Metabolic profiling of urine in young obese men using ultra performance liquid chromatography and Q-TOF mass spectrometry (UPLC/Q-TOF MS)

Obesity is currently epidemic in many countries worldwide. In the young adult, obesity often accompanies hyperlipemia, which is strongly related to the occurrence and development of obesity-related chronic diseases such as diabetes mellitus, hypertension and cardiovascular disease. This study investigated the differences in metabolomic profiling between obese (with hyperlipemia, n=30) and normal-weight (n=30) young men. Anthropometric parameters and conventional metabolites were measured. There were no significant differences between obese and normal-weight young men in age, height and fasting plasma glucose level. Obese young men showed increased weight, body mass index, fat mass, systolic blood pressure, and triglyeride, total cholesterol and insulin levels, and lower levels of testosterone. The endogenous metabolite profile of urine was investigated by UPLC/Q-TOF MS (ultra performance liquid chromatography and Q-TOF mass spectrometry) with electrospray ionization (ESI). Partial least squares (PLS) enabled clusters to be visualized. Eight urine principal metabolites contributing to the clusters were identified; these included increased L-prolyl-L-proline, leucyl-phenylalanine, and decanoylcarnitine in positive ESI mode (m/z 213.1267, 279.1715 and 316.2459, respectively) and N-acetylornithine, 17-hydroxypregnenolone sulfate, 11β-hydroxyprogesterone, 5a-dihydrotestosterone sulfate and glucosylgalactosyl hydroxylysine in negative ESI mode (m/z 173.0931, 411.1883, 331.185, 369.1751 and 485.1875, respectively). These metabolite changes in obese men suggested early changes of metabolism in young-male obesity with hyperlipemia. The study may further aid the clinical prevention and treatment of obesity and related chronic disease.     

Combining ion pairing agents for enhanced analysis of oligonucleotide therapeutics by reversed phase-ion pairing ultra performance liquid chromatography (UPLC)

The burgeoning field of oligonucleotide therapeutics is based upon synthetically derived biopolymers comprised of relatively simple RNA and DNA building blocks. Significant gains in knowledge around mechanisms of action (RNA interference, RNA splicing, etc.) and oligonucleotide design (ASO, siRNA, DsiRNA, miRNA, locked nucleic acid, etc.) have been the main drivers of recent investment for this field [1,2]. As therapeutics, there is currently great interest in oligonucleotides due to the reduced time required to achieve lead molecules and to their potential for treating previously untractable diseases. One of the more challenging areas for the field of oligonucleotide therapeutics is the development of high-quality analysis schemes for the determination of purity in drug substance and product. This, in part, is due to the fact that the synthesis of oligonucleotides results in a significant number of closely related impurities that are not easily removed during purification [1]. As a result, these macromolecules (4000-8000 MW on average, depending on chain length) and their soup of closely related impurities are typically not well resolved from one another via conventional chromatographic approaches. One of the more common chromatographic techniques used for oligonucleotide analysis is reversed phase-ion pairing liquid chromatography (RP-IP). Our research led us to the discovery that the use of multiple ion pairing agents combined in the mobile phase can improve the overall chromatographic resolution and peak shape of the oligonucleotide analytes over the use of a single ion pairing agent alone, resulting in enhanced purity analysis and the opportunity to identify related impurities with greater certainty. In addition, the use of combined ion pairing agents allowed for the development of a "universal" method which has provided superior chromatography for several different oligonucleotide compounds and their related impurities regardless of differences in nucleotide sequence. The RP-IP UPLC method conditions are ESI-MS compatible and have allowed for the mass identification of five positional isomeric impurities chromatographically resolved and present at less than 1% of the nominal parent peak area.     

Quantitative analysis of anandamide and related acylethanolamides in human seminal plasma by ultra performance liquid chromatography tandem mass spectrometry

