Analysis of human urine metabolites using SPE and NMR spectroscopy

Nuclear magnetic resonance (NMR) spectroscopic analysis of metabonome/metabolome has widespread applications in biomedical science researches. However, most of NMR resonances for urinary metabolites remain to be fully assigned. In the present study, human urine samples from two healthy volunteers were pre-treated with C18 solid-phase extraction and the resultant 5 sub-fractions were subjected to one- and two-dimensional NMR studies, including 1H J-Resolved, 1H-1H COSY, 1H-1H TOCSY, 1H-13C HSQC, and HMBC 2D NMR. More than 70 low molecular weight metabolites were identified, and complete assignments of 1H and 13C resonances including many complex coupled spin systems were obtained.     

Urinary metabolomic study of systemic lupus erythematosus based on gas chromatography/mass spectrometry

Systemic lupus erythematosus (SLE) is an autoimmune disease with heterogeneous organ and system manifestations. In this study, urinary metabolic alterations related to SLE were investigated by performing gas chromatography/mass spectrometry (GC/MS) based metabolomics and multivariate statistical analysis. Patients with SLE and healthy controls could be clearly differentiated in view of the metabolic abnormity in urine. Among 70 identified endogenous metabolites, 23 metabolites were dramatically increased in SLE patients, which involved in several key metabolic pathways including energy metabolism, nucleotide metabolism, oxidative stress and gut‐microbiome‐derived metabolism. This noninvasive and GC/MS‐based metabolomic technique is a promising and potent strategy for identifying novel biomarkers and understanding pathogenesis of SLE. Copyright © 2016 John Wiley & Sons, Ltd.     

Real‐Time Monitoring of Cancer Cell Metabolism and Effects of an Anticancer Agent using 2D In‐Cell NMR Spectroscopy

Altered metabolism is a critical part of cancer cell properties, but real‐time monitoring of metabolomic profiles has been hampered by the lack of a facile method. Here, we propose real‐time metabolomic monitoring of live cancer cells using ¹³C₆‐glucose and heteronuclear two‐dimensional (2D) NMR. The method allowed for metabolomic differentiation between cancer and normal cells on the basis of time‐dependent changes in metabolite concentrations. Cancer cells were found to have large in‐ and out‐flux of pyruvate as well as increased net production of alanine and acetate. The method also enabled evaluation of the metabolic effects of galloflavin whose anticancer effects have been attributed to its specific inhibition of lactate dehydrogenase. Our approach revealed previously unknown functional targets of galloflavin, which were further confirmed at the protein levels. Our method is readily applicable to the study of metabolic alterations in other cellular disease model systems.     

Urine metabolomic characteristics of female patients with occupational chronic cadmium poisoning after 15 years of treatment

Occupational chronic cadmium poisoning (OCCP) can cause irreversible organ damage. Currently, no effective treatment is available for OCCP, and effective and sensitive biomarkers for treatment evaluation are still lacking. In this study, metabolomics techniques were used to analyze changes in endogenous metabolites in the urine of patients with OCCP after 15 years of treatment. Thirty urine samples from female patients with OCCP and healthy female controls (n = 15 per group) were assessed using gas chromatography–time-of-flight mass spectrometry and ultra-high-performance liquid chromatography–Q-Exactive mass spectrometry. The OCCP group had higher concentrations of blood urea nitrogen and urinary cadmium but near-normal urinary concentrations of β₂-microglobulin and retinol-binding protein. Compared with the control group, the OCCP group had 66 significantly different metabolites with a variable importance in projection score >1 and p < 0.05. These differential metabolites were involved in various metabolic pathways, such as creatine metabolism, nicotinate and nicotinamide metabolism, the pentose phosphate pathway, d -glutamine and d -glutamate metabolism, and amino acid metabolism. Compared with the control group, the OCCP group had significantly higher urinary concentrations of creatine, glutamic acid, quinolinic acid and nicotinic acid. In a receiver operator characteristic analysis, the area under the curve of creatine was higher than those for glutamic acid, quinolinic acid and nicotinic acid, indicating that urinary concentrations of creatine could be used as a sensitive biomarker for the diagnosis and prognosis of OCCP and for monitoring its treatment.     

