Extracts Of Human Urine Research Articles

Integrated gene co-expression network analysis and experimental validation revealed potential targets of human urine extract CDA-II in treating chronic myeloid leukemia

BackgroundCell differentiation agent II (CDA-II) exhibits potent anti-proliferative and apoptosis-inducing properties against a variety of cancer cells. However, its mechanism of action in chronic myeloid leukemia (CML) remains unclear. MethodsCell counting Kit 8 (CCK-8) and flow cytometry were used to investigate the effects of CDA-II on the biological characteristics of K562 cells. Gene (mRNA and lncRNA) expression profiles were analyzed by bioinformatics to screen differentially expressed genes and to perform enrichment analysis. The Pearson correlation coefficients of lncRNAs and mRNAs were calculated using gene expression values, and a lncRNA/mRNA co-expression network was constructed. The MCODE and cytoHubba plugins were used to analyze the co-expression network. ResultsThe Results, derived from CCK-8 and flow cytometry, indicated that CDA-II exerts dual effects on K562 cells: it inhibits their proliferation and induces apoptosis. From bioinformatics analysis, we identified 316 mRNAs and 32 lncRNAs. These mRNAs were predominantly related to the meiotic cell cycle, DNA methylation, transporter complex and peptidase regulator activity, complement and coagulation cascades, protein digestion and absorption, and cell adhesion molecule signaling pathways. The co-expression network comprised of 163 lncRNA/mRNA interaction pairs. Notably, our analysis results implicated clustered histone gene families and five lncRNAs in the biological effects of CDA-II on K562 cells. ConclusionThis study highlights the hub gene and lncRNA/mRNA co-expression network as crucial elements in the context of CDA-II treatment of CML. This insight not only enriches our understanding of CDA-II's mechanism of action but also might provide valuable clues for subsequent experimental studies of CDA-II, and potentially contribute to the discovery of new therapeutic targets for CML.

A novel endogenous retention-index for minimizing retention-time variations in metabolomic analysis with reversed-phase ultrahigh-performance liquid-chromatography and mass spectrometry

Consistent retention time (tR) of metabolites is vital for identification in metabolomic analysis with ultrahigh-performance liquid-chromatography (UPLC). To minimize inter-experimental tR variations from the reversed-phase UPLC-MS, we developed an endogenous retention-index (endoRI) using in-sample straight-chain acylcarnitines with different chain-length (LC, C0–C26) without additives. The endoRI-corrections reduced the tR variations caused by the combined changes of mobile phases, gradients, flow-rates, elution time, columns and temperature from up to 5.1 min–0.2 min for most metabolites in a model metabolome consisting of 91 metabolites and multiple biological matrices including human serum, plasma, fecal, urine, A549 cells and rabbit liver extracts. The endoRI-corrections also reduced the inter-batch and inter-platform tR variations from 1.5 min to 0.15 min for 95 % of detected features in the above biological samples. We further established a quantitative model between tR and LC for predicting tR values of acylcarnitines when absent in samples. This makes it possible to compare metabolites’ tR from different tR databases and the UPLC-based metabolomic data from different batches.

Synthesis and Characterization of a New Molecularly Imprinted Polymer for Selective Extraction of Mandelic Acid Metabolite from Human Urine as a Biomarker of Environmental and Occupational Exposures to Styrene.

