NMR-based Metabonomics Research Articles

Hepatoprotective effects of Xiaoyao San formula on hepatic steatosis and inflammation via regulating the sex hormones metabolism.

The modified Xiaoyao San (MXS) formula is an adjuvant drug recommended by the National Health Commission of China for the treatment of liver cancer, which has the effect of preventing postoperative recurrence and metastasis of hepatocellular carcinoma and prolonging patient survival. However, the molecular mechanisms underlying that remain unclear. To investigate the role and mechanisms of MXS in ameliorating hepatic injury, steatosis and inflammation. A choline-deficient/high-fat diet-induced rat nonalcoholic steatohepatitis (NASH) model was used to examine the effects of MXS on lipid accumulation in primary hepatocytes. Liver tissues were collected for western blotting and immunohistochemistry (IHC) assays. Lipid accumulation and hepatic fibrosis were detected using oil red staining and Sirius red staining. The serum samples were collected for biochemical assays and NMR-based metabonomics analysis. The inflammation/lipid metabolism-related signaling and regulators in liver tissues were also detected to reveal the molecular mechanisms of MXS against NASH. MXS showed a significant decrease in lipid accumulation and inflammatory response in hepatocytes under metabolic stress. The western blotting and IHC results indicated that MXS activated AMPK pathway but inhibited the expression of key regulators related to lipid accumulation, inflammation and hepatic fibrosis in the pathogenesis of NASH. The metabonomics analysis systemically indicated that the arachidonic acid metabolism and steroid hormone synthesis are the two main target metabolic pathways for MXS to ameliorate liver inflammation and hepatic steatosis. Mechanistically, we found that MXS protected against NASH by attenuating the sex hormone-related metabolism, especially the metabolism of male hormones. MXS ameliorates inflammation and hepatic steatosis of NASH by inhibiting the metabolism of male hormones. Targeting male hormone related metabolic pathways may be the potential therapeutic approach for NASH.

Combination of peak-picking and binning for NMR-based untargeted metabonomics study

In NMR-based untargeted metabolomic studies, 1H NMR spectra are usually divided into equal bins/buckets to diminish the effects of peak shift caused by sample status or instrument instability, and to reduce the number of variables used as input for the multivariate statistical analysis. It was noticed that the peaks near bin boundaries may cause significant changes in integral values of adjacent bins, and the weaker peak may be obscured if it is allocated in the same bin with intense peaks. Several efforts have been taken to improve the performance of binning. Here we propose an alternative method, named P-Bin, based on the combination of the classic peak-picking and binning procedures. The location of each peak defined by peak-picking is used as the center of the individual bin. P-Bin is expected to keep all spectral information associated with the peaks and significantly reduce the data size as the spectral regions without peaks are not considered. In addition, both peak-picking and binning are routine procedures, making P-Bin easy to be implemented. To verify the performance, two sets of experimental data from human plasma and Ganoderma lucidum (G. lucidum) extracts were processed using the conventional binning method and the proposed method, before the principal component analysis (PCA) and the orthogonal projection to latent structures discriminant analysis (OPLS-DA). The results indicate that the proposed method has improved both the clustering performance of PCA score plots and the interpretability of OPLS-DA loading plots, and P-Bin could be an improved version of data preparation for metabonomic study.

Evaluation of aristolochic acid Ι nephrotoxicity in mice via 1H NMR quantitative metabolomics and network pharmacology approaches.

