A surgical method for continuous intraportal infusion of gut microbial metabolites in mice.

Gut microbe-derived metabolites influence human physiology and disease. However, establishing mechanistic links between gut microbial metabolites and disease pathogenesis in animal models remains challenging. The major route of absorption for microbe-derived small molecules is venous drainage via the portal vein to the liver. In the event of presystemic hepatic metabolism, the route of metabolite administration becomes critical. To our knowledge, we describe here a novel portal vein cannulation technique using a s.c. implanted osmotic pump to achieve continuous portal vein infusion in mice. We first administered the microbial metabolite trimethylamine (TMA) over 4 weeks, during which increased peripheral plasma levels of TMA and its host liver-derived cometabolite, trimethylamine-N-oxide, were observed when compared with a vehicle control. Next, 4-hydroxyphenylacetic acid (4-HPAA), a microbial metabolite that undergoes extensive presystemic hepatic metabolism, was administered intraportally to examine effects on hepatic gene expression. As expected, hepatic levels of 4-HPAA were elevated when compared with the control group while peripheral plasma 4-HPAA levels remained the same. Moreover, significant changes in the hepatic transcriptome were revealed by an unbiased RNA-Seq approach. Collectively, to our knowledge this work describes a novel method for administering gut microbe-derived metabolites via the portal vein, mimicking their physiologic delivery in vivo.

Pharmacokinetics, mass balance, and metabolism of [14C]vicagrel, a novel irreversible P2Y12 inhibitor in humans.

Vicagrel, a novel irreversible P2Y12 receptor inhibitor, is undergoing phase III trials for the treatment of acute coronary syndromes in China. In this study, we evaluated the pharmacokinetics, mass balance, and metabolism of vicagrel in six healthy male Chinese subjects after a single oral dose of 20 mg [14C]vicagrel (120 µCi). Vicagrel absorption was fast (Tmax = 0.625 h), and the mean t1/2 of vicagrel-related components was ~38.0 h in both plasma and blood. The blood-to-plasma radioactivity AUCinf ratio was 0.55, suggesting preferential distribution of drug-related material in plasma. At 168 h after oral administration, the mean cumulative excreted radioactivity was 96.71% of the dose, including 68.03% in urine and 28.67% in feces. A total of 22 metabolites were identified, and the parent vicagrel was not detected in plasma, urine, or feces. The most important metabolic spot of vicagrel was on the thiophene ring. In plasma pretreated with the derivatization reagent, M9-2, which is a methylated metabolite after thiophene ring opening, was the predominant drug-related component, accounting for 39.43% of the radioactivity in pooled AUC0-8 h plasma. M4, a mono-oxidation metabolite upon ring-opening, was the most abundant metabolite in urine, accounting for 16.25% of the dose, followed by M3-1, accounting for 12.59% of the dose. By comparison, M21 was the major metabolite in feces, accounting for 6.81% of the dose. Overall, renal elimination plays a crucial role in vicagrel disposition, and the thiophene ring is the predominant metabolic site.

Exploring the Biomarkers of Sepsis-Associated Encephalopathy (SAE): Metabolomics Evidence from Gas Chromatography-Mass Spectrometry.