The endocannabinoids anandamide, palmitoylethanolamide and oleoylethanolamide have been detected in human seminal plasma and are bioactive lipids implicated in regulation of sperm motility, capacitation and acrosome reaction. Several methods exist for endocannabinoid quantification but none have been validated for measurement in human seminal plasma. We describe sensitive, robust, reproducible solid phase and isotope-dilution UHPLC-ESI-MS/MS methods for the extraction and quantification of anandamide, palmitoylethanolamide and oleoylethanolamide in human seminal plasma. Precision and accuracy were evaluated using pooled seminal plasma over a 4 day period. For all analytes, the inter- and intraday precision (CV%) was between 6.6-17.7% and 6.3-12.5%, respectively. Analyses were linear over the range 0.237-19nM for anandamide and oleoylethanolamide and 0.9-76nM for PEA. Limits of detection (signal-to-noise >3) were 50, 100 and 100fmol/mL and limits of quantification (signal-to-noise >10) were 100, 200 and 200fmol/mL, respectively for anandamide, palmitoylethanolamide and oleoylethanolamide. Anandamide and oleoylethanolamide were stable at -80°C for up to 4 weeks, but palmitoylethanolamide declined significantly. We assessed seminal plasma from 40 human donors with normozoospermia and found mean (inter-quartile range) concentrations of 0.21nM (0.09-0.27), 1.785nM (0.48-2.32) and 15.54nM (7.05-16.31) for anandamide, oleoylethanolamide and palmitoylethanolamide, respectively. Consequently, this UHPLC-ESI-MS/MS method represents a rapid, reliable and reproducible technique for the analysis of these endocannabinoids in fresh seminal plasma.     

Quantitation of ursolic acid in human plasma by ultra performance liquid chromatography tandem mass spectrometry and its pharmacokinetic study

Ursolic acid is a hydroxy pentacyclic triterpene, which proved to have sedation, anti-inflammatory, antibacterial, antiulcer and anti-cancer activities. An ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) method with high selectivity, sensitivity and throughput has been established and validated for quantitation of total ursolic acid in human plasma. Plasma samples were pretreated by liquid-liquid extraction with ethyl acetate and were chromatographed by an ACQUITY UPLC BEH C(8) column (100 mm×2.1 mm, I.D., 1.7 μm) using mobile phase consisting of acetonitrile and 10 mM ammonium formate (90:10, v/v) at 0.2 mL/min. The duration of chromatography analysis was 3 min. The multiple reaction monitoring (MRM) was performed at m/z 455.1→455.0 for ursolic acid and m/z 469.3→425.2 for glycyrrhetinic acid (internal standard, IS) in the negative ion mode with electrospray ionization (ESI) source. The assay showed good linearity over the range of 10-5000 ng/mL for ursolic acid in human plasma with a lower limit of quantitation of 10 ng/mL. The mean extraction recovery was 73.2±4.5% and the matrix ion suppression ranged from -11.4% to -5.6%. The intra- and inter-day precisions were less than 7.0% and 7.2%, respectively, and the accuracy was within ±2.0%. Ursolic acid was stable during the analysis and the storage period. The validated method has been successfully applied to a pharmacokinetic study after intravenous infusion of Ursolic Acid Nano-liposomes to healthy volunteers.     

High resolution "ultra performance" liquid chromatography coupled to oa-TOF mass spectrometry as a tool for differential metabolic pathway profiling in functional genomic studies

The combination of a new 1.7 mum reversed-phase packing material, and a chromatographic system, operating at ca. 12,000 psi, (so-called ultra performance liquid chromatography, UPLC) has enabled dramatic increases in chromatographic performance to be obtained for complex mixture separation. This increase in performance is manifested in improved peak resolution, together with increased speed and sensitivity. Here, we show that UPLC offers significant advantages over conventional reversed-phase HPLC amounting to a more than doubling of peak capacity, an almost 10-fold increase in speed and a 3- to 5-fold increase in sensitivity compared to that generated with a conventional 3.5 microm stationary phase. The first functional genomic application of UPLC-MS technology is illustrated here with respect to multivariate metabolic profiling of urines from males and females of two groups of phenotypically normal mouse strains (C57BL19J and Alpk:ApfCD) and a "nude mouse" strain. We have also compared this technology to conventional HPLC-MS under similar analytical conditions and show improved phenotypic classification capability of UPLC-MS analysis together with increased ability to probe differential pathway activities between strains as a result of improved analytical sensitivity and resolution.     

Fluorometric analysis of pectenotoxin-2 in microalgal samples by high performance liquid chromatography

A rapid HPLC method with fluorescence detection of pectenotoxin-2 (PTX2), a polyether macrolide toxin, in microalgae is presented. A dienophile reagent, DMEQ-TAD, was used for precolumn fluorescence labeling. PTX2 could be quantitatively detected in the range 1-200 ng. This method confirmed the occurrence of PTX2 in net haul samples mostly composed of dinoflagellates Dinophysisspp. collected in the Adriatic Sea, Italy and Mutsu Bay, Japan.     

