Preparation of chitosan-modified magnetic Schiff base network composite nanospheres for effective enrichment and detection of hippuric acid and 4-methyl hippuric acid.

Chitosan-modified magnetic Schiff base network composite nanospheres (Fe3O4@SNW@Chitosan) were prepared for the enrichment and detection of hippuric acid (HA) and 4-methyl hippuric acid (4-MHA) via magnetic solid phase extraction (MSPE) connected with HPLC. The SNW was one of the covalent organic framework, which constructed through covalent bonds, shown comprising solvent stability, low density and accessible pores. The obtained Fe3O4@SNW@Chitosan has many merits as a magnetic sorbent, including a hydrophilic surface, uniform pore size, unique ordered channel structure, and superparamagnetism. The favourable linearity of this MSPE-HPLC method was in the range of 1-1000 µg L-1, and LODs of HA and 4-MHA were 0.3 µg L-1 and 0.2 µg L-1, respectively. The recoveries in urine samples were range from 95.3 to 109.0 % with the RSD less than 9.6 %. When employed for the enrichment of HA and 4-MHA, Fe3O4@SNW@Chitosan exhibited great potential as a candidate for preconcentration.

Spontaneous variability of pre-dialysis concentrations of uremic toxins over time in stable hemodialysis patients.

BACKGROUND AND AIM: Numerous outcome studies and interventional trials in hemodialysis (HD) patients are based on uremic toxin concentrations determined at one single or a limited number of time points. The reliability of these studies however entirely depends on how representative these cross-sectional concentrations are. We therefore investigated the variability of predialysis concentrations of uremic toxins over time. METHODS: Prospectively collected predialysis serum samples of the midweek session of week 0, 1, 2, 3, 4, 8, 12, and 16 were analyzed for a panel of uremic toxins in stable chronic HD patients (N = 18) while maintaining dialyzer type and dialysis mode during the study period. RESULTS: Concentrations of the analyzed uremic toxins varied substantially between individuals, but also within stable HD patients (intra-patient variability). For urea, creatinine, beta-2-microglobulin, and some protein-bound uremic toxins, Intra-class Correlation Coefficient (ICC) was higher than 0.7. However, for phosphorus, uric acid, symmetric and asymmetric dimethylarginine, and the protein-bound toxins hippuric acid and indoxyl sulfate, ICC values were below 0.7, implying a concentration variability within the individual patient even exceeding 65% of the observed inter-patient variability. CONCLUSION: Intra-patient variability may affect the interpretation of the association between a single concentration of certain uremic toxins and outcomes. When performing future outcome and interventional studies with uremic toxins other than described here, one should quantify their intra-patient variability and take into account that for solutes with a large intra-patient variability associations could be missed.

Urinary Metabolic Phenotyping of Women with Lower Urinary Tract Symptoms.

Lower urinary tract symptoms (LUTS), including urinary incontinence, urgency and nocturia, affect approximately half of women worldwide. Current diagnostic methods for LUTS are invasive and costly, while available treatments are limited by side effects leading to poor patient compliance. In this study, we aimed to identify urine metabolic signatures associated with LUTS using proton nuclear magnetic resonance (1H NMR) spectroscopy. A total of 214 urine samples were collected from women attending tertiary urogynecology clinics (cases; n = 176) and healthy control women attending general gynecology clinics (n = 36). Despite high variation in the urine metabolome across the cohort, associations between urine metabolic profiles and BMI, parity, overactive bladder syndrome, frequency, straining, and bladder storage were identified using KODAMA (knowledge discovery by accuracy maximization). Four distinct urinary metabotypes were identified, one of which was associated with increased urinary frequency and low BMI. Urine from these patients was characterized by increased levels of isoleucine and decreased levels of hippurate. Our study suggests that metabolic profiling of urine samples from LUTS patients offers the potential to identify differences in underlying etiology, which may permit stratification of patient populations and the design of more personalized treatment strategies.

New insights into the catalytic mechanism of human glycine N-acyltransferase.