BACKGROUND: Sepsis-associated encephalopathy (SAE) is a transient and reversible brain dysfunction, that occurs when the source of sepsis is located outside of the central nervous system; SAE affects nearly 30% of septic patients at admission and is a risk factor for mortality. In our study, we sought to determine whether metabolite changes in plasma could be a potential biomarker for the early diagnosis and/or the prediction of the prognosis of sepsis. METHOD: A total of 31 SAE patients and 28 healthy controls matched by age, gender, and body mass index (BMI) participated in our study. SAE patients were divided into four groups according to the Glasgow Coma Score (GCS). Plasma samples were collected and used to detect metabolism changes by gas chromatography-mass spectrometry (GC-MS). Analysis of variance was used to determine which metabolites significantly differed between the control and SAE groups. RESULTS: We identified a total of 63 metabolites that showed significant differences among the SAE and control groups. In particular, the 4 common metabolites in the four groups were 4-hydroxyphenylacetic acid; carbostyril, 3-ethyl-4,7-dimethoxy (35.8%); malic acid peak 1; and oxalic acid. The concentration of 4-hydroxyphenylacetic acid in sepsis patients decreased with a decrease of the GCS. CONCLUSIONS: According to recent research on SAE, metabolic disturbances in tissue and cells may be the main pathophysiology of this condition. In our study, we found a correlation between the concentration of 4-hydroxyphenylacetic acid and the severity of consciousness disorders. We suggest that 4-hydroxyphenylacetic acid may be a potential biomarker for SAE and useful in predicting patient prognosis.

Pharmacokinetics and pharmacokinetic/pharmacodynamic relationship of vicagrel, a novel thienopyridine P2Y12 inhibitor, compared with clopidogrel in healthy Chinese subjects following single oral dosing.

BACKGROUND AND OBJECTIVES: Vicagrel, a novel thienopyridine antiplatelet agent, is an analogue of clopidogrel in development for the treatment of acute coronary syndromes. This study investigated the pharmacokinetic properties of vicagrel after single oral dosing with a direct comparison with clopidogrel in healthy Chinese subjects in the first two phase I clinical studies. The relationship between the exposure to the active metabolite and the platelet reactivity was also assessed for vicagrel. METHODS: Study A was a single-ascending-dose study of vicagrel (5-75 mg) compared with clopidogrel (75 mg) in 67 healthy volunteers. Study B was a randomized, two-period, crossover, loading-dose study of vicagrel 20 mg compared with clopidogrel 300 mg in 12 healthy subjects. Plasma concentrations of three common metabolites of vicagrel and clopidogrel, the active thiol metabolite H4, the inactive thiol metabolite H3, and the S-methylated form of H3 (SM3, the major metabolite of vicagrel), were determined using a validated UHPLC-MS/MS method. The relationship between the AUC0-t of active H4 and the P2Y12 reaction units at 4 h after administration of vicagrel was investigated. Blood concentrations of vicagrel were determined after a single oral administration of vicagrel 25 mg to two healthy Chinese subjects. RESULTS: In the single-ascending-dose study, vicagrel was metabolized rapidly with the median tmax for the three metabolites, namely, H4, H3, and SM3, ranging from 0.25-1.75 h. The pharmacokinetics of the three metabolites for vicagrel were linear across the dose range of 5-75 mg, with the mean Cmax and AUCs for H4 and H3 increasing in an approximately 1:1 dose-proportional manner and for SM3 increasing in a <1:1 dose-proportional manner. The median tmax for active H4 in the vicagrel 5 mg group was slightly shorter than that in the clopidogrel 75 mg group (0.50 versus 0.75 h). The mean AUC0-t for H4 in the vicagrel 5 mg group was similar to that in the clopidogrel 75 mg group (11.7 versus 11.8 ng∙h/mL). The AUC0-t of active H4 was apparently associated with the P2Y12 reaction units at 4 h for vicagrel. In the loading-dose study, for active H4, the median tmax was slightly shorter (0.50 versus 0.75 h) and the mean AUC0-t was 29% higher in the vicagrel 20 mg group than those in the clopidogrel 300 mg group. After a single oral administration of vicagrel 25 mg to 2 subjects, vicagrel was detected in blood but in very low concentrations. CONCLUSIONS: Vicagrel was rapidly and extensively metabolized, and the levels of the parent drug in circulation were very low. The pharmacokinetics of the three metabolites of vicagrel were linear and predictable across the dose range of 5-75 mg. The AUC of active H4 was apparently associated with the P2Y12 reaction units for vicagrel. For active H4, vicagrel 5 mg produced similar exposure (AUC) with more rapid appearance compared with clopidogrel 75 mg, and vicagrel 20 mg produced even slightly higher exposure (AUC) with more rapid appearance compared with clopidogrel 300 mg in humans. TRIAL REGISTRATION: CTR20150346, CTR20160379.