Evaluation of analytical performance and reliability of direct nanoLC‐nanoESI‐high resolution mass spectrometry for profiling the (xeno)metabolome

Mass spectrometry (MS) profiling techniques are used for analysing metabolites and xenobiotics in biofluids; however, detection of low abundance compounds using conventional MS techniques is poor. To counter this, nanoflow ultra‐high‐pressure liquid chromatography‐nanoelectrospray ionization‐time‐of‐flight MS (nUHPLC‐nESI‐TOFMS), which has been used primarily for proteomics, offers an innovative prospect for profiling small molecules. Compared to conventional UHPLC‐ESI‐TOFMS, nUHPLC‐nESI‐TOFMS enhanced detection limits of a variety of (xeno)metabolites by between 2 and 2000‐fold. In addition, this study demonstrates for the first time excellent repeatability and reproducibility for analysis of urine and plasma samples using nUHPLC‐nESI‐TOFMS, supporting implementation of this platform as a novel approach for high‐throughput (xeno)metabolomics. Copyright © 2014 John Wiley & Sons, Ltd.     

NMR-based metabolomics to evaluate the milk composition from Friesian and autochthonous cows of Northern Italy at different lactation times

It is well established that different factors affect milk composition in cows and that milk composition, in turn, affect both technological and nutritional qualities. In this respect the comprehension of the metabolic variability of milk composition in relation to the lactation time as well as to the genetic background may be of paramount importance for the agri-food industries. In the present study we investigated the variations of the metabolic profiles during lactation in milks obtained from Friesian and autochthonous races from Northern Italy by ¹H NMR metabolomics. Furthermore, the external factors influencing the milk composition were minimized: the cows were breeded in the same farm, were fed with the same diet and were paired for the lactation interval and lactation stage. Our results showed a difference in milk composition between races and in relation to late lactation. The PLS-DA analysis permitted to distinguish the Friesian and autochthonous cow milks at the investigated different lactation times. Interestingly, the metabolites significantly involved into the discrimination between races appeared to be also technological property parameters, highlighting the importance of maintaining the biodiversity of cow breeds. Therefore, NMR-based metabolomics of milk could represent an informative tool to identify metabolites involved in milk quality both from a nutritional and industrial perspective.     

雷公藤甲素对大鼠尿样代谢物影响的核磁共振波谱研究

本文采用基于核磁共振的代谢组学方法研究了雷公藤甲素急性中毒大鼠尿样的代谢特征。结果表明给药后代表肝毒性生化指纹的creatine 和taurine相对含量升高,代表能量代谢的citrate, succinate, α-oxoglutarate相对含量下降,反应肠道菌群代谢状况的trimethylamine N-oxide,和hippurate等也异常,并且这些变化在的药物作用后16小时达到最大,56小时后大部分代谢物的浓度恢复正常。另外,在0~16小时实验组样品里观察到了被认为是一种新的急性肝中毒的生化标记物2’-deoxycytidine。以上NMR实验结果表明雷公藤甲素可能影响大鼠的肠道菌群和能量代谢,引发急性肝损伤和轻度肾衰竭。对注射组大鼠血浆的生化分析和肝组织的病理学切片也证明了实验大鼠肝功能的异常现象。以上结果说明NMR实验信息有助于进一步从分子水平上阐明雷公藤甲素的毒理学和药理学机制。     

Urinary metabolite profiling provides potential differentiation to explore the mechanisms of adjuvant‐induced arthritis in rats

To explore the pathogenesis of rheumatoid arthritis (RA) from the perspective of metabolomics, gas chromatography time‐of‐flight mass spectrometry (GC‐TOF/MS) technology was used to observe changes in the metabolic profiles of urine output from rats with adjuvant‐induced arthritis (AA). Sprague–Dawley rats were randomly divided into a control group and an experimental group, with eight in each. Rats in the experimental group were induced by intracutaneous innoculation of 0.1 mL Freund's complete adjuvant to right paws. On day 20 after immunization, the metabolic profiles between rat control and experimental groups were compared by combining GC‐TOF/MS technology with multivariate statistical approaches, including principal component analysis, partial least squares discriminant analysis and orthogonal projections to latent structures–discriminant analysis. Nine potential biomarkers were identified, including 2,2‐dimethylsuccinic acid, tartronic acid, dehydroshikimic acid, hippuric acid, adenine, phenaceturic acid, l ‐dopa, 1,4‐dihydroxy‐2‐naphthoic acid and melibiose. The findings indicate that the rats with AA are disturbed in metabolism of purine, amino acid, fat and energy. This study also demonstrates that the dysfunction in a range of biosynthetic and catabolic pathways, which leads to increased oxygen free radicals and inflammation, could cause underlying pathogenesis of RA. Copyright © 2016 John Wiley & Sons, Ltd.