4-Vinylpyridine molecularly imprinted polymer (4-VPMIP) microparticles for mandelic acid (MA) metabolite as a major biomarker of exposure to styrene (S) were synthesized by bulk polymerization with a noncovalent approach. A common mole ratio of 1:4:20 (i.e., metabolite template: functional monomer: cross-linking agent, respectively) was applied to allow the selective solid-phase extraction of MA in a urine sample followed by high-performance liquid chromatography-diode array detection (HPLC-DAD). In this research, the 4-VPMIP components were carefully selected: MA was used as a template (T), 4-Vinylpyridine (4-VP) as a functional monomer (FM), ethylene glycol dimethacrylate (EGDMA) as a cross-linker (XL), and azobisisobutyronitrile (AIBN) as an initiator (I) and acetonitrile (ACN) as a porogenic solvent. Non-imprinted polymer (NIP) which serves as a "control" was also synthesized simultaneously under the same condition without the addition of MA molecules. Fourier transform infrared (FT-IR) spectroscopy and scanning electron microscopy (SEM) were used to characterize the imprinted and nonimprinted polymer to explain the structural and morphological characteristics of the 4-VPMIP and surface NIP. The results obtained from SEM depicted that the polymers were irregularly shaped microparticles. Moreover, MIPs surfaces had cavities and were rougher than NIP. In addition, all particle sizes were less than 40 µm in diameter. The IR spectra of 4-VPMIPs before washing MA were a little different from NIP, while 4-VPMIP after elution had a spectrum that was almost identical to the NIP spectrum. The adsorption kinetics, isotherms, competitive adsorption, and reusability of 4-VPMIP were investigated. 4-VPMIP showed good recognition selectivity as well as enrichment and separation abilities for MA in the extract of human urine with satisfactory recoveries. The results obtained in this research imply that 4-VPMIP might be used as a sorbent for MA solid-phase extraction (MISPE), for the exclusive extraction of MA in human urine.

Rapid and simultaneous determination of histidine metabolism intermediates in human and mouse microbiota and biomatrices.

Histidine metabolism is a key pathway physiologically involved in satiety, recognition memory, skin, and neural protection and allergic diseases. Microbiologically-produced imidazole propionate induces type II diabetes and interferes with glucose lowering drugs. Despite their determinant health implications, no single method simultaneously assesses histidine metabolites in urine, feces, and microbiota. The aim of this study was to develop a simple, rapid, and sensitive method for the determination of histidine and its major bioactive metabolites histamine, N-acetylhistamine, imidazole-4-acetate, cis-urocanate, trans-urocanate, glutamate and imidazole propionate, using ultrahigh-performance liquid chromatography with electrospray ionization tandem mass spectrometry. An innovative simple extraction method from small aliquots of human and mice urine, feces and microbial cell extracts was coupled to separation in a 6.5min chromatographic run. The successful performance allowed accurate and precise quantification of all metabolites in mouse feces, suggesting broad exchange of histidine metabolites between the gut and mice. Higher urine histamine, histamine to histidine ratio, and imidazole-4-acetate pointed to an underlying inflammatory or allergic process in mice compared to human subjects. N-acetylhistamine and imidazole propionate were detected in human and mouse feces, confirming its origin from gut microbial metabolism. Our novel and robust analytical method captured histidine metabolism in a single assay that will facilitate broad and deep histidine metabolic phenotyping assessing the impact of microbiota on host health in large-scale human observational and interventional studies.

An exploratory approach for an oriented development of an untargeted hydrophilic interaction liquid chromatography-mass spectrometry platform for polar metabolites in biological matrices

The analysis of polar metabolites based on liquid chromatography-mass spectrometry (LC-MS) methods should take into consideration the complexity of interactions in LC columns to be able to cover a broad range of metabolites of key biological pathways. Therefore, in this study, different chromatographic columns were tested for polar metabolites including reversed-phase and hydrophilic interaction liquid chromatography (HILIC) columns. Based on a column screening, two new generations of zwitterionic HILIC columns were selected for further evaluation. A tree-based method optimization was applied to investigate the chromatographic factors affecting the retention mechanisms of polar metabolites with zwitterionic stationary phases. The results were evaluated based on a scoring system which was applied for more than 80 polar metabolites with a high coverage of key human metabolic pathways. The final optimized methods showed high complementarity to analyze a wide range of metabolic classes including amino acids, small peptides, sugars, amino sugars, phosphorylated sugars, organic acids, nucleobases, nucleosides, nucleotides and acylcarnitines. Optimized methods were applied to analyze different biological matrices, including human urine, plasma and liver cell extracts using an untargeted approach. The number of high-quality features (< 30% median relative standard deviation) ranged from 3,755 for urine to 5,402 for the intracellular metabolome of liver cells, showing the potential of the methods for untargeted purposes.