Aspirin Attenuates the Bioactivation of and Platelet Response to Vicagrel in Mice.

Vicagrel, a novel acetate analogue of clopidogrel, exerts more potent antiplatelet effect than clopidogrel in rodents. Relevant evidence indicated that aspirin and vicagrel are the drug substrate for carboxylesterase 2. Accordingly, it is deduced that concomitant use of aspirin could attenuate the bioactivation of and platelet response to vicagrel. To clarify whether there could be such an important drug-drug interaction, the differences in both the formation of vicagrel active metabolite H4 and the inhibition of adenosine diphosphate-induced platelet aggregation by vicagrel were measured and compared between mice treated with vicagrel alone or in combination with aspirin. The plasma H4 concentration was determined by liquid chromatography-tandem mass spectrometry, and the inhibition of platelet aggregation by vicagrel was assessed by whole-blood platelet aggregation. Compared with vicagrel (2.5 mg·kg) alone, concurrent use of aspirin (5, 10, or 20 mg·kg) significantly decreased systemic exposure of H4, an average of 38% and 41% decrease in Cmax and AUC0-∞ in mice when in combination with aspirin at 10 mg·kg, respectively. Furthermore, concomitant use of aspirin (10 mg·kg) and vicagrel (2.5 mg·kg) resulted in an average of 66% reduction in the inhibition of adenosine diphosphate-induced platelet aggregation by vicagrel. We conclude that aspirin significantly attenuates the formation of vicagrel active metabolite H4 and platelet response to vicagrel in mice, and that such an important drug-drug interaction would appear in clinical settings if vicagrel is taken with aspirin concomitantly when marketed in the future.

Comparative analysis of fecal phenolic content between normal and obese rats after oral administration of tea polyphenols.

Tea polyphenols (TP) have many health benefits, but most are metabolized into low molecular-weight phenolic acids after oral administration. In the present study, the absorption, metabolism, and excretion of catechins in rats fed a normal chow diet and in obese rats fed a high-fat and high-sugar (HFHS) diet were compared. After a ten-day oral administration of TP (500 mg per kg bw), the plasma levels of (-)-epigallocatechin gallate (EGCG) and (-)-gallocatechin gallate (GCG) in obese rats were significantly lower than those in the normal group. In obese rats, the fecal levels of EGCG, (-)-epicatechin gallate (ECG) and GCG were significantly enhanced. Ten phenolic metabolites of TP were quantitatively analyzed, and the results showed that 4-hydroxyphenylacetic acid was the primary metabolite in feces and plasma. The plasma and fecal concentrations of 4-hydroxyphenylacetic acid in the obese group were significantly lower than those in normal rats, but the levels of 4-hydroxyphenylpropionic acid in plasma and feces were increased. The content of other phenolic acids was also dramatically changed. These results suggested that a HFHS diet might influence the excretion of tea catechins, leading to insufficient metabolism of catechins by the gut microflora.

Dose-Dependent Increase in Unconjugated Cinnamic Acid Concentration in Plasma Following Acute Consumption of Polyphenol Rich Curry in the Polyspice Study.

