Min Ding

Simultaneous determination of tryptophan and kynurenine in plasma samples of children patients with Kawasaki disease by high-performance liquid chromatography with programmed wavelength ultraviolet detection

A simple, fast, sensitive and specific high-performance liquid chromatography (HPLC) method is developed for simultaneous determination of kynurenine (Kyn) and tryptophan (Trp) with ultraviolet (UV) detection setting programmed wavelength. The separation was carried out on an Agilent Hypersil ODS column (125 mm x 4.0 mm, 5 microm) in less than 6 min and the eluate was monitored by the programmed wavelength detection setting at 360 nm from 0 min to 4 min for Kyn, and at 278 nm from 4 min to 6 min for Trp in a single run with UV detector. The linearities of the method were from 0.20 micromol/L to 21.2 micromol/L for Kyn and 2.25-678.0 micromol/L for Trp, and the detection limits were 0.028 micromol/L for Kyn and 0.053 micromol/L for Trp, respectively. Satisfactory precisions and recoveries were obtained by this method. The assay was employed to analyze plasma samples of children patients with Kawasaki disease (KD). The result showed great difference between Kawasaki disease and control group.

Identification of potential microRNA-target pairs associated with osteopetrosis by deep sequencing, iTRAQ proteomics and bioinformatics.

MicroRNAs aberrantly express in many human diseases including some metabolic bone disorders. They have been found to be associated with osteoclast differentiation and function, which makes them attractive candidates for the therapy of bone. However, the potential clinical application of microRNAs in therapeutics rests heavily upon our in-depth understanding of microRNAs and their targets. To identify potential microRNA–target pairs associated with osteopetrosis, we performed a system approach including deep sequencing, iTRAQ quantitative proteomics, and bioinformatics in the peripheral blood mononuclear cells (PBMCs) taken from patients with osteopetrosis and health donors. Notably, 123 differently expressed microRNAs, 173 differently expressed proteins, and 117 computationally predicted microRNA–target pairs with reciprocally expressed level in PBMCs were found in the two sample groups. Functional annotation identified that the microRNA–target pairs were involved in cell growth, differentiation, cellular signaling network, and the network highlighted the microRNA–target pair of has-miR-320a and ADP ribosylation factor 1 (Arf1) potentially associated with CLCN7 mutations in osteopetrosis. The pair of has-miR-320a and Arf1 was further verified by real-time PCR, western blot, and the interaction between has-miR-320a and its targeted sequence on the Arf1 mRNAs was confirmed by luciferase assay. Collectively, the present study established a new system approach for the investigation of microRNAs, and the microRNA–target pairs, particular has-miR-320a and Arf1, may have important roles in osteopetrosis.

Association between serum bile acid profiles and gestational diabetes mellitus: A targeted metabolomics study

BACKGROUNDnGiven the potential influence of aberrant bile acid metabolism on glucose homeostasis, we hypothesized that serum bile acid metabolism is altered in gestational diabetes mellitus (GDM). We characterized the metabolic profiling changes of serum bile acids in GDM and to find the potential biomarkers for the diagnosis and differential diagnosis of GDM.nnnMETHODSnBased on ultrahigh performance liquid chromatography/hybrid quadrupole time-of-flight mass spectrometry, a targeted metabolomics study that involved targeted and untargeted screening techniques was performed to explore the changes in serum bile acid metabolism of GDM cases, intrahepatic cholestasis of pregnancy (ICP) cases and healthy controls.nnnRESULTSnThere were 3 significantly different profiling of serum bile acids for GDM, ICP and controls. Compared to the controls, GDM individuals demonstrated significant increases in 8 bile acid species, including 2 dihydroxy conjugated, 1 trihydroxy unconjugated and 5 sulfated bile acids. β-muricholic acid (β-MCA) and di-2 were well-suited to use as the metabolic markers for the diagnosis and differential diagnosis of GDM, respectively.nnnCONCLUSIONSnThese preliminary findings revealed the protective effect of body against cytotoxicity via elimination of increased sulfated bile acids and aberrant enzyme activity participated in the cycle β-MCA→hyodeoxycholic acid (HDCA) of the bile acid metabolism pathway for the women with GDM, which gave us further insights into the etiology and pathophysiology of GDM.