Nano-sized Fe3O4@SiO2-molecular imprinted polymer as a sorbent for dispersive solid-phase microextraction of melatonin in the methanolic extract of Portulaca oleracea, biological, and water samples

In this study, a magnetic molecularly imprinted polymer (MMIP (Fe3O4@SiO2-MIP)) was used for the dispersive magnetic solid-phase microextraction (d-MSP-μ-E) to design an easy and effective method for melatonin (MLT) extraction in the methanolic extract of Portulaca oleracea, human urine and plasma, and water samples. HPLC with UV detection was utilized, and pH, the type and volume of eluent, MMIP mass, and contact time were considered as effective factors in the study of MLT separation and pre-concentration. These factors were optimized by Plackett–Burman and multi-objective response surface methodology (RSM). The values were 10 mg, 14 min, 4.2, methanol, 0.180 mL, 2.5 min, for the MMIP mass, time of sorption, sample pH, eluent type, eluent volume, and time of elution, respectively. At the optimum conditions, the limit of detection (LOD) was 0.046 ng mL−1, and the limit of quantification (LOQ) was 0.156 ng mL−1. The sorption capacity of the proposed MMIP sorbent was 109.1 mg g−1 at the optimum conditions. Besides, linear dynamic range (LDR) was 0.2–4200 ng mL−1, and the precision of the method (RSD %) for triplicate measurements was <6.1%. The MMIP showed saturation magnetization of 19.75 emu g−1, resulting in fast separation of the sorbent. The sorption test revealed the high sorption capacity of the MMIP for MLT and its homogeneous binding sites. In all spiked levels (50, 100, 200, and 500 ng mL−1), 93.07–104.1% was the range obtained for the recovery of MLT. The relative selectivity factor (β) values of MLT/tryptophan, MLT/serotonin, MLT/ferulic acid, MLT/mefenamic acid, MLT/quercetin, MLT/luteolin, and MLT/chlorogenic acid were 1.60, 1.68, 2.02, 2.38, 2.32, 2.40, and 2.50, respectively. The results of desorption–regeneration cycles (seven times) by employing the MMIP showed the high stability of the resultant material. In conclusion, the MMIP combined with the magnetic separation showed a specific sorption behavior for MLT and suggested a simple, flexible, selective, and powerful analytical tool.

Use of ammonium sulfite as a post-column derivatization reagent for rapid detection and quantification of aldehydes by LC-MS

Derivatization is frequently used in liquid chromatography–mass spectrometry (LC-MS) to improve the ionization characteristics of analytes, and facilitate their structural elucidation and quantification. However, the off-line derivatization process usually requires more analysis time. Therefore, the present work develops a novel LC/post-column derivatization (PCD)/MS (LC-PCD-MS) method for rapid detection of aldehydes in complex samples. The PCD method is based on a bisulfite addition reaction with aldehydes that can be easily conducted by mixing the LC elute with an (NH4)2SO3 solution via a tee. The resulting derivatives readily produce diagnostic HSO3− ions under collision induced dissociation (CID) in the negative ion mode of MS, even at an extremely low CID energy such as 1 eV, which can significantly improve the detection selectivity of aldehydes and reduce the occurrence of false positives. The limits of detection (LODs) of hexanal and heptanal were 3 nM and 2 nM, respectively. Moreover, good linearity in the concentration range of 0.01–1 μM for hexanal and heptanal was obtained with the determination coefficient (R2) being greater than 0.996 and the intra- and inter-day relative standard deviations (RSDs) were lower than 13.5% and 17.9%, respectively, indicating the acceptable reproducibility of the method. The LC-PCD-MS method was successfully applied for the rapid quantification of aldehydes in human urine and solvent extracts of traditional Chinese medicines (TCMs). Furthermore, a precursor ion scan strategy based on the diagnostic HSO3− ion was also developed and applied for the preliminary screening of aldehydes in complex samples of cinnamon and lemongrass.