Spices that are rich in polyphenols are metabolized to a convergent group of phenolic/aromatic acids. We conducted a dose-exposure nutrikinetic study to investigate associations between mixed spices intake and plasma concentrations of selected, unconjugated phenolic/aromatic acids. In a randomized crossover study, 17 Chinese males consumed a curry meal containing 0 g, 6 g, and 12 g of mixed spices. Postprandial blood was drawn up to 7 h at regular intervals and plasma phenolic/aromatic acids were quantified via liquid chromatography tandem mass spectrometry (LC-MS/MS). Cinnamic acid (CNA, p < 0.0001) and phenylacetic acid (PAA, p < 0.0005) concentrations were significantly increased with mixed spices consumption, although none of the other measured phenolic/aromatic acids differ significantly between treatments. CNA displayed a high dose-exposure association (R² > 0.8, p < 0.0001). The adjusted mean area under the plasma concentration-time curve until 7 h (AUC0⁻7 h) for CNA during the 3 increasing doses were 8.4 ± 3.4, 376.1 ± 104.7 and 875.7 ± 291.9 nM.h respectively. Plasma CNA concentration may be used as a biomarker of spice intake.

Cortisol, cortisone, and 4-methoxyphenylacetic acid as potential plasma biomarkers for early detection of non-small cell lung cancer.

BACKGROUND: Lung cancer is the most common cause of cancer-related deaths in men and women worldwide. Novel diagnostic biomarkers are urgently required to enable the early detection and treatment of lung cancer, and using novel methods to explore tumor-related biomarkers is a hot topic in lung cancer research. The purpose of this study was to use ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS) metabolomics analysis technology combined with multivariate data processing methods to identify potential plasma biomarkers for non-small cell lung cancer (NSCLC). METHODS: Plasma samples from 99 NSCLC patients and 112 healthy controls were randomly divided into the screening group and the validation group, respectively. UPLC-MS metabolomics analysis technology combined with multivariate data processing methods were used to identify potential plasma biomarkers for NSCLC. RESULTS: A total of 254 metabolites were detected and validated in plasma. Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA) modeling indicated that 28 endogenous metabolites were present at significantly different levels in patients with NSCLC than healthy controls (variable importance in projection (VIP)>1 and P<0.001 (independent samples t-test) in both the screening group and the validation group). Further analysis revealed that cortisol, cortisone, and 4-methoxyphenylacetic acid had high sensitivity and specificity values as biomarkers for discriminating between NSCLC and healthy controls. Significant associations between specific plasma metabolites and the pathological type or stage of NSCLC were also observed. CONCLUSIONS: Metabolomics has the potential to distinguish between NSCLC patients and healthy controls, and may reveal new plasma biomarkers for the early detection of NSCLC.

Self-rated health in Brazilian adults and elderly: Data from the National Household Sample Survey 2008 / Percepción de la salud en adultos y ancianos brasileños: datos de la Encuesta Nacional de Hogares 2008

Objective. To investigate prevalence of poor self-rated health and its association with individual and household-level characteristics among adults and elderly in Brazil. Materials and methods. Cross-sectional study with Brazilian National Household Sample Survey 2008 (n=257 816). Crude and multilevel-adjusted Poisson regression models were fitted. Results. After adjusted analysis, poor self-rated health was significantly associated with higher household income, living alone, not having piped water nor garbage collection, lower education, not having health insurance, female sex, higher age, being a current or previous smoker, physical inactivity, having chronic diseases, having physical impairment. Subjects living in rural areas also had higher prevalence of poor self-rated health. The factors most strongly associated with the outcome were physical impairment and reporting three or more chronic diseases. Conclusions. Socioeconomic, health related behaviors, and physical health were associated with poor self-rated health.

Objetivo. Investigar la prevalencia de la percepción negativa de salud y su asociación con características individuales a nivel de los hogares en adultos y adultos mayores de Brasil. Material y métodos. Estudio transversal con datos de la Encuesta Nacional de Hogares de 2008 (n=257 816). Se estimaron modelos de regresión de Poisson multinivel crudos y ajustados. Resultados. Después del análisis ajustado, la autopercepción negativa de salud se asoció significativamente con mayor ingreso, vivir solo, no tener agua corriente ni recolección de basura, baja educación, carecer de seguro de salud, sexo femenino, mayor edad, tabaquismo, inactividad física, enfermedades crónicas y deterioro físico. Los habitantes de zonas rurales también tuvieron mayor prevalencia de percepción negativa. Los factores más fuertemente asociados fueron impedimento físico y presentación de tres o más enfermedades crónicas. Conclusiones. Factores socioeconómicos, comportamientos relacionados con la salud y salud física se asociaron con la percepción negativa.

