chronic diseases.

Acute Consumption of Flavan-3-ol-Enriched Dark Chocolate Affects Human Endogenous Metabolism.

Flavan-3-ols and methylxanthines have potential beneficial effects on human health including reducing cardiovascular risk. We performed a randomized controlled crossover intervention trial to assess the acute effects of consumption of flavan-3-ol-enriched dark chocolate, compared with standard dark chocolate and white chocolate, on the human metabolome. We assessed the metabolome in urine and blood plasma samples collected before and at 2 and 6 h after consumption of chocolates in 42 healthy volunteers using a nontargeted metabolomics approach. Plasma samples were assessed and showed differentiation between time points with no further separation among the three chocolate treatments. Multivariate statistics applied to urine samples could readily separate the postprandial time points and distinguish between the treatments. Most of the markers responsible for the multivariate discrimination between the chocolates were of dietary origin. Interestingly, small but significant level changes were also observed for a subset of endogenous metabolites. 1H NMR revealed that flavan-3-ol-enriched dark chocolate and standard dark chocolate reduced urinary levels of creatinine, lactate, some amino acids, and related degradation products and increased the levels of pyruvate and 4-hydroxyphenylacetate, a phenolic compound of bacterial origin. This study demonstrates that an acute chocolate intervention can significantly affect human metabolism.

Urinary Time- or Dose-Dependent Metabolic Biomarkers of Aristolochic Acid-Induced Nephrotoxicity in Rats.

The metabolic mechanisms underlying aristolochic acid (AA)-induced nephrotoxicity are inconclusive. A Gas Chromatography-Mass Spectrometer (GC-MS)-based metabolomic study was performed to analyze urinary metabolites in AA-treated rats at different dosages (10, 20, and 40 mg/kg) and time points (2, 4, and 6 days). Serum blood urea nitrogen (BUN), creatinine, and kidney injury were significantly changed only on the 6th day in 40 mg/kg AA group, whereas metabolic alternation appeared even on the 2nd day in 10 mg/kg AA group. A total of 84 differential metabolites were identified in 40 mg/kg AA groups time-dependently and 81 in 10, 20, and 40 mg/kg AA groups dose-dependently (6 days) compared with control group. Eight metabolites were selected as potential metabolic biomarkers including methylsuccinic acid, nicotinamide, 3-hydroxyphenylacetic acid, citric acid, creatinine, uric acid, glycolic acid, and gluconic acid. Four of them were dose-dependently altered including methylsuccinic acid, citric acid, creatinine, and 3-hydroxyphenylacetic acid, which were defined as "early metabolic biomarker." The alteration of nicotinamide, uric acid, and gluconic acid was time- and dose-dependent, whereas the change of glycolic acid was time- or dose-independent. The latter 4 metabolites were defined as "late metabolic biomarker" because of the obvious reduction on the 6th day in 40 mg/kg AA group. In summary, the urinary metabolic alterations were more sensitive than conventional biomarkers of renal injury. The identified metabolites suggested pathways of energy metabolism, gut microbiota, and purine metabolism were associated with AA-induced nephrotoxicity time- or dose-dependently. Further investigation was warranted to determine the roles of the 8 potential metabolic biomarkers in AA-induced nephrotoxicity.

Urinary 3-(3-Hydroxyphenyl)-3-hydroxypropionic Acid, 3-Hydroxyphenylacetic Acid, and 3-Hydroxyhippuric Acid Are Elevated in Children with Autism Spectrum Disorders.

Autism spectrum disorders (ASDs) are a group of mental illnesses highly correlated with gut microbiota. Recent studies have shown that some abnormal aromatic metabolites in autism patients are presumably derived from overgrown Clostridium species in gut, which may be used for diagnostic purposes. In this paper, a GC/MS based metabolomic approach was utilized to seek similar biomarkers by analyzing the urinary information in 62 ASDs patients compared with 62 non-ASDs controls in China, aged 1.5-7. Three compounds identified as 3-(3-hydroxyphenyl)-3-hydroxypropionic acid (HPHPA), 3-hydroxyphenylacetic acid (3HPA), and 3-hydroxyhippuric acid (3HHA) were found in higher concentrations in autistic children than in the controls (p < 0.001). After oral vancomycin treatment, urinary excretion of HPHPA (p < 0.001), 3HPA (p < 0.005), and 3HHA (p < 0.001) decreased markedly, which indicated that these compounds may also be from gut Clostridium species. The sensitivity and specificity of HPHPA, 3HPA, and 3HHA were evaluated by receiver-operating characteristic (ROC) analysis. The specificity of each compound for ASDs was very high (>96%). After two-regression analysis, the optimal area under the curve (AUC, 0.962), sensitivity (90.3%), and specificity (98.4%) were obtained by ROC curve of Prediction probability based on the three metabolites. These findings demonstrate that the measurements of the three compounds are strong predictors of ASDs and support the potential clinical utility for identifying a subgroup of ASDs subjects.