DQF J-RES NMR: Suppressing the singlet signals for improving the J-RES spectra from complex mixtures.

Two-dimensional J-RESolved spectroscopy (J-RES) finds routine use in metabolomics for reducing signal overlap as it separates chemical shift and multiplet information along two frequency axes. However, only magnitude mode of the experiment is practical which prevents exploitation of its full resolving power. Tailing from high-intensity metabolite peaks often obscure nearby low-intensity metabolite peaks which leads to ambiguity in assignment of metabolites. Absorptive mode J-RES spectroscopy offers better-resolving power but comes at the cost of either sensitivity or complicated post-processing. Quite often for certain complex mixtures such as bio-fluids some components of the mixture display intense singlet signals which dominate the whole spectrum resulting in less reliable detection of weaker metabolite signals. Multi-frequency presaturation could suppress these intense singlets but will also remove the useful weaker multiplet peaks which are either totally eclipsed with the intense singlets or very close in frequency. We show that by using a double quantum filter (DQF) in magnitude mode J-RES technique, the intensity of the strong singlet metabolite peaks can be reduced relative to the intensity of the sparsely present multiplet metabolite signals. This approach leads to the identification of many weak intensity multiplet peaks which are otherwise undetected due to their overlap with intense singlet peaks in regular J-RES as well as 1D 1H spectra. Although the improved intensity of most of the weaker peaks relative to the strong singlet peaks is observed, some multiplets can disappear due to the delay-dependent modulation of the signals by the DQF. A few DQF J-RES spectra recorded with different DQF delays, therefore, produce better assignment when analyzed together. The technique is demonstrated on a mixture of eight compounds, human urine, and plant extract samples.

Human Urine Extract Cell Differentiation Agent 2 Protects PC12 Cells from Serum Deprivation-Induced Apoptosis Accompanied with Priming of Extracellular Signal-Regulated Kinase Activation and Differentiation Induction.

To investigate the potential neuroprotective effect of human urine extract cell differentiation agent 2 (CDA-2) by the model of serum deprivation-induced apoptosis of PC12 cells and study the underlying molecular mechanisms. Apoptosis of PC12 cells was induced by serum deprivation. CDA-2 at doses of 0.5-4 mg/mL was used to treat the serum-deprived PC12 cells. The cellular viability was measured by sulforhodamine B binding assay and the cell apoptosis was determined by flow cytometer. Western blot was used to analyze the expression of differentiation markers and activity of extracellular signal-regulated kinase (ERK). The cellular morphology was examined under an inverted microscope. CDA-2 inhibited apoptotic cell death of serum-deprived PC12 cells in a dose-dependent manner. Expression of low- and mid-sized neurofilaments was observed in serum-deprived PC12 cells treated with CDA-2 or nerve growth factor (NGF). However, CDA-2 did not induce proliferation of these cells like NGF. The morphology of CDA-2 treated cells was changed from rounded to neuron-like flat polygonal shape in contrast to the extensive neurite outgrowth induced by NGF. CDA-2 transiently induced the phosphorylation of ERK in serum deprived-PC12 cells and the expression of neurofilaments induced by CDA-2 was attenuated by mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) inhibitor PD98059. Human urine extract CDA-2 showed a potential neuroprotective activity which may correlate with ERK activation and differentiation induction.