Protein-phenolic interactions and inhibition of glycation - combining a systematic review and experimental models for enhanced physiological relevance.

BACKGROUND: while antiglycative capacity has been attributed to (poly)phenols, the exact mechanism of action remains unclear. Studies so far are often relying on supra-physiological concentrations and use of non-bioavailable compounds. METHODS: to inform the design of a physiologically relevant in vitro study, we carried out a systematic literature review of dietary interventions reporting plasma concentrations of polyphenol metabolites. Bovine Serum Albumin (BSA) was pre-treated prior to in vitro glycation: either no treatment (native), pre-oxidised (incubated with 10 nM H2O2, for 8 hours) or incubated with a mixture of phenolic acids at physiologically relevant concentrations, for 8 hours). In vitro glycation was carried out in the presence of (i) glucose only (0, 5 or 10 mM), (ii) glucose (0, 5 or 10 mM) plus H2O2 (10 nM), or (iii) glucose (0, 5 or 10 mM) plus phenolic acids (10-160 nM). Fructosamine was measured using the nitro blue tetrazolium method. RESULTS: following (high) dietary polyphenol intake, 3-hydroxyphenylacetic acid is the most abundant phenolic acid in peripheral blood (up to 338 µM) with concentrations of other phenolic acids ranging from 13 nM to 200 µM. The presence of six phenolic acids with BSA during in vitro glycation did not lower fructosamine formation. However, when BSA was pre-incubated with phenolic acids, significantly lower concentration of fructosamine was detected under glycoxidative conditions (glucose 5 or 10 mM plus H2O2 10 nM) (p < 0.001 vs. native BSA). CONCLUSION: protein pre-treatment, either with oxidants or phenolic acids, is an important regulator of subsequent glycation in a physiologically relevant system. High quality in vitro studies under conditions closer to physiology are feasible and should be employed more frequently.

Preparation, characterization and pharmacokinetics of Amoitone B-loaded long circulating nanostructured lipid carriers.

Amoitone B, chemically synthesized as the derivative of Cytosporone B, is a powerful agonist for Nur77 receptor. It has outstanding anticancer activity in vivo. However, the water-insolubility and short biological half-life lead to poor bioavailability, which limits its application. The aim of this study was to develop polyethylene glycol-coated Amoitone B-loaded nanostructured lipid carriers (AmB-PEG-NLC) for parenteral delivery of Amoitone B to prolong drug circulation time in body and enhance the bioavailability. AmB-PEG-NLC were prepared by emulsion-evaporation and low temperature-solidification method, while Amoitone B-loaded NLC (AmB-NLC) were also prepared as control. The characteristics of AmB-PEG-NLC and AmB-NLC such as particle size, zeta potential, entrapment efficiency and drug loading were investigated in detail. The mean particle size was about 200 nm and the zeta potential value was about -15 mV. The X-ray diffraction analysis demonstrated that Amoitone B was not in crystalline state in NLC (AmB-PEG-NLC and AmB-NLC). Drug release pattern with burst release initially and prolonged release afterwards was obtained in vitro for AmB-PEG-NLC. Furthermore, AmB-PEG-NLC exhibited prolonged MRT (mean residence time) and higher AUC (area under drug concentration-time curve) compared with AmB-NLC as well as Amoitone B solution. These results indicated that AmB-PEG-NLC could be a promising delivery system for Amoitone B to prolong the circulation time in body and thus improve its bioavailability.

Elevated phenylacetic acid levels do not correlate with adverse events in patients with urea cycle disorders or hepatic encephalopathy and can be predicted based on the plasma PAA to PAGN ratio.