Although many studies have shown that herbs containing aristolochic acids can treat various human diseases, AAΙ in particular has been implicated as a nephrotoxic agent. Here, we detail the nephrotoxic effect of AAΙ via an approach that integrated 1H NMR-based metabonomics and network pharmacology. Our findings revealed renal injury in mice after the administration of AAΙ. Metabolomic data confirmed significant differences among the renal metabolic profiles of control and model groups, with significant reductions in 12 differential metabolites relevant to 23 metabolic pathways. Among them, there were seven important metabolic pathways: arginine and proline metabolism; glycine, serine, and threonine metabolism; taurine and hypotaurine metabolism; ascorbate and aldehyde glycolate metabolism; pentose and glucosinolate interconversion; alanine, aspartate, and glutamate metabolism; and glyoxylate and dicarboxylic acid metabolism. Relevant genes, namely, nitric oxide synthase 1 (NOS1), pyrroline-5-carboxylate reductase 1 (PYCR1), nitric oxide synthase 3 (NOS3) and glutamic oxaloacetic transaminase 2 (GOT2), were highlighted via network pharmacology and molecular docking techniques. Quantitative real-time PCR findings revealed that AAI administration significantly downregulated GOT2 and NOS3 and significantly upregulated NOS1 and PYCR1 expression and thus influenced the metabolism of arginine and proline. This work provides a meaningful insight for the mechanism of AAΙ renal injury.

Metabolic profiling of HIV infected individuals on an AZT-based antiretroviral treatment regimen reveals persistent oxidative stress

Human immunodeficiency viral (HIV) infection and long-term use of combination antiretroviral therapy (cART) have both been associated with the development of metabolic and immunological complications. Despite having many markers for HIV disease progression, the reliability of these markers remains debatable and most of these cannot be used as valid markers for treatment response. As such, it remains important to discover and develop biological markers, which will aid in monitoring disease progression, treatment response and the diagnoses of HIV-related metabolic disorders. Previous HIV-metabonomics studies unravelled the ability to detect and measure potential biological markers of HIV disease progression and treatment response. Several significantly differing metabolites were identified, however, only a small number of studies have investigated the link of specific metabolic disorders to an exact antiretroviral regimen. Here, an NMR-based untargeted metabonomic approach was used to profile metabolic changes in the sera of 24 HIV+ cART+ individuals receiving Zidovudine-based combination antiretroviral therapy compared to their 15 HIV+ ART- and 38 HIV- counterparts. Chemometric analysis identified significant differences in metabolic features related to glutamine, glutamate, glutathione, glucose and arginine. Pathway analysis also revealed the glutamine and glutamate metabolism pathway as the most significantly altered pathway between the HIV+ cART+ and HIV+ cART- group. Findings from this study further confirm the reliability of NMR-based metabonomics in HIV biomarker discovery. In addition, this study contributes to our understandings of the metabolic effect of antiretroviral therapy.

NMR-based metabonomics reveals the dynamic effect of electro-acupuncture on central nervous system in gastric mucosal lesions (GML) rats

BackgroundGastric mucosal lesions (GML) are common in gastric diseases and seriously affect the quality of life. There are inevitable side effects in drug therapy. Acupuncture is an important part of traditional Chinese medicine. Electro-acupuncture (EA) has unique curative effect in treatment of GML. However, there are still few reports on the central mechanism of electro-acupuncture in treatment of GML. In this study, NMR metabonomics was used to explore the central metabolic change mechanism of electro-acupuncture in treatment of GML.MethodsSD rats were randomly divided into Control, GML and EA groups. According to different intervention time, each group was further divided into 3 subgroups. This study mainly established GML model rats by 75% ethanol. Dynamic expressions of metabolites in cerebral cortex and medulla were observed by 1D 1H Nuclear Magnetic Resonance (NMR) metabolomics, combined with gastric mucosal histopathological examination to evaluate the time-effect relationship of electro-acupuncture at Zusanli (ST36) and Liangmen (ST21) points for 1 day, 4 days and 7 days treatment of GML.ResultsThe results showed that the repair effect of electro-acupuncture on gastric mucosal injury was the most obvious in 4 days and stable in 7 days, and 4 days electro-acupuncture can effectively inhibit GML gastric mucosal inflammation and the expression of inflammatory cells. Meanwhile, the NMR spectrum results of medulla and cerebral cortex showed that, 21 potential metabolites were identified to participate in the mechanism of pathogenesis of GML and the regulation of electro-acupuncture, including 15 in medulla and 10 in cerebral cortex. Metabolic pathway analysis showed that the differential metabolites involved 19 metabolic pathways, which could be divided into energy, neurotransmitters, cells and cell membrane and antioxidation according to their functions. The correlation analysis of stomach, medulla and cerebral cortex shows that the stimulation signal of GML may reach the cerebral cortex from the stomach through medulla, and electro-acupuncture can treat GML by regulating the central nervous system (CNS).Conclusions4 days electro-acupuncture treatment can significantly improve gastric mucosal injury, and the curative effect tends to be stable in 7 days treatment. Meanwhile, the pathogenesis of GML and the efficacy of electro-acupuncture involve metabolic pathways such as energy, neurotransmitters, cells and antioxidation, and electro-acupuncture can treat GML by regulating CNS.