Retention behavior of lipids in reversed-phase ultrahigh-performance liquid chromatography–electrospray ionization mass spectrometry

Reversed-phase ultrahigh-performance liquid chromatography (RP-UHPLC) method using two 15cm sub–2μm particles octadecylsilica gel columns is developed with the goal to separate and unambiguously identify a large number of lipid species in biological samples. The identification is performed by the coupling with high-resolution tandem mass spectrometry (MS/MS) using quadrupole – time-of-flight (QTOF) instrument. Electrospray ionization (ESI) full scan and tandem mass spectra are measured in both polarity modes with the mass accuracy better than 5ppm, which provides a high confidence of lipid identification. Over 400 lipid species covering 14 polar and nonpolar lipid classes from 5 lipid categories are identified in total lipid extracts of human plasma, human urine and porcine brain. The general dependences of relative retention times on relative carbon number or relative double bond number are constructed and fit with the second degree polynomial regression. The regular retention patterns in homologous lipid series provide additional identification point for UHPLC/MS lipidomic analysis, which increases the confidence of lipid identification. The reprocessing of previously published data by our and other groups measured in the RP mode and ultrahigh-performance supercritical fluid chromatography on the silica column shows more generic applicability of the polynomial regression for the description of retention behavior and the prediction of retention times. The novelty of this work is the characterization of general trends in the retention behavior of lipids within logical series with constant fatty acyl length or double bond number, which may be used as an additional criterion to increase the confidence of lipid identification.

An alternative approach for assessment of liquid chromatography-mass spectrometry matrix effects using auto-sampler programmed co-injection.

We report the use of auto-sampler programmable functions to co-inject analyte standard solution and matrix extract to assess ion enhancement and suppression (matrix effects) in LC-MS. This is effectively an automated post-extraction addition (APEA) procedure, emulating the manual post-extraction addition (PEA) approach widely adopted for assessment of matrix effects. To verify that APEA was comparable to the conventional PEA approach, matrix effects were determined using both methods for a selection of 31 illicit and pharmaceutical drugs in 10 different human urine extracts. Matrix effects measured using APEA were statistically indistinguishable from manual PEA methodology for 27 of the 31 drugs. Of the four drugs that showed significant differences using the two methods, three differed by less than 2 %, which is within the expected accuracy limits required for matrix effect determinations. The remaining analyte, trimeprazine, was found to degrade in the spiked PEA matrix extract, accounting for the difference between matrix effects measured by the PEA and APEA approaches. APEA enables a single matrix extract to be assessed at multiple analyte concentrations, resulting in a considerable reduction in sample preparation time. In addition, APEA can reduce the quantity of analyte-free sample matrix required for matrix effect assessment, which is an important consideration in certain analytical and bioanalytical fields. This work shows that APEA may be considered as an acceptable alternative to PEA for the assessment of matrix effects in LC-MS method validation and may be applicable to a variety of matrices such as environmental samples.

Targeting tryptophan and tyrosine metabolism by liquid chromatography tandem mass spectrometry

An imbalance in tryptophan (Trp) and tyrosine (Tyr) metabolites is associated with neurological and inflammatory disorders. The accurate and precise measurement of these compounds in biological specimens is a powerful tool to understand the biochemical state in several diseases. In this study, a rapid, accurate and sensitive method based on liquid chromatography–tandem mass spectrometry (LC–MS/MS) for the targeted analysis of the metabolism of Trp and Tyr has been developed and validated. The method allows for the adequate quantification of Trp, Tyr and, eight Trp metabolites, three Tyr metabolites, together with four competitive large neutral amino acids. Serotonin, 5-hydroxyindoleacetic acid, kynurenine, kynurenic acid, dopamine, and homovanilic acid were among the targeted compounds. Sample preparation, chromatographic separation and mass spectrometric detection were optimized in human urine, human plasma and mice prefrontal cortex extracts. The method was shown to be linear (r>0.98) in the range of endogenous concentrations for all studied metabolites. In general, the limits of detection were suitable for the detection of the endogenous levels. Intra- and inter-assay precisions below 25% and accuracies ranging from 80 to 120% were found for most of the analytes. The use of labeled internal standards corrected the moderate matrix effect observed for some compounds. The applicability of the method was confirmed by analyzing urine samples collected from 13 healthy volunteers and comparing the results with previously established normal ranges. In addition, urine samples from two patients and a heterozygous carrier of a family with disturbed monoamine metabolism due to a loss of function mutation in the MAOA gene (X-linked) were analyzed and compared with samples from controls. All data together show the potential of the developed approach for targeted metabolomic studies.