Simultaneous Detection of 3-Nitrotyrosine and 3-Nitro-4-hydroxyphenylacetic Acid in Human Urine by Online SPE LC-MS/MS and Their Association with Oxidative and Methylated DNA Lesions.

Reactive nitrogen species (RNS) can modify proteins at tyrosine and tryptophan residues, and they are involved in the pathogenesis of various human diseases. In this study, we present the first liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method that enables the simultaneous measurement of urinary 3-nitrotyrosine (3-NTYR) and its metabolite 3-nitro-4-hydroxyphenylacetic acid (NHPA). After the addition of stable isotope-labeled internal standards, urine samples were purified and enriched using manual solid-phase extraction (SPE) and HPLC fractionation followed by online SPE LC-MS/MS analysis. The limits of quantification in urine were 3.1 and 2.5 pg/mL for 3-NTYR and NHPA, respectively. Inter- and intraday imprecision was <15%. The mean relative recoveries of 3-NTYR and NHPA in urine were 89-98% and 90-98%, respectively. We further applied this method to 65 urinary samples from healthy subjects. Urinary samples were also analyzed for N-nitrosodimethylamine (NDMA) as well as oxidative and methylated DNA lesions, namely, 8-oxo-7,8-dihydroguanine (8-oxoGua), 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), N7-methylguanine (N7-MeG), and N3-methyladenine (N3-MeA), using reported LC-MS/MS methods. Urinary 3-NTYR and NHPA levels were measured at concentrations of 63.2 ± 51.5 and 77.4 ± 60.8 pg/mL, respectively. Urinary 3-NTYR and NHPA levels were highly correlated with each other and with 8-oxoGua and 8-oxodGuo. Our findings demonstrated that a relationship exists between oxidative and nitrative stress. However, 3-NTYR and NHPA were correlated with N7-MeG and N3-MeA but not with NDMA, suggesting that NDMA may not be a representative biomarker of N-nitroso compounds that are induced by RNS.

Qualitative and quantitative analysis of felbinac and its major metabolites in human plasma and urine by liquid chromatography tandem mass spectrometry and its application after intravenous administration of felbinac trometamol injection.

We present a method for the qualitative and quantitative analysis of felbinac and its major metabolites in human plasma and urine by HPLC-MS/MS and its application. Qualitative analysis through LC-Triple-TOF-MS/MS indicated that oxidization was the main phase-I metabolic pathway of felbinac in human, conjugation with sulfate and glucuronide groups produced at least 7 phase-II metabolites. Quantitative analysis through HPLC-MS/MS in MRM mode was developed and validated for the quantification of felbinac and its major metabolite (4'-hydroxyfelbinac) in human plasma and urine. Linear calibration curves were obtained for felbinac and 4'-hydroxyfelbinac in plasma and urine (r>0.996); intra- and inter-day precision values (RSD%) obtained were ranged from 1.13 to 6.49%, and the accuracy were between 95.9% and 108.6% for the two analytes. The pharmacokinetics and excretion analysis showed that the t1/2 of 4'-hydroxyfelbinac (8.25 ± 4.15 h) is a litter longer than that of felbinac (6.13 ± 2.01 h), but the mean AUC(0-t) value of felbinac was about 20 times higher than that of 4'-hydroxyfelbinac; excretion of felbinac and 4'-hydroxyfelbinac reached their peak values at about 3-6h after intravenous administration of felbinac trometamol in human.

Alterations of urinary metabolite profile in model diabetic nephropathy.