Even though the glycine conjugation pathway was one of the first metabolic pathways to be discovered, this pathway remains very poorly characterized. The bi-substrate kinetic parameters of a recombinant human glycine N-acyltransferase (GLYAT, E.C. 2.3.1.13) were determined using the traditional colorimetric method and a newly developed HPLC-ESI-MS/MS method. Previous studies analyzing the kinetic parameters of GLYAT, indicated a random Bi-Bi and/or ping-pong mechanism. In this study, the hippuric acid concentrations produced by the GLYAT enzyme reaction were analyzed using the allosteric sigmoidal enzyme kinetic module. Analyses of the initial rate (v) against substrate concentration plots, produced a sigmoidal curve (substrate activation) when the benzoyl-CoA concentrations was kept constant, whereas the plot with glycine concentrations kept constant, passed through a maximum (substrate inhibition). Thus, human GLYAT exhibits mechanistic kinetic cooperativity as described by the Ferdinand enzyme mechanism rather than the previously assumed Michaelis-Menten reaction mechanism.

Association between exposure to o-chlorobenzylidene malononitrile (CS riot control agent) and urinary metabolite 2-chlorohippuric acid in U.S. Army Mask Confidence Training.

This study was conducted among U.S. Army soldiers to evaluate the association between exposure to o-chlorobenzylidene malononitrile (CS riot control agent) and urinary metabolite 2-chlorohippuric acid (CHA) detected in test subjects (n = 87) after completion of Mask Confidence Training. CS exposures ranged 0.086-4.9 mg/m³ ([Formula: see text] = 2.7 mg/m³). CHA levels (corrected for creatinine) at 2-, 8-, 24-, and 30-hr post-exposure resulted in ranges of 94.6-1120 µg/g-cr ([Formula: see text] = 389 µg/g-cr), 15.80-1170 µg/g-cr ([Formula: see text] = 341 µg/g-cr), 4.00-53.1 µg/g-cr ([Formula: see text] = 19.3 µg/g-cr), and 1.99-28.4 µg/g-cr ([Formula: see text] = 10.6 µg/g-cr), respectively. Spearman's correlation revealed CHA levels strongly correlated with time sampled (r = -0.748, p < 0.05) and weakly correlated with CS concentration (r = 0.270, p < 0.05). A linear relationship was observed between CHA, CS concentration, and time of urine sample according to the following regression equation: ln(CHA, µg/g-cr) = 5.423 + 0.316 (CS conc., mg/m³) - 0.002 (time sampled), (R = 0.910, R² = 0.827, p < 0.05). This relationship suggests that CHA has the potential to be an effective retrospective indicator of CS exposure in future biomarker developments.

New nanostructured support for carrier-mediated electromembrane extraction of high polar compounds.

A new support has been proposed to be used for carrier-mediated electromembrane extraction purposes. The new support (Tiss®-OH) is a 100µm thickness sheet nanofiber membrane manufactured by electrospinning and composed by acrylic nanofibers. It has been used in an electromembrane extraction (EME) combined with a HPLC procedure using diode array detection. The proposed method has been used for the extraction of four high polarity acidic compounds: nicotinic acid, amoxicillin, hippuric acid and salicylic acid. Analytes were extracted from an aqueous sample solution (pH 4) (donor phase) using a Tiss®-OH sheet that supports a 5% (w/v) Aliquat®336 in 1-octanol liquid membrane. Aqueous solution (pH 6) was used as acceptor phase. The electrical field was generated from a d.c. electrical current of 100V through two spiral shaped platinum wires placed into donor and acceptor phases. Analytes were extracted in 10min with recoveries in the 60-85% range. The proposed EME procedure has been successfully applied to the determination of the target analytes in human urine samples.

Biomarker discovery for drug-induced phospholipidosis: phenylacetylglycine to hippuric acid ratio in urine and plasma as potential markers.

CONTEXT: Drug-induced phospholipidosis is one of the significant concerns in drug development, especially in safety assessment and noninvasive diagnostic tool is highly desirable. OBJECTIVE: The objective of this study is to explored novel biomarkers for phospholipidosis using a metabolomic approach. METHOD: NMR spectrometry and LC/MS/MS analyses were applied to urine and plasma of rats administrated cationic amphiphilic drugs. RESULTS: The phenylacetylglycine to hippuric acid ratio in plasma was increased in time and dose-dependent manners; and it was well correlated with histopathological observation. CONCLUSION: The plasma phenylacetylglycine to hippuric acid ratio is a potential marker in monitoring drug-induced phospholipidosis.