BACKGROUND: Phenylacetic acid (PAA) is the active moiety in sodium phenylbutyrate (NaPBA) and glycerol phenylbutyrate (GPB, HPN-100). Both are approved for treatment of urea cycle disorders (UCDs) - rare genetic disorders characterized by hyperammonemia. PAA is conjugated with glutamine in the liver to form phenylacetyleglutamine (PAGN), which is excreted in urine. PAA plasma levels ≥ 500 µg/dL have been reported to be associated with reversible neurological adverse events (AEs) in cancer patients receiving PAA intravenously. Therefore, we have investigated the relationship between PAA levels and neurological AEs in patients treated with these PAA pro-drugs as well as approaches to identifying patients most likely to experience high PAA levels. METHODS: The relationship between nervous system AEs, PAA levels and the ratio of plasma PAA to PAGN were examined in 4683 blood samples taken serially from: [1] healthy adults [2], UCD patients of ≥ 2 months of age, and [3] patients with cirrhosis and hepatic encephalopathy (HE). The plasma ratio of PAA to PAGN was analyzed with respect to its utility in identifying patients at risk of high PAA values. RESULTS: Only 0.2% (11) of 4683 samples exceeded 500 µg/ml. There was no relationship between neurological AEs and PAA levels in UCD or HE patients, but transient AEs including headache and nausea that correlated with PAA levels were observed in healthy adults. Irrespective of population, a curvilinear relationship was observed between PAA levels and the plasma PAA:PAGN ratio, and a ratio>2.5 (both in µg/mL) in a random blood draw identified patients at risk for PAA levels>500 µg/ml. CONCLUSIONS: The presence of a relationship between PAA levels and reversible AEs in healthy adults but not in UCD or HE patients may reflect intrinsic differences among the populations and/or metabolic adaptation with continued dosing. The plasma PAA:PAGN ratio is a functional measure of the rate of PAA metabolism and represents a useful dosing biomarker.

Comparison of two mitotane starting dose regimens in patients with advanced adrenocortical carcinoma.

CONTEXT: Mitotane is the only approved drug for treatment of adrenocortical carcinoma. Its pharmacokinetic properties are not fully elucidated and different dosing regimens have never been compared head to head. OBJECTIVE: The objective of the study was to investigate the relationship between mitotane dose and plasma concentration comparing two dosing regimens. DESIGN/SETTING: This was a prospective, open-label, multicenter trial of a predefined duration of 12 weeks. PATIENTS/INTERVENTIONS: Forty mitotane-naïve patients with metastatic adrenocortical carcinoma were assigned to a predefined low- or high-dose regimen by the local investigator. Thirty-two patients could be evaluated in detail. MAIN OUTCOME MEASURE: The difference in median mitotane plasma levels between both treatment groups was measured. RESULTS: Despite a difference in mean cumulative dose (440 ± 142 g vs 272 ± 121 g), median maximum plasma levels were not significantly different between the two groups [high dose 14.3 mg/L (range 6.3-29.7, n = 20) vs 11.3 mg/L (range 5.5-20.0, n = 12), P = .235]. Ten of 20 patients on the high-dose regimen reached plasma concentrations of 14 mg/L or greater after 46 days (range 18-81 d) compared with 4 of 12 patients on the low-dose regimen after 55 days (range 46-74 d, P = .286). All patients who reached 14 mg/L at 12 weeks displayed a level of 4.1 mg/L or greater on day 33 (100% sensitivity). There were no significant differences in frequency and severity of adverse events. Among patients not receiving concomitant chemotherapy mitotane exposure was higher in the high-dose group: 1013 ± 494 mg/L · d vs 555 ± 168 mg/L · d (P = .080). CONCLUSIONS: The high-dose starting regimen resulted in neither significantly different mitotane levels nor a different rate of adverse events, but concomitant chemotherapy influenced these results. Thus, for mitotane monotherapy the high-dose approach is favorable, whereas for combination therapy a lower dose seems reasonable.