Countering the diabetes pandemic and consequent complications, such as nephropathy, will require better understanding of disease mechanisms and development of new diagnostic methods. Animal models can be versatile tools in studies of diabetic renal disease when model pathology is relevant to human diabetic nephropathy (DN). Diabetic models using endothelial nitric oxide synthase (eNOS) knock-out mice develop major renal lesions characteristic of human disease. However, it is unknown whether they can also reproduce changes in urinary metabolites found in human DN. We employed Type 1 and Type 2 diabetic mouse models of DN, i.e. STZ-eNOS(-/-) C57BLKS and eNOS(-/-) C57BLKS db/db, with the goal of determining changes in urinary metabolite profile using proton nuclear magnetic resonance (NMR). Six urinary metabolites with significantly lower levels in diabetic compared to control mice have been identified. Specifically, major changes were found in metabolites from tricarboxylic acid (TCA) cycle and aromatic amino acid catabolism including 3-indoxyl sulfate, cis-aconitate, 2-oxoisocaproate, N-phenyl-acetylglycine, 4-hydroxyphenyl acetate, and hippurate. Levels of 4-hydroxyphenyl acetic acid and hippuric acid showed the strongest reverse correlation to albumin-to-creatinine ratio (ACR), which is an indicator of renal damage. Importantly, similar changes in urinary hydroxyphenyl acetate and hippurate were previously reported in human renal disease. We demonstrated that STZ-eNOS(-/-) C57BLKS and eNOS(-/-) C57BLKS db/db mouse models can recapitulate changes in urinary metabolome found in human DN and therefore can be useful new tools in metabolomic studies relevant to human pathology.

Entre o campo e o laboratório: a dinâmica de produção de conhecimento no Ambulatório de Menopausa do Caism/Unicamp / Between the country and the laboratory: the dynamics of the production of knowledge at the Menopause Outpatients Clinic at Caism, Unicamp

Este estudo investiga as práticas de produção de conhecimento sobre a menopausa no Caism/Unicamp, centro de referência para políticas públicas em saúde da mulher. Foram realizadas observações de consultas ginecológicas, entrevistas com mulheres e médicos e observação de reuniões de apoio psicológico, buscando identificar os discursos que circulam no lugar e o processo de alistamento de diferentes atores para que os conhecimentos ali produzidos alcancem credibilidade e “viajem” além dos limites do hospital-escola, tornando-se “universais”. A análise baseia-se nos “estudos localistas”, alinhados aos estudos sociais de ciência e tecnologia.

This study investigates the practices involved in the production of knowledge about menopause at Caism, Unicamp, a reference center for public policies for women’s health. Gynecological appointments and psychological support meetings were observed, and women and doctors were interviewed in order to identify what discourse circulates there and how different actors are brought in to ensure that the knowledge produced attains credibility and “travels” beyond the boundaries of the teaching hospital to become “universal”. The analysis is based on localized studies aligned with social studies of science and technology.

Noninvasive diagnosis and evaluation of curative surgery for gastric cancer by using NMR-based metabolomic profiling.

BACKGROUND: Mass screening for gastric cancer (GC), particularly using endoscopy, may not be the most practical approach as a result of its high cost, lack of acceptance, and poor availability. Thus, novel markers that can be used in cost-effective diagnosis and noninvasive screening for GC are needed. METHODS: A total of 154 urine samples from GC patients and healthy individuals and 30 pairs of matched tumor and normal stomach tissues were collected. Multivariate analysis was performed on urinary and tissue metabolic profiles acquired using (1)H nuclear magnetic resonance and (1)H high-resolution magic angle spinning spectroscopy, respectively. In addition, metabolic profiling of urine from GC patients after curative surgery was performed. RESULTS: Multivariate statistical analysis showed significant separation in the urinary and tissue data of GC patients and healthy individuals. The metabolites altered in the urine of GC patients were related to amino acid and lipid metabolism, consistent with changes in GC tissue. In the external validation, the presence of GC (early or advanced) from the urine model was predicted with high accuracy, which showed much higher sensitivity than carbohydrate antigen 19-9 and carcinoembryonic antigen. Furthermore, 4-hydroxyphenylacetate, alanine, phenylacetylglycine, mannitol, glycolate, and arginine levels were significantly correlated with cancer T stage and, together with hypoxanthine level, showed a recovery tendency toward healthy controls in the postoperative samples compared to the preoperative samples. CONCLUSIONS: An urinary metabolomics approach may be useful for the effective diagnosis of GC.

Antitumor activity of CDA-â ¡, a urinary preparation, on human multiple myeloma cell lines via the mitochondrial pathway.