Expanding Biosensing Abilities through Computer-Aided Design of Metabolic Pathways.

Detection of chemical signals is critical for cells in nature as well as in synthetic biology, where they serve as inputs for designer circuits. Important progress has been made in the design of signal processing circuits triggering complex biological behaviors, but the range of small molecules recognized by sensors as inputs is limited. The ability to detect new molecules will increase the number of synthetic biology applications, but direct engineering of tailor-made sensors takes time. Here we describe a way to immediately expand the range of biologically detectable molecules by systematically designing metabolic pathways that transform nondetectable molecules into molecules for which sensors already exist. We leveraged computer-aided design to predict such sensing-enabling metabolic pathways, and we built several new whole-cell biosensors for molecules such as cocaine, parathion, hippuric acid, and nitroglycerin.

The role of sodium in the salty taste of permeate.

Many food companies are trying to limit the amount of sodium in their products. Permeate, the liquid remaining after whey or milk is ultrafiltered, has been suggested as a salt substitute. The objective of this study was to determine the sensory and compositional properties of permeates and to determine if elements other than sodium contribute to the salty taste of permeate. Eighteen whey (n=14) and reduced-lactose (n=4) permeates were obtained in duplicate from commercial facilities. Proximate analyses, specific mineral content, and nonprotein nitrogen were determined. Organic acids and nucleotides were extracted followed by HPLC. Aromatic volatiles were evaluated by gas chromatography-mass spectrometry. Descriptive analysis of permeates and model solutions was conducted using a trained sensory panel. Whey permeates were characterized by cooked/milky and brothy flavors, sweet taste, and low salty taste. Permeates with lactose removed were distinctly salty. The organic acids with the highest concentration in permeates were lactic and citric acids. Volatiles included aldehydes, sulfur-containing compounds, and diacetyl. Sensory tests with sodium chloride solutions confirmed that the salty taste of reduced-lactose permeates was not solely due to the sodium present. Permeate models were created with NaCl, KCl, lactic acid, citric acid, hippuric acid, uric acid, orotic acid, and urea; in addition to NaCl, KCl, lactic acid, and orotic acid were contributors to the salty taste.

Second-order multivariate models for the processing of standard-addition synchronous fluorescence-pH data. Application to the analysis of salicylic acid and its major metabolite in human urine.

In the present work, we describe the determination of salicylic acid and its major metabolite, salicyluric acid, in spiked human urine samples, using synchronous fluorescence spectra measured in a flow-injection system with a double pH gradient. Because the fluorescent urine background constitutes a potentially interfering signal, it becomes necessary to achieve the second-order advantage. Moreover, due to significant changes in the signal of the analytes in the presence of the urine matrix, mainly for salicyluric acid, standard addition was required in order to obtain appropriate quantifications. Several second-order multivariate calibration models were evaluated for this purpose: PARAFAC and MCR-ALS in two different modes, and PLS/RBL.

Lonicera caerulea fruits reduce UVA-induced damage in hairless mice.

UVA photons are less energetic than UVB photons but they are more abundant in solar radiation. Modern tools have shown that UVA light has serious adverse effects on the skin. We investigated the effect of consuming Lonicera caerulea berries on UVA-induced damage in SKH-1 mice. The mice were fed a diet containing L. caerulea berries (10%, w/w) for 14 days before a single UVA (30 J/cm(2)) treatment. Effects on haematological and antioxidant parameters were evaluated 4 and 24h after irradiation. The bioavailability of L. caerulea phenolics was also assessed. Consuming the L. caerulea berry-enriched diet caused reduced malondialdehyde production and increased catalase activity and glutathione levels were found in skin and erythrocytes. UVA-induced NADPH:quinone oxidoreductase-1 and gamma-L-glutamate-L-cysteine ligase protein in skin were reduced in mice fed L. caerulea berries. Enhanced heme oxygenase-1 level in skin, interleukin-17 in plasma and reduced interleukin-12 levels in plasma were found in the mice on the experimental diet. Histological (pyknotic) changes in the nuclei of basal cells induced by UVA exposure were reduced in L. caerulea berry consuming animals. HLPC-MS analysis showed high concentrations of hippuric acid, one of the main metabolites of aromatic amino acids and phenolic compounds, in skin, liver, urine and faeces of mice consuming the berries. Taken together, consumption of L. caerulea berries affords protection from the adverse effects of a single UVA exposure mainly via modulation of antioxidant parameters.