An integrated metabonomics study to reveal the inhibitory effect and metabolism regulation of taurine on breast cancer

Breast cancer is a common metastatic malignant tumor in women. Taurine has been found to have anti-tumor effects on a variety of cancers. However, to the best of our knowledge, the role of taurine in the metastasis of breast cancer has not been reported. Thus, this study examined the effects of taurine on the growth and lung metastasis of breast cancer. Furthermore, the metabolism of serum, tumor tissue, and lung metastasis tissue were studied in a 4T1 subcutaneously transplanted breast cancer model through the integration of a 1H NMR-based metabonomics approach and histopathological assessments. The results showed that taurine significantly attenuated the tumor growth and lung metastasis, improved the pathological structure of tumor and lung tissue, and improved the metabolic disorders in 4T1 breast cancer mice. Additionally, taurine reversed the changes in serum lactate, creatine, and choline caused by the progression of breast cancer tumors. The levels of leucine/isoleucine, valine, alanine, arginine, methionine, glutamate, histidine, trimethylamine oxide (TMAO), taurine, and glucose in tumor tissues decreased, with an increment in lipids, lactate, and N-acetyl glycoprotein. Also, there was a reversal of leucine/isoleucine, valine, lactate, arginine, N-acetyl glycoprotein, glutamate, histidine, choline, and glycerophosphocholine/phosphocholine (GPC/PC) in the lung tissues. These metabolites changes were involved in the metabolic pathways of glycolysis, choline, amino acid, and lipid, suggesting that taurine exerted anti-breast cancer effects through the regulation of the underlying metabolism. This study provides a scientific basis for the adoption of taurine in the treatment of breast cancer metastasis.

Phase coherent excitation of SABRE permits simultaneous hyperpolarization of multiple targets at high magnetic field.

Hyperpolarization methods in magnetic resonance overcome sensitivity limitations, especially for low-γ nuclei such as 13C and 15N. Signal Amplification By Reversible Exchange (SABRE) and extended SABRE (X-SABRE) are efficient and low-cost methods for generating large polarizations on a variety of nuclei, but they most commonly use low magnetic fields (μT-mT). High field approaches, where hyperpolarization is generated directly in the spectrometer, are potentially much more convenient but have been limited to selectively hyperpolarize single targets. Here we introduce a new pulse sequence-based approach that affords broadband excitation of SABRE hyperpolarization at high magnetic fields without having to tailor pulse sequence parameters to specific targets. This permits simultaneous hyperpolarization of multiple targets for the first time at high field and offers a direct approach to integration of high-field SABRE hyperpolarization into routine NMR applications, such as NMR-based metabonomics and biomolecular NMR.

1H NMR-based metabonomics for infertility diagnosis in men with varicocele.