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.

GC-MS methods for metabolic profiling of microbial fermentation products of dietary polyphenols in human and in vitro intervention studies.

Flavonoids, a subclass of polyphenols, are major constituents of many plant-based foods and beverages, including tea, wine and chocolate. Epidemiological studies have shown that a flavonoid-rich diet is associated with reduced risk of cardiovascular diseases. The majority of the flavonoids survive intact until they reach the colon where they are then extensively metabolized into smaller fragments. Here, we describe the development of GC-MS-based methods for the profiling of phenolic microbial fermentation products in urine, plasma, and fecal water. Furthermore, the methods are applicable for profiling products obtained from in vitro batch culture fermentation models. The methods incorporate enzymatic deconjugation, liquid-liquid extraction, derivatization, and subsequent analysis by GC-MS. At the level of individual compounds, the methods gave recoveries better than 80% with inter-day precision being better than 20%, depending on the matrix. Limits of detection were below 0.1 microg/ml for most phenolic acids. The newly developed methods were successfully applied to samples from human and in-vitro intervention trials, studying the metabolic impact of flavonoid intake. In conclusion, the methods presented are robust and generally applicable to diverse biological fluids. Its profiling character is useful to investigate on a large scale the gut microbiome-mediated bioavailability of flavonoids.

Gas chromatographic--mass spectrometric determination of phenylacetic acid in human blood.

Phenyl acetic acid, a metabolite of 2-phenyl ethylamine, acts as a neuromodulator in the nigrostriatal dopaminergic pathway stimulating the release of dopamine. The evaluation of phenyl acetic acid concentration in the biological fluid reflects phenyl ethylamine levels thus allowing the assessment of the modulatory role of this endogenous substance. Changes in biological fluids levels of 2-phenylethylamine and/or in its metabolite have been reported in affective disorders, such as depression and schizophrenia. Recently, the occurrence of the "attention deficit hyperactivity syndrome" has been frequently reported in childhood population and involvement of dopaminergic dysfunction in this disease has been suspected. A fast, reliable and reproducible method for the determination of phenyl acetic acid in human blood, is therefore needed in order to have a screening tool for monitoring both healthy childhood population and suspected "attention deficit hyperactivity syndrome" patients. The gas chromatographic-mass spectrometric method here described makes use of a deuterated internal standard in order to overcome problems related to the lack of reproducibility often encountered when a derivatization step is performed.

Increased plasma phenylacetic acid in patients with end-stage renal failure inhibits iNOS expression.

NO prevents atherogenesis and inflammation in vessel walls by inhibition of cell proliferation and cytokine-induced endothelial expression of adhesion molecules and proinflammatory cytokines. Reduced NO production due to inhibition of either eNOS or iNOS may therefore reinforce atherosclerosis. Patients with end-stage renal failure show markedly increased mortality due to atherosclerosis. In the present study we tested the hypothesis that uremic toxins are responsible for reduced iNOS expression. LPS-induced iNOS expression in mononuclear leukocytes was studied using real-time PCR. The iNOS expression was blocked by addition of plasma from patients with end-stage renal failure, whereas plasma from healthy controls had no effect. Hemofiltrate obtained from patients with end-stage renal failure was fractionated by chromatographic methods. The chromatographic procedures revealed a homogenous fraction that inhibits iNOS expression. Using gas chromatography/mass spectrometry, this inhibitor was identified as phenylacetic acid. Authentic phenylacetic acid inhibited iNOS expression in a dose-dependent manner. In healthy control subjects, plasma concentrations were below the detection level, whereas patients with end-stage renal failure had a phenylacetic acid concentration of 3.49 +/- 0.33 mmol/l (n = 41). It is concluded that accumulation of phenylacetic acid in patients with end-stage renal failure inhibits iNOS expression. That mechanism may contribute to increased atherosclerosis and cardiovascular morbidity in patients with end-stage renal failure.