Novel electroanalyical method for the determination of andrographolide from Andrographis paniculata extract and urine samples

This paper presents, for the first time, electrochemical behavior of diterpene lactone andrographolide and its voltammetric determination using differential pulse voltammetry (DPV) at boron-doped diamond electrode, in human urine and oil extract from Andrographis paniculata seeds. Andrographolide, due to its chemical structure, provides well defined peak at +1.55V vs. Ag/AgCl electrode (3M) in Britton–Robinson buffer solution pH 12. The proposed method, under optimum experimental parameters displays excellent selectivity towards andrographolide in the presence of some interference, as ascorbic acid, uric acid and dopamine, with detection limit of 0.75μM. Calibration curves were found to be linear in the range from 2 to 40μM, with two linear ranges, first from 2 to 10μM and second from 10 to 40μM with corresponding equations I (μA)=(4.00±0.12) c (μM)+(13.42±0.66) (R2=0.9975) and I (μA)=(0.87±0.05) c (μM)+(46.33±1.53) (R2=0.9767), respectively. Relative standard deviation of 7 measurements was 4% which verified excellent repeatability of proposed method and this method was successfully applied for the determination of andrographolide in human urine sample and extract from the A. paniculata seeds. The Prussian blue method was used for validation of the proposed elactroanalytical method.

Enhanced acylcarnitine annotation in high-resolution mass spectrometry data: fragmentation analysis for the classification and annotation of acylcarnitines.

Metabolite annotation and identification are primary challenges in untargeted metabolomics experiments. Rigorous workflows for reliable annotation of mass features with chemical structures or compound classes are needed to enhance the power of untargeted mass spectrometry. High-resolution mass spectrometry considerably improves the confidence in assigning elemental formulas to mass features in comparison to nominal mass spectrometry, and embedding of fragmentation methods enables more reliable metabolite annotations and facilitates metabolite classification. However, the analysis of mass fragmentation spectra can be a time-consuming step and requires expert knowledge. This study demonstrates how characteristic fragmentations, specific to compound classes, can be used to systematically analyze their presence in complex biological extracts like urine that have undergone untargeted mass spectrometry combined with data dependent or targeted fragmentation. Human urine extracts were analyzed using normal phase liquid chromatography (hydrophilic interaction chromatography) coupled to an Ion Trap-Orbitrap hybrid instrument. Subsequently, mass chromatograms and collision-induced dissociation and higher-energy collisional dissociation (HCD) fragments were annotated using the freely available MAGMa software1. Acylcarnitines play a central role in energy metabolism by transporting fatty acids into the mitochondrial matrix. By filtering on a combination of a mass fragment and neutral loss designed based on the MAGMa fragment annotations, we were able to classify and annotate 50 acylcarnitines in human urine extracts, based on high-resolution mass spectrometry HCD fragmentation spectra at different energies for all of them. Of these annotated acylcarnitines, 31 are not described in HMDB yet and for only 4 annotated acylcarnitines the fragmentation spectra could be matched to reference spectra. Therefore, we conclude that the use of mass fragmentation filters within the context of untargeted metabolomics experiments is a valuable tool to enhance the annotation of small metabolites.

MyCompoundID: Using an Evidence-Based Metabolome Library for Metabolite Identification

Identification of unknown metabolites is a major challenge in metabolomics. Without the identities of the metabolites, the metabolome data generated from a biological sample cannot be readily linked with the proteomic and genomic information for studies in systems biology and medicine. We have developed a web-based metabolite identification tool ( http://www.mycompoundid.org ) that allows searching and interpreting mass spectrometry (MS) data against a newly constructed metabolome library composed of 8,021 known human endogenous metabolites and their predicted metabolic products (375,809 compounds from one metabolic reaction and 10,583,901 from two reactions). As an example, in the analysis of a simple extract of human urine or plasma and the whole human urine by liquid chromatography-mass spectrometry and MS/MS, we are able to identify at least two times more metabolites in these samples than by using a standard human metabolome library. In addition, it is shown that the evidence-based metabolome library (EML) provides a much superior performance in identifying putative metabolites from a human urine sample, compared to the use of the ChemPub and KEGG libraries.