"Omics" techniques have been used to understand and to identify biomarkers of male infertility. We report on the first metabonomics models created to diagnose varicocele and infertility among men with varicocele. We recruited 35 infertile men with varicocele (VI group), 21 fertile men with varicocele (VF group) and 24 fertile men without varicocele (C group). All men underwent standard semen analysis, scrotal duplex ultrasonography, and sexual hormone level measurement. Hydrogen-1 nuclear magnetic resonance (1H NMR) spectra of seminal plasma were used to create metabonomics models to discriminate between men with and without varicocele, and between fertile and infertile men with varicocele. Using the statistical formalisms partial least square discriminants analysis and genetic algorithm-based linear discriminant analysis (GA-LDA), we created two models that discriminated the three groups from each other with accuracy of 92.17%. We also created metabonomics models using orthogonal partial least square discriminants analysis and GA-LDA that discriminated VF group from VI group, with an accuracy of 94.64% and 100% respectively. We identified 19 metabolites that were important in group segregation: caprate, 2-hydroxy-3-methylvalerate, leucine, valine, 3-hydroxybutyrate, lactate, alanine, 4-aminobutyrate, isoleucine, citrate, methanol, glucose, glycosides, glycerol-3-phosphocoline, n-acetyltyrosine, glutamine, tyrosine, arginine, and uridine. 1HNMR-based metabonomics of seminal plasma can be used to create metabonomics models to discriminate between men with varicocele from those without varicocele, and between fertile men with varicocele from those infertile with varicocele. Furthermore, the most important metabolites for group segregation are involved in the oxidative stress caused by varicocele.

Daily mycoprotein consumption for 1 week does not affect insulin sensitivity or glycaemic control but modulates the plasma lipidome in healthy adults: a randomised controlled trial.

Mycoprotein consumption has been shown to improve acute postprandial glycaemic control and decrease circulating cholesterol concentrations. We investigated the impact of incorporating mycoprotein into the diet on insulin sensitivity (IS), glycaemic control and plasma lipoprotein composition. Twenty healthy adults participated in a randomised, parallel-group trial in which they consumed a 7 d fully controlled diet where lunch and dinner contained either meat/fish (control group, CON) or mycoprotein (MYC) as the primary source of dietary protein. Oral glucose tolerance tests were performed pre- and post-intervention, and 24 h continuous blood glucose monitoring was applied throughout. Fasting plasma samples were obtained pre- and post-intervention and were analysed using quantitative, targeted NMR-based metabonomics. There were no changes within or between groups in blood glucose or serum insulin responses, nor in IS or 24 h glycaemic profiles. No differences between groups were found for 171 of the 224 metabonomic targets. Forty-five lipid concentrations of different lipoprotein fractions (VLDL, LDL, intermediate-density lipoprotein and HDL) remained unchanged in CON but showed a coordinated decrease (7-27 %; all P < 0·05) in MYC. Total plasma cholesterol, free cholesterol, LDL-cholesterol, HDL2-cholesterol, DHA and n-3 fatty acids decreased to a larger degree in MYC (14-19 %) compared with CON (3-11 %; P < 0·05). Substituting meat/fish for mycoprotein twice daily for 1 week did not modulate whole-body IS or glycaemic control but resulted in changes to plasma lipid composition, the latter primarily consisting of a coordinated reduction in circulating cholesterol-containing lipoproteins.

Quantitative Metabonomic Phenotypes in Different Structures of Mung Bean (Vigna radiata) Seeds and Their Germination-Associated Dynamic Changes.

Plant seed germination involving dynamic water uptakes and biochemical changes is essential for preservation of plant germplasm resource and worldwide food supply. To understand the germination-associated compartmental biochemistry changes, we quantitatively analyzed the metabolite composition (metabonome) for embryonic axes, cotyledons, and testae of mung bean (Vigna radiata) seeds in three germination phases using the NMR-based metabonomics approach. We found that three structures of mung bean seeds had distinct metabonomic phenotypes dominated by 53 metabolites including amino acids, carbohydrates, organic acids, choline metabolites, nucleotides/nucleosides, and shikimate-mediated secondary metabolites together with calcium and magnesium cations. During germination, all three seed structures had outstanding but distinct metabonomic changes. Both embryonic axis and cotyledon showed remarkable metabolic changes related to degradation of carbohydrates and proteins, metabolism of amino acids, nucleotides/nucleosides, and choline together with energy metabolism and shikimate-mediated plant secondary metabolism. The metabonomic changes in these two structures were mostly related to multiple functions for biochemical activities in the former and nutrient mobilizations in the latter. In contrast, testa metabonomic changes mainly reflected the metabolite leakages from the other two structures. Phase 1 of germination was featured with degradation of oligosaccharides and proteins and recycling of stored nucleic acids together with anaerobic metabolisms, whereas phase 2 was dominated by energy metabolism, biosynthesis of osmolytes, and plant secondary metabolites. These provided essential metabolic information for understanding the biochemistry associated with early events of seed germination and possible metabolic functions of different seed structures for plant development.