Cell differentiation agent II (CDA­II) is a DNA methyltransferase inhibitor isolated from healthy human urine. In the present study, the antitumor activity of CDA­II on human multiple myeloma (MM) cell lines via the mitochondrial pathway was first revealed. The human MM cell lines were exposed to CDA­II. Cytotoxicity, caspase activation, apoptosis and the effects on the mitochondrial pathway were assessed. CDA­â…¡ was capable of decreasing the depolarized mitochondrial membranes and activating caspase­3 and ­9 and poly (ADP­ribose) polymerase in MM cells treated with CDA­II. CDA­II induced caspase­dependent cell death accompanied by a significant decrease in X-linked inhibitor of apoptosis protein (XIAP), survivin and Mcl­1 levels. The caspase­3 inhibitor, Z­DEVD­FMK, inhibited CDA­II­induced apoptosis. CDA­II potently increased the Bax levels, decreased the Bcl­2/Bax ratio and decreased the expression of the downstream targets of NF­κB. In conclusion, the results of the present study demonstrated that CDA­II treatment leads to the inhibition of p65 nuclear localization and potently induces caspase­dependent apoptosis in MM cells mediated through the mitochondrial pathway at low nanomolar concentrations. These results indicate that CDA­II is a novel inhibitor of NF­κB activity, with notable antimyeloma efficacy. This study provides a rationale for the clinical investigation of CDA­â…¡ in human MM.

Phenolic acid concentrations in plasma and urine from men consuming green or black tea and potential chemopreventive properties for colon cancer.

SCOPE: Tea polyphenols are metabolized by the colonic microflora yielding phenolic metabolites, which may contribute to the health benefits of tea. We determined the serum and urine concentrations of phenolic acids, hippuric acid, and polyhydroxyphenyl-γ-valerolactones during green tea (GT) and black tea (BT) administration. The effects of (-)-epigallocatechin gallate (EGCG) and 3,4-dihydroxyphenylacetic acid (3,4-DHPAA) alone and in combination on bioavailability, intracellular metabolism, and antiproliferative activity were determined in HCT-116 colon cancer cells. METHODS AND RESULTS: The concentration of phenolic metabolites was quantified by HPLC with electrochemical detection and MS. Urine concentrations of 4-hydroxyphenylacetic acid (4-HPAA), 3-hydroxyphenylacetic acid (3-HPAA), and polyhydroxy-γ-valerolactones were increased significantly in men drinking GT compared to control. Urine concentration of 3-O-methylgallic acid (3OMGA) was significantly increased in men drinking BT compared to control. Serum 3,4-DHPAA was significantly increased after consumption of GT and BT and 4-HPAA after GT consumption. In vitro treatment of HCT-116 colon cancer cells with 3,4-DHPAA and EGCG exhibited an additive antiproliferative effect, while methylation of 3,4-DHPAA was significantly decreased. 3OMGA exhibited the strongest antiproliferative activity among the phenolic acids. CONCLUSION: The consumption of both, GT and BT, was associated with a significant increase in urinary and serum phenolic acids.

Analysis of 4-bromo-2,5-dimethoxyphenethylamine abuser's urine: identification and quantitation of urinary metabolites.

The metabolites of 4-bromo-2,5-dimethoxyphenethylamine (2C-B), a psychoactive drug with hallucinogenic activity, were investigated in a urine sample from a user of 2C-B. The urine sample was deconjugated enzymatically and the metabolites were recovered by liquid-liquid extraction. The extract was analyzed by gas chromatography/mass spectrometry after derivatization, and the results were used to identify and quantitate the metabolites. 4-Bromo-2,5-dimethoxyphenylacetic acid was the most abundant metabolite of 2C-B in human urine and accounted for 73% of the total amount of detected metabolites, followed by 4-bromo-2-hydroxy-5-methoxyphenylacetic acid (13%) and 4-bromo-2,5-dimethoxyphenylethyl alcohol (4.5%). According to the literature, the main metabolites of 2C-B in rat urine are N-(4-bromo-2-methoxy-5-hydroxyphenylethyl)acetamide and N-(4-bromo-2-hydroxy-5-methoxyphenylethyl)acetamide. However, these metabolites accounted for only a small proportion of the total amount of detected metabolites in human urine, which indicates that there are significant species-specific differences in the metabolism of 2C-B. 4-Bromo-2,5-dimethoxyphenylacetic acid, which was the most abundant metabolite in human urine, is thought to be generated by deamination of 2C-B by monoamine oxidase (MAO) followed by oxidation by aldehyde dehydrogenase. Our results suggest that MAO plays a crucial role in the metabolism of 2C-B in humans.