Rapid separation of seed gliadins by reversed-phase ultra performance liquid chromatography (RP-UPLC) and its application in wheat cultivar and germplasm identification

To separate gliadin from wheat flour, a novel and stability-indicating reversed-phase ultra performance liquid chromatography (RP-UPLC) method is established and optimized. A comparative analysis of routine capillary electrophoresis (CE), reversed-phase high-performance liquid chromatography (RP-HPLC), and RP-UPLC was performed and the results showed that the resolution and efficiency of RP-UPLC were significantly higher than those of CE and RP-HPLC. Characteristic RP-UPLC patterns of different bread wheat variety and related species were readily identified. These results demonstrated that our RP-UPLC procedure resulted in significant improvements in sensitivity, speed, and resolution, and thus is highly useful in wheat cultivar and germplasm identification.     

Application of faecal metabonomics on an experimental model of tubulointerstitial fibrosis by ultra performance liquid chromatography/high-sensitivity mass spectrometry with MSE data collection technique

Chronic renal failure (CRF) is a major challenge for the public healthcare problem. A novel UPLC Q-TOF/MS method with MS^E data collection mode was developed as a very effective biochemical analytical tool for precise identification of important biomarkers in the adenine-induced CRF rats. Nine endogenous metabolites were identified by using metabonomic method combined with multivariate data analysis, the accurate mass, isotopic pattern, MS^E fragments information and MassLynx i-FIT algorithm. The identified metabolites indicated the perturbations of bile acid and phospholipid metabolism are related to CRF rats. This work shows that metabonomics method is a valuable tool in CRF mechanism study.     

Serum metabonomics study of adenine-induced chronic renal failure in rats by ultra performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry

An ultra performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC Q-TOF MS) metabonomics approach was employed to study the serum metabolic profiling of adenine-induced chronic renal failure (CRF) rats. Acquired data were subjected to principal component analysis (PCA) for differentiating the CRF and the normal control groups. Potential biomarkers were screened by using S-plot and were identified by the accurate mass, isotopic pattern and MS/MS fragments information obtained from UPLC Q-TOF MS analysis. Significant differences in the serum level of creatinine, amino acids and LysoPCs were observed, indicating the perturbations of amino acid metabolism and phospholipid metabolism in adenine-induced CRF rats. This research proved that metabonomics is a promising tool for disease research.     

A comparative multidimensional LC-MS proteomic analysis reveals mechanisms for furan aldehyde detoxification in <Emphasis Type="Italic">Thermoanaerobacter pseudethanolicus</Emphasis> 39E

Background

Chemical and physical pretreatment of lignocellulosic biomass improves substrate reactivity for increased microbial biofuel production, but also restricts growth via the release of furan aldehydes, such as furfural and 5-hydroxymethylfurfural (5-HMF). The physiological effects of these inhibitors on thermophilic, fermentative bacteria are important to understand; especially as cellulolytic strains are being developed for consolidated bioprocessing (CBP) of lignocellulosic feedstocks. Identifying mechanisms for detoxification of aldehydes in naturally resistant strains, such as Thermoanaerobacter spp., may also enable improvements in candidate CBP microorganisms.

Results

Thermoanaerobacter pseudethanolicus 39E, an anaerobic, saccharolytic thermophile, was found to grow readily in the presence of 30 mM furfural and 20 mM 5-HMF and reduce these aldehydes to their respective alcohols in situ. The proteomes of T. pseudethanolicus 39E grown in the presence or absence of 15 mM furfural were compared to identify upregulated enzymes potentially responsible for the observed reduction. A total of 225 proteins were differentially regulated in response to the 15 mM furfural treatment with 152 upregulated versus 73 downregulated. Only 87 proteins exhibited a twofold or greater change in abundance in either direction. Of these, 54 were upregulated in the presence of furfural and 33 were downregulated. Two oxidoreductases were upregulated at least twofold by furfural and were targeted for further investigation. Teth39_1597 encodes a predicted butanol dehydrogenase (BdhA) and Teth39_1598, a predicted aldo/keto reductase (AKR). Both genes were cloned from T. pseudethanolicus 39E, with the respective enzymes overexpressed in E. coli and specific activities determined against a variety of aldehydes. Overexpressed BdhA showed significant activity with all aldehydes tested, including furfural and 5-HMF, using NADPH as the cofactor. Cell extracts with AKR also showed activity with NADPH, but only with four-carbon butyraldehyde and isobutyraldehyde.

Conclusions

T. pseudethanolicus 39E displays intrinsic tolerance to the common pretreatment inhibitors furfural and 5-HMF. Multidimensional proteomic analysis was used as an effective tool to identify putative mechanisms for detoxification of furfural and 5-HMF. T. pseudethanolicus was found to upregulate an NADPH-dependent alcohol dehydrogenase 6.8-fold in response to furfural. In vitro enzyme assays confirmed the reduction of furfural and 5-HMF to their respective alcohols.