1H NMR-based metabolomics analyses in children with Helicobacter pylori infection and the alteration of serum metabolites after treatment

Background and aimsHelicobacter pylori (H. pylori) infection can occur in early childhood, without eradication therapies such infection can persist throughout life and cause many different diseases. This study investigated the metabolic characteristics and explored the underlying mechanism of children with H. pylori infection, and identified potential biomarkers for evaluating the efficacy of H. pylori eradication therapies. MethodsWe performed 1H NMR-based metabonomics coupled with multivariate analysis to investigate the metabolic profiling of serum samples between Children with and without H. pylori infection. In the same manner, we compared the alternations of metabolites in H. pylori-infected children before and after H. pylori eradication therapies. Results21 metabolites from serum in H. pylori-infected and H. pylori-uninfected children were identified, which were mainly involved in energy, amino acid, lipid and microbial metabolism. We found that the serum levels of trimethylamine N-oxide and alanine were significantly higher in H. pylori-infected children compared to uninfected sera, whereas lactate was significantly lower. We also found that the levels of trimethylamine N-oxide and creatine in H. pylori-infected children was significantly decreased after H. pylori eradication therapies, whereas lactate and low-density lipoprotein/very low-density lipoprotein was significantly increased. ConclusionsThis is the first study using 1H NMR-based metabolomics approach to explore the effects of H. pylori infection in children. Our results demonstrated that the disturbances of metabolism in energy, amino acids, lipids and microbiota could play an important role in the pathogenesis of gastrointestinal and extragastric diseases caused by H. pylori infection. Trimethylamine N-oxide and lactate might serve as potential serum biomarkers for evaluating the efficacy of H. pylori eradication therapies.

Quantitative Metabonomic Analysis Reveals the Germination-Associated Dynamic and Systemic Biochemical Changes for Mung-Bean (Vigna radiata) Seeds.

Seed germination is essential for plant survival, germplasm resource preservation, and worldwide food supplies, although the germination-associated seed biochemical variations are not fully understood. With the NMR-based metabonomics, we quantitatively analyzed the comprehensive metabolite composition (metabonome) of mung-bean (Vigna radiata) seeds at eight time points of germination covering all three phases. We found that mung-bean seed metabonomes were dominated by 63 metabolites including lipids, amino acids, oligo-/monosaccharides, cyclitols, cholines, organic acids, nucleotides/-sides, nicotinates, and the shikimate pathway-mediated secondary metabolites. During germination, metabolic changes included mainly the degradation of proteins and raffinose family oligosaccharides, glycolysis, tricarboxylic acid (TCA) cycle, anaerobic respiration, biosynthesis of osmolytes and antioxidants together with the metabolisms of nucleotides/-sides, nicotinates, and amino acids. Oligosaccharide degradation was the primary energy source for germination, which coupled with the mobilization of starch and protein storages to produce sugars and amino acids for biomaterial and energy generations. Osmotic and redox regulations were prerequisites for seed germination together with mitochondrial reparations and generations to enable TCA cycle. During the postgermination growth stage (phase-3), the use of small molecules including amino acids and saccharides was switched to meet the growth demands of radicle cells. Small metabolites passed freely through seed testa leaking into the culture media during early germination but were reabsorbed by seed cells around the postgermination growth stage. Extra after-ripening accelerated these metabolic processes of seeds in phase-1, especially the biosynthesis of cyclitols, choline, and nicotinates, increasing the germination uniformity in terms of speed and percentage. Germination-resistant seeds were incapable of activating the germination-associated metabolic processes.

Dynamic Analysis of the Biochemical Changes in Rats with Polycystic Ovary Syndrome (PCOS) Using Urinary 1H NMR-Based Metabonomics.