An electrochemical sensor for indole in plasma based on MWCNTs-chitosan modified screen-printed carbon electrode

Indole is an essential metabolite in intestinal tract. The dysregulation of plasma indole concentration occurred in various diseases. In this study, the indole in plasma was determined directly using electrochemical sensor with multiwall carbon nanotubes-chitosan (MWCNTs-CS) modified screen-printed carbon electrode (SPCE). The electrochemical behavior of indole was elucidated by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) on the MWCNTs-CS composites modified SPCE (MWCNTs-CS/SPCE). The results showed that the current responses of indole improved greatly due to the high catalytic activity and electron transfer reaction of nano-composites. Under the optimized conditions, the linear range of indole was from 5 to 100μgL-1 with the detection limit of 0.5μgL-1 (S/N = 3). This novel electrochemical sensor exhibited acceptable accuracies and precisions with the variations less than 7.3% and 9.0%, respectively. Furthermore, high performance liquid chromatography (HPLC) method was utilized to compare with the established electrochemical method for the determination of indole in plasma. The results showed a high correlation between the two methods. At last, the electrochemical sensor was successfully applied to detect the level of indole in plasma samples with satisfactory selectivity and sensitivity. The concentrations of plasma indole in healthy pregnant women and gestational diabetes mellitus (GDM) patients were 5.3 (4.1-7.0)μgL-1 and 7.2 (4.5-9.4)μgL-1, respectively.

Feasibility of urinary microRNA profiling detection in intrahepatic cholestasis of pregnancy and its potential as a non-invasive biomarker.

Intrahepatic cholestasis of pregnancy (ICP), a pregnancy-related liver disease, leads to complications for both mother and fetus. Circulating microRNAs (miRNAs) have emerged as candidate biomarkers for many diseases. So far, the circulating miRNAs profiling of ICP has not been investigated. To assess the urinary miRNAs as non-invasive biomarkers for ICP, a differential miRNA profiling was initially analyzed by individual quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assay in urinary samples from a screening set including 10 ICP and 10 healthy pregnancies. The selected candidate miRNAs were then validated by a validation set with 40 ICP and 50 healthy pregnancies using individual qRT-PCR assay. Compared with the expression in urine of healthy pregnant women, the expression levels of hsa-miR-151-3p and hsa-miR-300 were significantly down-regulated, whereas hsa-miR-671-3p and hsa-miR-369-5p were significantly up-regulated in urine from ICP patients (pu2009<u20090.05 and false discovery rateu2009<u20090.05). A binary logistic regression model was constructed using the four miRNAs. The area under the receiver operating characteristic curve was 0.913 (95% confidence intervalu2009=u20090.847 to 0.980; sensitivityu2009=u200982.9%, specificityu2009=u200987.0%). Therefore, urinary microRNA profiling detection in ICP is feasible and maternal urinary miRNAs have the potential to be non-invasive biomarkers for the diagnosis of ICP.

A highly sensitive method for simultaneous determination of the quaternary ammonium pesticides chlormequat and mepiquat in pears and potatoes by modified QuEChERS-high performance liquid chromatography-tandem mass spectrometry

A highly sensitive high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for simultaneous determination of the quaternary ammonium pesticides chlormequat (CQ) and mepiquat (MQ) in pears and potatoes was developed and fully validated. The modified QuEChERS method was employed for sample preparation. A hydrophilic interaction liquid chromatography (HILIC) column was used for the chromatographic separation of highly polar analytes. The detection was performed by a triple quadrupole mass spectrometer with an electrospray ionization (ESI) source in positive ion mode by multiple reaction monitoring (MRM). The detailed fragmentation mechanisms of targeted analytes in MS/MS system were studied on the theoretical level using density functional theory (DFT) calculations. The limits of detection (LODs) were 0.021 μg kg−1 and 0.21 μg kg−1 for CQ and MQ, respectively. The mean recoveries of CQ and MQ were in the range of 83.4–119.4% with RSD less than 7.0%. The developed method was applied to the analysis of CQ and MQ in actual samples from different retail outlets in China, implying its potential in fast monitoring of CQ and MQ residues.

Capillary electrophoresis with electrochemiluminescence detection for the simultaneous determination of cisatracurium besylate and its degradation products in pharmaceutical preparations

Capillary electrophoresis with electrochemiluminescence detection for the simultaneous analysis of cisatracurium besylate and its degradation products (laudanosine, quaternary monoacrylate) in pharmaceutical preparation was developed and fully validated. The significant parameters that influence capillary electrophoresis separation and electrochemiluminescence detection were optimized. The total analysis time of the analytes was 15 min. The linearities of the method were 0.1∼40.0 μg/mL for cisatracurium besylate and 0.04∼8.00 μg/mL for laudanosine, with correlation coefficients (r) of 0.999 and 0.998, respectively. The detection limits (S/N = 3) were 83.0 ng/mL for cisatracurium besylate and 32.0 ng/mL for laudanosine. The intraday relative standard deviations of the analytes were <3.0%, and the interday relative standard deviations were <8.0%. The developed method was cost-effective, sensitive, fast, and resource-saving, which was suitable for the ingredient analysis in pharmaceutical preparation.