     

Simultaneous analysis of multiple aminothiols in human plasma by high performance liquid chromatography with fluorescence detection

Aminothiols serve numerous vital functions in biochemistry, including detoxification and regulation of cellular metabolism, enzymatic activity, and protein trafficking and degradation. Plasma aminothiol concentrations are frequently measured for clinical and translational research investigating oxidative stress, and for routine clinical diagnosis and monitoring of vascular injury. Although a variety of techniques are available to measure aminothiol concentrations in plasma, high performance liquid chromatography with fluorescence detection (HPLC–FD) is the most widely used. This review summarizes HPLC–FD methods, including pre-analytical considerations, procedures for sample reduction, derivatization, and chromatographic separation of the primary biological aminothiols cysteine, homocysteine, cysteinylglycine, and glutathione in human plasma.     

Application of high performance liquid chromatography and gas chromatography-mass spectrometry to analysis of prostaglandin E1 in biological media.

A method for the quantitation of prostaglandin (PG) E₁ in biological samples of gas chromatography—mass spectrometry has been developed. PGE₁ was separated from PGE₂, 13,14-dihydro-PGE₂, and other potentially interfering prostaglandins by reversed phase high performance liquid chromatography. After conversion of PGE₁ to PGB₁ by treatment with methanolic KOH, PGB₁ was derivatized to the methyl ester trimethylsilyl ether and analyzed by selected ion monitoring using hexadeutero-PGE₁ as an internal standard. Measurable levels of PGE₁ were found in human and rat urine and in incubates of rat and rabbit renal papilla. PGE₁ excretion and production by renal slices was blocked by treatment with indomethacin. A complete mass spectrum of derivatized PGE₁ was obtained from PGE₁ generated by rabbit renal papillary slices.     

Measurement of cyst(e)amine in physiological samples by high performance liquid chromatography

Two methods for measurement of cyst(e)amine in physiological samples are described. One method involves reduction of disulfides present in the sample with tributylphosphine, reversed phase chromatography of thiols, and electrochemical detection of cysteamine and other thiols. The other method involves reduction of disulfides with dithiothreitol, derivatization of thiols with 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin, separation of these derivatives by reversed phase chromatography, and fluorometric detection of the thiol adducts. The endogenous concentration of cysteamine in rat liver was estimated to be less than 2.5 nmol/g. Cysteamine is produced in tissues postmortem; rapid sampling/freezing of tissues and rapid inactivation of enzymes during tissue preparation are essential for accurate measurement of endogenous cysteamine concentrations.     

Honeydew sugars in wild-caught Phlebotomus ariasi detected by high performance liquid chromatography (HPLC) and gas chromatography (GC)

Phlebotomus ariasi Tonnoir sandflies were caught in light traps hung in oak trees and in a house in the Cévennes focus of leishmaniasis in the South of France. The flies were cryopreserved either immediately on removal from the traps, or after starvation for 6-7 days, or after 6-7 days starvation followed by exposure to oak infested with the aphid genera Lachnus or Thelaxes. After transportation to the laboratory, the sandflies were thawed and aqueous extracts of the crushed flies were analysed for their carbohydrate content using high performance liquid chromatography (HPLC) and gas chromatography (GC). Starved female sandflies lacked significant amounts of any saccharides. Four types of sugar, melezitose and its hydrolysis products turanose, glucose and fructose, were observed in flies which had been starved previously and then placed with Lachnus infested oak. The results also indicate the presence of hydrolysis products of melezitose: (a) in flies previously starved and placed with Thelaxes infested oak, (b) in P.ariasi cryopreserved direct from the light traps hung in oak trees infested with Lachnus and Thelaxes, and (c) flies caught in a house. Unidentifiably small quantities of a trisaccharide were also detected in the latter groups of flies. In previous tests, sugars were detected in P.ariasi after their exposure to aphid-infested oak (Quercus ilex L.), but not when P.ariasi females were exposed to washed oak leaves without aphids. The results indicate that P.ariasi feed on melezitose and/or turanose, the main local source of which is aphid honeydew. A better understanding of sugar meal sources of sandflies using HPLC and GC techniques will assist in our understanding of sandfly/Leishmania relationships, parasite transmission and epidemiology.     