Evaluation of hippuric acid content in goat milk as a marker of feeding regimen.

Organic producers, traders, and consumers must address 2 issues related to milk: authentication of the production system and nutritional differentiation. The presence of hippuric acid (HA) in goat milk samples has been proposed as a possible marker to differentiate the feeding regimen of goats. The objective of this work is to check the hypothesis that HA could be a marker for the type of feeding regimen of goats by studying the influence of production system (conventional or organic) and feeding regimen (with or without grazing fodder). With this purpose, commercial cow and goat milk samples (n=27) and raw goat milk samples (n=185; collected from different breeds, localizations, and dates) were analyzed. Samples were grouped according to breed, feeding regimen, production system, and origin to compare HA content by ANOVA and honestly significant difference Tukey test at a confidence level of ≥95%. Hippuric acid content was obtained by analyzing milk samples with capillary electrophoresis. This method was validated by analyzing part of the samples with HPLC as a reference technique. Sixty-nine raw goat milk samples (of the total 158 samples analyzed in this work) were quantified by capillary electrophoresis. In these samples, the lowest average content for HA was 7±3 mg/L. This value corresponds to a group of conventional raw milk samples from goats fed with compound feed. The highest value of this group was 28±10 mg/L, corresponding to goats fed compound feed plus grass. Conversely, for organic raw goat milk samples, the highest concentration was 67±14 mg/L, which corresponds to goats fed grass. By contrast, the lowest value of this organic group was 26±10 mg/L, which belongs to goats fed organic compounds. Notice that the highest HA average content was found in samples from grazing animals corresponding to the organic group. This result suggests that HA is a good marker to determine the type of goats feeding regimen; a high content of HA represents a diet based mainly or exclusively on eating green grass (grazing), independently of the production system. Hence, this marker would not be useful for the actual organic policies to distinguish organic milk under the current regulations, because organic dairy ruminants can be fed organic compound feed and conserved fodder without grazing at all.

Comparative study of Mg/Al- and Zn/Al-layered double hydroxide-perindopril erbumine nanocomposites for inhibition of angiotensin-converting enzyme.

The intercalation of a drug active, perindopril, into Mg/Al-layered double hydroxide for the formation of a new nanocomposite, PMAE, was accomplished using a simple ion exchange technique. A relatively high loading percentage of perindopril of about 36.5% (w/w) indicates that intercalation of the active took place in the Mg/Al inorganic interlayer. Intercalation was further supported by Fourier transform infrared spectroscopy, and thermal analysis shows markedly enhanced thermal stability of the active. The release of perindopril from the nanocomposite occurred in a controlled manner governed by pseudo-second order kinetics. MTT assay showed no cytotoxicity effects from either Mg/Al-layered double hydroxide or its nanocomposite, PMAE. Mg/Al-layered double hydroxide showed angiotensin-converting enzyme inhibitory activity, with 5.6% inhibition after 90 minutes of incubation. On incubation of angiotensin-converting enzyme with 0.5 µg/mL of the PMAE nanocomposite, inhibition of the enzyme increased from 56.6% to 70.6% at 30 and 90 minutes, respectively. These results are comparable with data reported in the literature for Zn/Al-perindopril.

Study of acute biochemical effects of thallium toxicity in mouse urine by NMR spectroscopy.