Urinary phenylacetylglutamine as dosing biomarker for patients with urea cycle disorders.

UNLABELLED: We have analyzed pharmacokinetic data for glycerol phenylbutyrate (also GT4P or HPN-100) and sodium phenylbutyrate with respect to possible dosing biomarkers in patients with urea cycle disorders (UCD). STUDY DESIGN: These analyses are based on over 3000 urine and plasma data points from 54 adult and 11 pediatric UCD patients (ages 6-17) who participated in three clinical studies comparing ammonia control and pharmacokinetics during steady state treatment with glycerol phenylbutyrate or sodium phenylbutyrate. All patients received phenylbutyric acid equivalent doses of glycerol phenylbutyrate or sodium phenylbutyrate in a cross over fashion and underwent 24-hour blood samples and urine sampling for phenylbutyric acid, phenylacetic acid and phenylacetylglutamine. RESULTS: Patients received phenylbutyric acid equivalent doses of glycerol phenylbutyrate ranging from 1.5 to 31.8 g/day and of sodium phenylbutyrate ranging from 1.3 to 31.7 g/day. Plasma metabolite levels varied widely, with average fluctuation indices ranging from 1979% to 5690% for phenylbutyric acid, 843% to 3931% for phenylacetic acid, and 881% to 1434% for phenylacetylglutamine. Mean percent recovery of phenylbutyric acid as urinary phenylacetylglutamine was 66.4 and 69.0 for pediatric patients and 68.7 and 71.4 for adult patients on glycerol phenylbutyrate and sodium phenylbutyrate, respectively. The correlation with dose was strongest for urinary phenylacetylglutamine excretion, either as morning spot urine (r = 0.730, p < 0.001) or as total 24-hour excretion (r = 0.791 p<0.001), followed by plasma phenylacetylglutamine AUC(24-hour), plasma phenylacetic acid AUC(24-hour) and phenylbutyric acid AUC(24-hour). Plasma phenylacetic acid levels in adult and pediatric patients did not show a consistent relationship with either urinary phenylacetylglutamine or ammonia control. CONCLUSION: The findings are collectively consistent with substantial yet variable pre-systemic (1st pass) conversion of phenylbutyric acid to phenylacetic acid and/or phenylacetylglutamine. The variability of blood metabolite levels during the day, their weaker correlation with dose, the need for multiple blood samples to capture trough and peak, and the inconsistency between phenylacetic acid and urinary phenylacetylglutamine as a marker of waste nitrogen scavenging limit the utility of plasma levels for therapeutic monitoring. By contrast, 24-hour urinary phenylacetylglutamine and morning spot urine phenylacetylglutamine correlate strongly with dose and appear to be clinically useful non-invasive biomarkers for compliance and therapeutic monitoring.

Simultaneous determination of aromatic acid metabolites of styrene and styrene-oxide in rat urine by gas chromatography-flame ionization detection.

A convenient and reliable gas chromatographic method was developed for the simultaneous determination of six aromatic acid metabolites of styrene and styrene-oxide in rat urine; i.e., benzoic (BA), phenylacetic (PAA), mandelic (MA), phenylglyoxylic (PGA), hippuric (HA) and phenylaceturic (PAUA) acids. The method involves a one-pot esterification-extraction procedure, performed directly on urine without prior treatment. Analyses were performed on a RTX-1701 capillary column and the recovered isopropyl esters derivatives were detected by flame ionization detection. The analytical method was validated for selectivity, linearity, detection and quantification limits, recovery and intra-day and inter-day precisions. Calibration curves showed linearity in the range of 8-800 mg/L, except for HA and PAUA (40-800 mg/L). Limits of detection were between 0.2 (PPA) and 7.0 (PAUA) mg/L. The intra-day precisions determined at three concentrations levels were less than 5% for BA, PAA, MA and PGA and 9% for HA and PAUA, respectively. The corresponding mean inter-day precisions for these two groups were 8 and 16%, respectively. The method was successfully applied to quantitatively analyze styrene, styrene-oxide, ethylbenzene and toluene metabolites in urine samples from rats exposed by inhalation to these compounds at levels close to the occupational threshold limit values. Provided that this method can be transposed to human urine, it could have applications as part of biological monitoring for workers exposed to styrene or related compounds.