Interaction ofγ-Glutamyltranspeptidase with Ibuprofen-S-Acyl-Glutathione In Vitro and In Vivo in Human

Ibuprofen is metabolized to chemically reactive acyl glucuronide and S-acyl-CoA metabolites that are proposed to transacylate glutathione (GSH) forming ibuprofen-S-acyl-GSH (I-SG) in vivo. Herein, we report the detection of novel metabolites of ibuprofen, namely ibuprofen-N-acyl-cysteinylglycine (I-N-CG), ibuprofen-N-acyl-cysteine (I-N-C), and the mercapturic acid conjugate, ibuprofen-S-acyl-N-acetylcysteine (I-S-NAC), in urine from an ibuprofen-dosed volunteer. Thus, analysis of ibuprofen-dosed (Advil, 800 mg, Pfizer, Madison, NJ) human urine extracts by sensitive liquid chromatography tandem mass spectrometric detection resulted in the identification of I-N-CG, I-N-C, and I-S-NAC derivatives as minor metabolites (6.0, 1.7, and 0.2 µg excreted 10-hours postadministration, respectively). I-N-CG is proposed to be formed from the degradation of I-SG by γ-glutamyltranspeptidase (γ-GT)-mediated cleavage of the γ-glutamyl group, leading to an unstable ibuprofen-S-acyl-cysteinylglycine (I-S-CG) intermediate that undergoes spontaneous S to N intramolecular rearrangement. Then, dipeptidase-mediated cleavage of glycine from I-N-CG leads to the formation of I-N-C. Treatment of racemic I-SG (100 µM) in vitro with commercially available bovine kidney γ-GT (0.1 units/ml) in buffer at pH 7.4 and 37°C resulted in its complete degradation, yielding (R)- and (S)-I-N-CG after 15 minutes of incubation. In vitro enzyme kinetic studies with bovine kidney γ-GT incubated separately with (R)- and (S)-I-SG isomers revealed no enantioselective degradation. Results from these studies provided evidence that ibuprofen is metabolized in human to reactive transacylating-type intermediates that react with GSH, forming I-SG thioester that, following degradation by γ-GT and dipeptidase enzymes and following S to N intramolecular rearrangement, leads to the urinary excretion of the I-N-CG and I-N-C amide-linked conjugates, respectively.

Study of the matrix effect on the determination of nonconjugated xenobiotics in human urine by high-performance liquid chromatography/tandem mass spectrometry

Factors affecting the sensitivity and selectivity of the determination of diuretics, anabolic steroids, central nervous system stimulants, and narcotics in the analysis of human urine extracts by high-performance liquid chromatography-tandem mass spectrometry with atmospheric pressure electrospray ionization and recording of positive ions were investigated. Mass spectra were obtained for all of the test compounds; the characteristic ions, retention times, detection limits, degree of ionization suppression by the matrix, the extraction of the analytes from human biological fluids were determined for all analytes; the selectivity and specificity of determination were evaluated.

Laser desorption/ionization mass spectrometry on nanostructured semiconductor substrates: DIOS™ and QuickMass™