Polycystic ovarian syndrome (PCOS) is the most common endocrine disease that causes reproductive abnormalities in fertile women. It is closely related to the persistent anovulatory, insulin resistance, and high androgen. However, the molecular mechanisms underlying the pathological development of PCOS are still unclear. In this study, we aimed to explore the distinctive metabolic patterns in insulin combined with human chorionic gonadotrophin induced PCOS. The dynamic changes of endogenous metabolites in the development of PCOS were studied using untargeted metabolomic approaches based on nuclear magnetic resonance. The results showed that the degree of PCOS disorder metabolites at different periods was not exactly the same. Twelve significantly differential endogenous metabolites from different time points were selected as potential biomarkers relate to pathological process of PCOS. Among them, six metabolites showed a good diagnostic accuracy with PCOS model. The arginine and proline metabolic pathway was considered as one of the most crucial pathways that affects occurrence and development of PCOS. In addition, IRS-1, Akt, PI3K, IκB, and NF-κB (p65) were significantly changed in the ovary tissue of PCOS rats, which revealed that the IRS-1-PI3K/Akt and NF-κB signal pathway may be involved in the development of PCOS. This study demonstrated that metabolomic analysis is a powerful tool for providing novel insight into understanding the pathogenesis of PCOS and provide a basic reference for the diagnosis of PCOS at the onset stage.

Analysis of Metabolic Characteristics of SIRT7 Low Expression Glioma Cell Line Using NMR-Based Metabonomics

Analysis of Metabolic Characteristics of SIRT7 Low Expression Glioma Cell Line Using NMR-Based Metabonomics

Effects of Tianshu Capsule on Spontaneously Hypertensive Rats as Revealed by 1H-NMR-Based Metabolic Profiling.

Hypertension is one of the most common cardiovascular diseases, resulting in serious complications such as cardiovascular damage and chronic kidney disease. Tianshu capsule (TSC), composed of Chuanxiong (Ligusticum chuanxiong Hort) and Tianma (Gastrodiaelata Blume), has been widely used to treat the blood stasis type of headache and migraine in clinic. Results of previous research showed its antihypertensive effects, but the underlying mechanisms were still unclear. The purpose of this study was to evaluate the antihypertensive effect of TSC on spontaneously hypertensive rats by 1H NMR-based metabonomics and enzyme-linked immunosorbent assay (ELIAS), explore potential biomarkers and targets, and probe the potential mechanism of TSC on antihypertensive treatment. The results showed that TSC could decrease the product of oxidative stress (MDA) and enhance the activities of SOD and GSH-Px, down-regulate the expression of enzymes (LDHA, PKM2 and HK2) related to glycolysis, and perturb the levels of a series of amino acids (isoleucine, alanine, asparagine, citrate, etc.) and pathways. Multivariate statistical analyses showed remarkable changes in some endogenous metabolites after administrating TSC related to oxidative stress, amino acid metabolism and energy metabolism disturbances. Some enzymes (alanine-glyoxylate aminotransferase-2, tyrosine hydroxylase, dopa decarboxylase, etc.) related to metabolic biomarkers were predicted as the potential targets of TSC treatment on SHRs. The discoveries are helpful to understand the antihypertensive mechanism of TSC and provide theoretical evidence for its future research, development and clinical use.

Deciphering the Mechanical Network of Chronic Atrophic Gastritis: A Urinary Time-Dependent Metabonomics-Based Network Pharmacology Study.

Chronic atrophic gastritis (CAG) is one of the most important pre-cancerous states with a high prevalence. Deciphering its mechanical network is of significant importance for its diagnosis and treatment. The time-series factor associated with CAG progression specially needs to be considered together with its biological condition. In the present work, 1H NMR-based dynamic metabonomics was firstly performed to analyze the urinary metabolic features of CAG coupled with ANOVA-simultaneous component analysis (ASCA). As results, 4 (alanine, lipids, creatine, and dimethylglycine), 2 (α-ketoglutarate and alanine) and 5 (succinate, α-ketoglutarate, alanine, hippurate, and allantoin) urine metabolites were finally selected as the candidate biomarkers related to phenotype, time, and their interaction, respectively. Mechanistically, the network pharmacology analysis further revealed these metabolites were involved into mitochondrial function, oxidation reduction, cofactor binding, generation of precursor metabolites and energy, nucleotide binging, coenzyme metabolic process, cofactor metabolic process, cellular respiration, and tricarboxylic acid cycle. Especially, mitochondria were the most important targeted organelle referred 30 targeted proteins. The present work provided a novel network pharmacology approach for elucidating the mechanisms underlying the pathogenesis of CAG based on urinary time dependent metabonomics.