Evaluation of a protocol for metabolic profiling studies on human blood plasma by combined ultra-performance liquid chromatography/mass spectrometry: From extraction to data analysis

The investigation presented here describes a protocol designed to perform high-throughput metabolic profiling analysis on human blood plasma by ultra-performance liquid chromatography/mass spectrometry (UPLC/MS). To address whether a previous extraction protocol for gas chromatography (GC)/MS-based metabolic profiling of plasma could be used for UPLC/MS-based analysis, the original protocol was compared with similar methods for extraction of low-molecular-weight compounds from plasma via protein precipitation. Differences between extraction methods could be observed, but the previously published extraction method was considered the best. UPLC columns with three different stationary phases (C8, C18, and phenyl) were used in identical experimental runs consisting of a total of 60 injections of extracted male and female plasma samples. The C8 column was determined to be the best for metabolic profiling analysis on plasma. The acquired UPLC/MS data of extracted male and female plasma samples was subjected to principal component analysis (PCA) and orthogonal projections to latent structures discriminant analysis (OPLS-DA). Furthermore, a strategy for compound identification was applied here, demonstrating the strength of high-mass-accuracy time-of-flight (TOF)/MS analysis in metabolic profiling.     

Identification of biomarkers for melamine-induced nephrolithiasis in young children based on ultra high performance liquid chromatography coupled to time-of-flight mass spectrometry (U-HPLC-Q-TOF/MS)

Milk products contaminated with melamine caused renal disease in young children in mainland China in 2008. The present study was designed to identify potential markers and assess the underlying metabolomic mechanisms of melamine-induced nephrolithiasis in young children. Urine samples were collected from healthy children (n=74) and from children diagnosed with nephrolithiasis (n=73) with either a positive (n=40) or a negative (n=33) history of melamine exposure. Ultra high-performance liquid chromatography coupled to time of flight mass spectrometry (U-HPLC-MS/MS) was applied to profile the abundances of metabolites. Partial least squares-discriminant analysis (PLS-DA) was used to discriminate between the samples. Seven compounds were found to highly discriminate between healthy controls and nephrolithiasis patients with a history of melamine exposure. The critical markers such as proline and 5C-aglycone were the predominant markers in the control group and detected only rarely in nephrolithiasis patients with a history of melamine exposure. In contrast, hypoxanthine at was the most significant compound that distinguished nephrolithiasis patients with a history of melamine exposure. It was increased to 116.12±23.34 μg/L (mean±S.D.) in the melamine-induced nephrolithiasis group, whereas the non-melamine group was at the level of 67.47±9.33 μg/L (p<0.001). The biomarkers for melamine-induced nephrolithiasis identified by this study may have clinical application in determining the aetiology of renal disease in young children.     

Characterization by high performance liquid chromatography (HPLC) of the solubilization of phosphorus in iron ore by a fungus

Summary The value of iron ore is adversely affected by phosphorus in concentrations over 0.03% by weight. The present research concerns the use of metabolic products of aPenicillium-like fungus to leach insoluble phosphates (hydroxyapatite) from ores. Ion chromatography was used to measure metabolism of glucose into acidic fragments. The rate and products of glucose degradation depended on both the chemical composition of the growth medium (buffered or not) and incubation conditions (shaken or quiescent). The principal products were identified as oxalic acid and isomers of propylene dicarboxylic acid, mainly itaconic acid. Continued, slow metabolism of itaconic acid generates more oxalic acid. Aliphatic acids were not detected. Both iron ore phosphate and calcium phosphate were partially solubilized by either the spent broth or aqueous oxalic acid. Solubilization of ore phosphorus was greatly assisted by hydrochloric acid added to the spent broth in small increments. The data suggest biological alternatives to costly leaching procedures that use only mineral acids.     

High performance liquid chromatography analysis of 2-mercaptoethylamine (cysteamine) in biological samples by derivatization with N-(1-pyrenyl) maleimide (NPM) using fluorescence detection

2-Mercaptoethylamine (cysteamine) is an aminothiol compound used as a drug for the treatment of cystinosis, an autosomal recessive lysosomal storage disorder. Because of cysteamine's important role in clinical settings, its analysis by sensitive techniques has become pivotal. Unfortunately, the available methods are either complex or labor intensive. Therefore, we have developed a new rapid, sensitive, and simple method for determining cysteamine in biological samples (brain, kidney, liver, and plasma), using N-(1-pyrenyl) maleimide (NPM) as the derivatizing agent and reversed-phase high performance liquid chromatography (HPLC) with a fluorescence detection method (lambda(ex)=330 nm, lambda(em)=376 nm). The mobile phase was acetonitrile and water (70:30) with acetic acid and o-phosphoric acid (1 mL/L). The calibration curve for cysteamine in serine borate buffer (SBB) was found to be linear over a range of 0-1200 nM (r(2)=0.9993), and in plasma and liver matrix, the r(2) values were 0.9968 and 0.9965, respectively. The coefficients of the variation for the within-run and between-run precisions ranged from 0.68 to 9.90% and 0.63 to 4.17%, respectively. The percentage of relative recovery ranged from 94.1 to 98.6%.