A Phase I clinical and pharmacological evaluation of sodium phenylbutyrate on an 120-h infusion schedule.

PURPOSE: Sodium phenylbutyrate (PB) demonstrates potent differentiating capacity in multiple hematopoietic and solid tumor cell lines. We conducted a Phase I and pharmacokinetic study of PB by continuous infusion to characterize the maximum tolerated dose, toxicities, pharmacokinetics, and antitumor effects in patients with refractory solid tumors. PATIENTS AND METHODS: Patients were treated with a 120-h PB infusion every 21 days. The dose was escalated from 150 to 515 mg/kg/day. Pharmacokinetics were performed during and after the first infusion period using a validated high-performance liquid chromatographic assay and single compartmental pharmacokinetic model for PB and its principal metabolite, phenylacetate. RESULTS: A total of 24 patients were enrolled on study, with hormone refractory prostate cancer being the predominant tumor type. All patients were evaluable for toxicity and response. A total of 89 cycles were administered. The dose-limiting toxicity (DLT) was neuro-cortical, exemplified by excessive somnolence and confusion and accompanied by clinically significant hypokalemia, hyponatremia, and hyperuricemia. One patient at 515 mg/kg/day and another at 345 mg/kg/day experienced this DLT. Toxicity resolved < or =12 h of discontinuing the infusion. Other toxicities were mild, including fatigue and nausea. The maximum tolerated dose was 410 mg/kg/day for 5 days. Pharmacokinetics demonstrated that plasma clearance of PB increased in a continuous fashion beginning 24 h into the infusion. In individuals whose V(max) for drug elimination was less than their drug-dosing rate, the active metabolite phenylacetate accumulated progressively. Plasma PB concentrations (at 410 mg/kg/day) remained above the targeted therapeutic threshold of 500 micromol/liter required for in vitro activity. CONCLUSION: The DLT in this Phase I study for infusional PB given for 5 days every 21 days is neuro-cortical in nature. The recommended Phase II dose is 410 mg/kg/day for 120 h.

A phase I dose escalation and bioavailability study of oral sodium phenylbutyrate in patients with refractory solid tumor malignancies.

PURPOSE: Phenylbutyrate (PB) is an aromatic fatty acid with multiple mechanisms of action including histone deacetylase inhibition. Preclinically, PB demonstrates both cytotoxic and differentiating effects at a concentration of 0.5 mM. We conducted a Phase I trial of p.o. PB patients with refractory solid tumor malignancies to evaluate toxicity, pharmacokinetic parameters, and feasibility of p.o. administration. EXPERIMENTAL DESIGN: Twenty-eight patients with refractory solid tumor malignancies were enrolled on this dose-escalation to maximally tolerated dose trial. Five dose levels of PB were studied: 9 g/day (n = 4), 18 g/day (n = 4), 27 g/day (n = 4), 36 g/day (n = 12), and 45 g/day (n = 4). Pharmacokinetic studies were performed and included an p.o. bioavailability determination. Compliance data were also collected. RESULTS: The recommended Phase II dose is 27 g/day. Overall the drug was well tolerated with the most common toxicities being grade 1-2 dyspepsia and fatigue. Nonoverlapping dose-limiting toxicities of nausea/vomiting and hypocalcemia were seen at 36 g/day. The p.o. bioavailability of PB was 78% for all dose levels, and the biologically active concentration of 0.5 mM was achieved at all dose levels. Compliance was excellent with 93.5% of all possible doses taken. No partial remission or complete remission was seen, but 7 patients had stable disease for more than 6 months while on the drug. CONCLUSIONS: PB (p.o.) is well tolerated and achieves the concentration in vivo that has been shown to have biological activity in vitro. PB may have a role as a cytostatic agent and should be additionally explored in combination with cytotoxics and other novel drugs.