Thallium (Tl) is a toxic heavy metal and its exposure to the human body causes physiological and biochemical changes due to its interference with potassium-dependent biological reactions. A high-resolution (1)H NMR spectroscopy based metabonomic approach has been applied for investigating acute biochemical effects caused by thallium sulfate (Tl(2)SO(4)). Male strain A mice were divided in three groups and received three doses of Tl(2)SO(4) (5, 10 and 20 mg kg(-1) b.w., i.p.). Urine samples collected at 3, 24, 72 and 96 h post-dose time points were analyzed by (1)H NMR spectroscopy. NMR spectral data were processed and analyzed using principal components analysis to represent biochemical variations induced by Tl(2)SO(4). Results showed Tl-exposed mice urine to have distinct metabonomic phenotypes and revealed dose- and time-dependent clustering of treated groups. The metabolic signature of urine analysis from Tl(2)SO(4)-treated animals exhibited an increase in the levels of creatinine, taurine, hippurate and ß-hydroxybutyrate along with a decrease in energy metabolites trimethylamine and choline. These findings revealed Tl-induced disturbed gut flora, membrane metabolite, energy and protein metabolism, representing physiological dysfunction of vital organs. The present study indicates the great potential of NMR-based metabonomics in mapping metabolic response for toxicology, which could ultimately lead to identification of potential markers for Tl toxicity.

Differentiation of organic goat's milk based on its hippuric acid content as determined by capillary electrophoresis.

Organic foods have lately aroused interest by virtue of their quality and their essential, exclusive characteristics. In this study, we assessed the potential of CE to detect a marker that is able to identify the particular food given to the goats and develop an effective method to determine directly the most abundant organic acid in goat's milk (viz. hippuric acid). In addition, we examined the use of the hippuric acid content of goat's milk as a general marker for authenticating organic goat's milk. The feeding of these goats can be influenced by the time of the year. For this reason, we have collected samples from 20 conventionally fed goats and 20 organically fed goats over a period of 2 months. A threshold value dependent on the hippuric acid content was thus established for the first time with a view to discriminating between conventional and organic goat's milk. Organic acids in goat's milk were separated in a running buffer consisting of 120 mM sodium tetraborate decahydrate and 0.5 mM CTAB set to pH 8. The precision of the ensuing method is acceptable for hippuric acid; thus, the RSD for peak area and migration time was less than 10 and 4%, respectively. Also, calibration curves were linear throughout the studied concentration range.

HPLC study of uremic fluids related to optical dialysis adequacy monitoring.

PURPOSE: The aim of this study was to investigate uremia-related high-performance liquid chromatography (HPLC) ultraviolet (UV) absorbance profiles of serum and spent dialysate and to study the removal of uremic retention solutes in connection with optical dialysis adequacy monitoring. METHODS: 10 uremic patients were investigated using online spectrophotometry at a wavelength of 280 nm over the course of 30 hemodialysis treatments. The dialysate and blood samples were taken and analyzed simultaneously using standard biochemical methods and reversed-phase HPLC. Filters with cutoff at 3 kDa and 70 kDa were used for the pre-treatment of the serum. The chromatographic peaks were detected by a UV detector at wavelengths of 254 and 280 nm. RESULTS: This study indicated that the main solute responsible for UV absorbance in the spent dialysate is a low-molecular-weight, water-soluble, non-protein-bound compound uric acid (UA). Three additional uremic retention solutes - creatinine (CR), indoxyl sulphate (IS) and hippuric acid (HA) - were identified from the HPLC profiles. The number of detected HPLC peaks was not significantly different for a serum filtered through the 3 kDa or 70 kDa cutoff filters, and was lower for the spent dialysate, indicating that the molecular weight (MW) of the main UV chromophores in the uremic fluids did not exceed 3 kDa. The reduction ratio (RR) estimated by the total area of HPLC peaks at 254 nm and 280 nm in the serum and by the online UV absorbance at 280 nm was best related to the removal of small water-soluble non-protein bound solutes like urea (UR), CR and UA. CONCLUSIONS: The present study contributes new information on the removal of uremic retention solutes during hemodialysis and on the origin of the optical dialysis adequacy monitoring signal.

A rapid assay for angiotensin-converting enzyme activity using ultra-performance liquid chromatography-mass spectrometry.