In the era of systems biology, new analytical platforms are under demand. Desorption/ionization on silicon mass spectrometry (DIOS-MS) is a promising high throughput laser mass spectrometry approach that has attracted a lot of attention, and has been commercialized. Another substrate material manufactured by physical method has also been made commercially available under the trade name of QuickMass™. These two commercial substrates, DIOS™ and QuickMass™, were investigated independently from the manufacturers and were characterized by a number of advanced surface techniques. This work determined (1) the correlation between the substrate physicochemical properties and their LDI activity, (2) the feasibility of metabolic profiling from complex biological matrices and (3) the laser desorption/ionization mechanism. The DIOS™ substrate was characterized with a thick nano-sized porous layer, a high surface concentration of fluorocarbon and silicon oxides and super-hydrophobicity. In contrast, the QuickMass™ substrate consisted of a non-porous germanium thin-film. The relatively high ionization efficiency obtained from the DIOS™ substrate was contributed to the fluorosilane manufacturing processes and its porous morphology. Despite the QuickMass™ substrate being less effective, it was noted that the use of germanium affords a self-cleaning mechanism and suppresses background interference of mass spectra. The suitability of DIOS™ substrates for metabolic profiling of complex biological matrices was demonstrated. DIOS mass spectra of human blood plasma, human urine and animal liver tissue extracts were produced. Suitable extraction methods were found to be important, but relatively simplified approaches were sufficient. Further investigations of the DIOS desorption/ionization mechanism were carried out. The previously proposed sub-surface state reaction could be a molten-solid interfacial state reaction of the substrate and this had a significant effect toward the protonation reaction of amines.

Abstract C189: Active compound isolated from human urine induced differentiation and apoptosis in K562 chronic myelogenous leukemia cells and enhanced the effects of imatinib

Abstract Leukemia is a serious disease that affects both children and adults. Although all-trans retinoic acid (ATRA) and targeted drug such as imatinib could effectively treat acute promyelocytic (APL) and chronic myelogenous leukemia (CML), respectively, rare therapeutics is available when drug resistance or recurrence occurred. Searching for alternative agent for refractory patient is urgently needed. Our previous studies had found that a human urine extract CDA-2 could induce differentiation of HL60 APL cells as well as potentiate the effects of ATRA and imatinib on HL60 and K562 CML cells, respectively. Purification of active compound possessing these anti-leukemic effects from CDA-2 was thus performed. Recently, an active compound named as P18.9 was isolated. It induced differentiation of K562 CML cells as evidenced by the nitroblue-tetrazolium (NBT) reduction test. At higher dose, flow cytometry analysis showed that P18.9 induced sub-G1 apoptotic fraction of K562 cells and enhanced the sub-G1 fraction induced by imatinib at a scale of about two folds. Although P18.9 did not induce differentiation of HL60 APL cells, it substantially enhanced the ATRA-induced differentiation. This is the first study to isolate an active compound from human urine, which exerts anti-leukemic effects alone or cooperatively with clinical used drugs. The chemical structure of P18.9 had been identified and further subsequent structure modification is ongoing. Continuing the effort to develop P18.9 and its derivatives as novel therapeutics for refractory APL or CML patients is warranted. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C189.

De novo synthesis, uptake and proteolytic processing of lipocalin‐type prostaglandin D synthase, β‐trace, in the kidneys

Lipocalin-type prostaglandin D synthase (L-PGDS) is a multifunctional protein that produces prostaglandin D(2) and binds and transports various lipophilic substances after secretion into various body fluids as beta-trace. L-PGDS has been proposed to be a useful diagnostic marker for renal injury associated with diabetes or hypertension, because the urinary and plasma concentrations are increased in patients with these diseases. However, it remains unclear whether urinary L-PGDS is synthesized de novo in the kidney or taken up from the blood circulation. In crude extracts of monkey kidney and human urine, we found L-PGDS with its original N-terminal sequence starting from Ala23 after the signal sequence, and also its N-terminal-truncated products starting from Gln31 and Phe34. In situ hybridization and immunohistochemical staining with monoclonal antibody 5C11, which recognized the amino-terminal Ala23-Val28 loop of L-PGDS, revealed that both the mRNA and the intact form of L-PGDS were localized in the cells of Henle's loop and the glomeruli of the kidney, indicating that L-PGDS is synthesized de novo in these tissues. However, truncated forms of L-PGDS were found in the lysosomes of tubular cells, as visualized by immunostaining with 10A5, another monoclonal antibody, which recognized the three-turn alpha-helix between Arg156 and Thr173. These results suggest that L-PGDS is taken up by tubular cells and actively degraded within their lysosomes to produce the N-terminal-truncated form.