High-performance liquid chromatography assay with programmed flow elution for cisatracurium in human plasma: application to pharmacokinetics in infants and children.

A high-performance liquid chromatography (HPLC) assay with fluorescence detection (FLD) for quantification of cisatracurium in human plasma was developed and fully validated. Liquid-liquid extraction was employed for sample preparation. The separation was carried out on a C18 column with ternary mobile phase composed of 30mmolL(-1) phosphate buffer (pH 3.0), acetonitrile and methanol (60:35:5, v/v/v). Verapamil was used as the internal standard. The isocratic elution with programmed flow rate was employed by setting at 0.8mLmin(-1) from 0 to 3.5min, 0.5mLmin(-1) from 3.5 to 6min, and 1.0mLmin(-1) from 6 to 10min. The fluorescence detection was performed at 236nm for excitation and 324nm for emission. The assay was linear from 50 to 2800ngmL(-1), with a detection limit of 12ngmL(-1). The correlation coefficient (r) for linear regression was 0.9997. The intra-day coefficients of variation (CVs) were less than 2.0%, and the inter-day CVs were less than 4.0%. The mean recoveries were in the range of 92.1-100.4%. The total HPLC run time was less than 10min. The developed HPLC method was fast, simple, sensitive, accurate and suitable for studying the pharmacokinetics of cisatracurium in infants and children after intravenous administration.

Indirect electrochemical detection for total bile acids in human serum.

Bile acids level in serum is a useful index for screening and diagnosis of hepatobiliary diseases. As bile acids concentration is closely related to the degree of hepatobiliary diseases, detecting it is a vital factor to understand the stage of the diseases. The prevalent determination for bile acids is the enzymatic cycling method which has low sensitivity while reagent-consuming. It is desirable to develop a new method with lower cost and higher sensitivity. An indirect electrochemical detection (IED) for bile acids in human serum was established using the screen printed carbon electrode (SPCE). Since bile acids do not show electrochemical signals, they were converted to 3-ketosteroids by 3-α-hydroxysteroid dehydrogenase (3α-HSD) in the presence of nicotinamide adenine dinucleotide (NAD(+)), which was reduced to NADH. NADH could then be oxidized on the surface of SPCE, generating a signal that was used to calculate the total bile acids (TBA) concentration. A good linear calibration for TBA was obtained at the concentration range from 5.00μM to 400μM in human serum. Both the precisions and recoveries were sufficient to be used in a clinical setting. The TBA concentrations in 35 human serum samples by our IED method didnt show significant difference with the result by enzymatic cycling method, using the paired t-test. Moreover, our IED method is reagent-saving, sensitive and cost-effective.

[Simultaneous determination of tryptophan and its metabolites in plasma by high performance liquid chromatography with on-column derivatization].

A high performance liquid chromatography-ultraviolet/fluorescence detection (HPLC-UV/FLD) with on-column derivatization was established to simultaneously determine tryptophan (Trp), kynurenine (Kyn), 5-hydroxyindole acetic acid (5-Hiaa) and kynurenic acid (Kyna). A Hypersil C-18 column (250 mm x 4.0 mm, 5 microm) was used for the analysis at 30 degrees C. The separation was carried out with the mobile phase consisting of 250 mmol/L zinc acetate (pH 5.5) and acetonitrile (95: 5, v/v) at a flow rate of 0.8 mL/min using 3-nitrotyrosine as internal standard (IS). The excitation (Ex) and emission (Em) wavelengths were set at 278 nm (lambda(ex))/343 nm (lambda(em)) for 5-Hiaa and 244 nm (lambda(ex))/400 nm (lambda(em)) for Kyna, while the wavelengths of ultraviolet detection were set at 360 nm for Kyn and IS, 302 nm for Trp. The recoveries were in the range of 91.62% to 114.17%. The linearities were from 2.50 micromol/L to 320.00 micromol/L for Trp, 0.32 micromol/L to 15.36 micromol/L for Kyn, 3.27 nmol/L to 104.60 nmol/L for 5-Hiaa, and 14.00 nmol/L to 464.80 nmol/L for Kyna. The detection limits were 0.078 micromol/L, 0.056 micromol/L, 0.690 nmol/L and 1.290 nmol/L for Trp, Kyn, 5-Hiaa, and Kyna, respectively. Thirty plasma samples of normal pregnant women and 28 plasma samples of healthy controls were tested, and the results exhibited that the concentrations of Trp, Kyn and Kyna in the plasma of the normal pregnant women were significantly different from those of the control group (all P < 0.01). The method is simple and sensitive with good reproducibility, and it is suitable for clinical measurements.