Multi-platform metabonomics unravel amino acids as markers of HIV/combination antiretroviral therapy-induced oxidative stress

Infection by the human immunodeficiency virus (HIV) elicits an immune response wherein neutrophils produce reactive oxygen species (ROS) to defend against pathogen invasion. Consequently, disproportionate levels of ROS in relation to antioxidants lead to oxidative stress (OS), which plays a key role in HIV disease progression and pathogenesis. There is a close relationship between oxidative stress status and HIV infection/progression, both separately and in the presence of combination antiretroviral therapy (cART). Biomarkers of oxidative stress present an additional means of monitoring HIV disease progression and/or management. Thus, the objective of this study was to apply untargeted nuclear magnetic resonance (NMR)-based metabonomics followed by targeted quantitative gas chromatography-mass spectrometry (GC/MS) analyses to identify predictors of oxidative stress in HIV infected individuals, with or without cART. Untargeted NMR-based metabonomics allowed a global profiling of metabolic perturbations in HIV-infected sera. The cohort consisted of 21 HIV-negative control subjects (HIV−) and 113 HIV-infected individuals, of which 100 were on cART. Significant differences in metabolic features corresponding to changes in glucose, lipids, phenylalanine, glutamic acid, aspartic acid and branched amino acids were observed, which point to oxidative stress and insulin resistance. To further confirm oxidative stress, targeted GC/MS-based metabonomics, performed in succession, allowed for a quantitative description of a total of 9 oxidative stress-related metabolites. Significant up-regulation of aspartic acid, phenylalanine and glutamic acid were observed in the HIV-infected cohorts as compared to controls. Tryptophan and tyrosine were down-regulated whereas cystine levels were increased in HIV-infected and untreated individuals as compared to both HIV treated and negative control subjects. Pathway analysis also revealed 11 metabolic pathways to be significantly altered by infection and/or treatment. These pathways included aminoacyl-tRNA biosynthesis, nitrogen metabolism and phenylalanine, tyrosine and tryptophan biosynthesis. This pilot study demonstrated the use of multiplatform metabonomic strategies to elucidate metabolic markers that would be essential in predicting HIV/cART-induced oxidative stress. This could aid and contribute in HIV treatment and management programmes.

Integrated Systems Pharmacology, Urinary Metabonomics, and Quantitative Real-Time PCR Analysis to Uncover Targets and Metabolic Pathways of the You-Gui Pill in Treating Kidney-Yang Deficiency Syndrome.