Angiotensin-converting enzyme (ACE) plays an important role in the renin-angiotensin system and ACE activity is usually assayed in vitro by monitoring the transformation from a substrate to the product catalyzed by ACE. A rapid and sensitive analysis method or ACE activity by quantifying simultaneously the substrate hippuryl-histidyl-leucine and its product hippuric acid using an ultra-performance liquid chromatography coupled with electrospray ionization-mass spectrometry (UPLC-MS) was first developed and applied to assay the inhibitory activities against ACE of several natural phenolic compounds. The established UPLC-MS method showed obvious advantages over the conventional HPLC analysis in shortened running time (3.5 min), lower limit of detection (5 pg) and limit of quantification (18 pg), and high selectivity aided by MS detection in selected ion monitoring (SIM) mode. Among the six natural products screened, five compounds, caffeic acid, caffeoyl acetate, ferulic acid, chlorogenic acid and resveratrol indicated potent in vitro ACE inhibitory activity with IC(50) values of 2.527 +/- 0.032, 3.129 +/- 0.016, 10.898 +/- 0.430, 15.076 +/- 1.211 and 6.359 +/- 0.086 mm, respectively. A structure-activity relationship estimation suggested that the number and the situation of the hydroxyls on the benzene rings and the acrylic acid groups may play the most predominant role in their ACE inhibitory activity.

The autocorrelation matrix probing biochemical relationships after metabolic fingerprinting with CE.

Fingerprinting together with statistical analysis is often employed to compare samples in metabonomic studies of a disease. Correlation algorithms can aid by extracting information based on the variation patterns of key metabolites. This information can be linked to metabolite identification or to specific up/down-regulated biochemical pathways. Matlab-based software employing the Pearson's correlation algorithm was applied to urine electropherograms from 20 mice infected with the schistosoma parasite. The fingerprints were the sum of electropherograms analysed with normal and reverse polarity, in two different modes MEKC and CZE and with two different capillaries (uncoated and polyacrylamide coated) to provide a broad picture of the samples. Hippurate, a metabolite that was depleted in the infected group and is present in both polarities, was chosen as a test variable; it correlated with itself to a p value of <0.000. Phenylacetylglycine, a metabolite shown as over expressed in the disease, was positively correlated to three metabolites in its same pathway with a correlation coefficient of 0.7 and p<0.000 to phenylalanine, 0.7 and p<0.000 to 2-hydroxyphenylacetic and 0.55 and p<0.003 to phenylacetate. The study shows that the autocorrelation matrix is able to provide extra information from data files acquired by CE analyses. It underlined an up-regulated metabolic path by association in the schistosoma infection model.

[Autopsy case of aspirin intoxication: distribution of salicylic acid and salicyluric acid in body fluid and organs].

A 52 year-old woman ingested approximately 300 tablets (325 mg) of aspirin in a suicide attempt. We analyzed the concentrations of salicylic acid (SA) and salicyluric acid (SUA) in body fluids and organs using a modified previous high-performance liquid chromatographic method. The concentrations of SA in heart and femoral blood were 1.1 mg/mL and 1.3 mg/mL, respectively; the results were far higher than the lethal level. The concentration of SA was 0.3-0.4 mg/g in brain, 0.9-1.4 mg/g in lung, 0.6-0.8 mg/g in liver and 0.9 mg/mL in kidney.

Quantitation of phenylalanine and its trans-cinnamic, benzoic and hippuric acid metabolites in biological fluids in a single GC-MS analysis.

We describe a sensitive, simple and convenient stable isotope dilution assay developed to study endogenous metabolism of administered stable isotope-labeled phenylalanine (Phe) in phenylketonuric (PKU) mice treated experimentally with phenylalanine ammonia lyase (PAL). Mouse urine and plasma containing endogenous and administered labeled Phe together with internal standard Phe bearing a different pattern of labeling are converted by in situ diazotization to 2-chloro-3-phenylpropionic acid (CPP). A single solvent extraction is then used to isolate the isotopomers of CPP along with the trans-cinnamic acid (TCA) produced from Phe by PAL, as well as the TCA metabolites benzoic and hippuric acids. This procedure eliminates the need for a separate ion-exchange isolation step for Phe on a second sample aliquot and separate GC-MS analysis. Extracted CPP and the Phe metabolites are then measured by conversion to the pentafluorobenzyl esters and a single analysis by electron capture negative ion GC-MS. The estimated lower limit of quantitation is 0.1 microM.