Kidney-yang deficiency syndrome (KYDS) is a metabolic disease caused by a neuro-endocrine disorder. The You-gui pill (YGP) is a classic traditional Chinese medicine (TCM) formula for the treatment of KYDS and has been widely used to warm and recuperate KYDS clinically for hundreds of years in China. However, it is unknown whetherthe corresponding targets and metabolic pathways can also be found via using metabonomics based on one platform (e.g., 1H NMR) to study different biological samples of KYDS. At the same time, relevant reports on further molecular verification (e.g., RT-qPCR analysis) of these targets associated with biomarkers and metabolic pathways have not yet, to our knowledge, been seen in KYDS’s research. In the present study, a comprehensive strategy integrating systems pharmacology and 1H NMR-based urinary metabonomics analysis was proposed to identify the target proteins and metabolic pathways that YGP acts on KYDS. Thereafter, further validation of target proteins in kidney tissue was performed through quantitative real-time PCR analysis (RT-qPCR). Furthermore, biochemical parameters and histopathological analysis were studied. As a result, seven target proteins (L-serine dehydratase; phosphoenolpyruvate carboxykinase; spermidine synthase; tyrosyl-tRNA synthetase, glutamine synthetase; 3-hydroxyacyl-CoA dehydrogenase; glycine amidinotransferase) in YGP were discovered to play a therapeutic role in KYDS via affecting eight metabolic pathways (glycine, serine and threonine metabolism; butanoate metabolism; TCA cycle, etc.). Importantly, three target proteins (i.e., 3-hydroxyacyl-CoA dehydrogenase; glutamine synthetase; and glycine amidinotransferase) and two metabolic pathways (butanoate metabolism and dicarboxylate metabolism) related to KYDS, to our knowledge, had been newly discovered in our study. The mechanism of action mainly involved energy metabolism, oxidative stress, ammonia metabolism, amino acid metabolism, and fatty acid metabolism. In short, our study demonstrated that targets and metabolic pathways for the treatment of KYDS by YGP can be effectively found via combining with systems pharmacology and urinary metabonomics. In addition to this, common and specific targets and metabolic pathways of KYDS treated by YGP can be found effectively by integration with the analysis of different biological samples (e.g., serum, urine, feces, and tissue). It is; therefore, important that this laid the foundation for deeper mechanism research and drug-targeted therapy of KYDS in future.

Osteopenia-osteoporosis discrimination in postmenopausal women by 1H NMR-based metabonomics.

This is a report on how 1H NMR-based metabonomics was employed to discriminate osteopenia from osteoporosis in postmenopausal women, identifying the main metabolites associated to the separation between the groups. The Assays were performed using seventy-eight samples, being twenty-eight healthy volunteers, twenty-six osteopenia patients and twenty-four osteoporosis patients. PCA, LDA, PLS-DA and OPLS-DA formalisms were used. PCA discriminated the samples from healthy volunteers from diseased patient samples. Osteopenia-osteoporosis discrimination was only obtained using Analysis Discriminants formalisms, as LDA, PLS-DA and OPLS-DA. The metabonomics model using LDA formalism presented 88.0% accuracy, 88.5% specificity and 88.0% sensitivity. Cross-Validation, however, presented some problems as the accuracy of modeling decreased. LOOCV resulted in 78.0% accuracy. The OPLS-DA based model was better: R2Y and Q2 values equal to 0.871 (p<0.001) and 0.415 (p<0.001). LDA and OPLS-DA indicated the important spectral regions for discrimination, making possible to assign the metabolites involved in the skeletal system homeostasis, as follows: VLDL, LDL, leucine, isoleucine, allantoin, taurine and unsaturated lipids. These results indicate that 1H NMR-based metabonomics can be used as a diagnosis tool to discriminate osteoporosis from osteopenia using a single serum sample.

A NMR-Based Metabonomics Approach to Determine Protective Effect of a Combination of Multiple Components Derived from Naodesheng on Ischemic Stroke Rats.

Naodesheng (NDS) is a widely used traditional Chinese medicine (TCM) prescription for the treatment of ischemic stroke. A combination of 10 components is derived from NDS. They are: Notoginsenoside R1, ginsenoside Rg1, ginsenoside b1, ginsenoside Rd, hydroxysafflor yellow A, senkyunolide I, puerarin, daidzein, vitexin, and ferulic acid. This study aimed to investigate the protective effect of the ten-component combination derived from NDS (TCNDS) on ischemic stroke rats with a middle cerebral artery occlusion (MCAO) model by integrating an NMR-based metabonomics approach with biochemical assessment. Our results showed that TCNDS could improve neurobehavioral function, decrease the cerebral infarct area, and ameliorate pathological features in MCAO model rats. In addition, TCNDS was found to decrease plasma lactate dehydrogenase (LDH) and malondialdehyde (MDA) production and increase plasma superoxide dismutase (SOD) production. Furthermore, 1H-NMR metabonomic analysis indicated that TCNDS could regulate the disturbed metabolites in the plasma, urine, and brain tissue of MCAO rats, and the possible mechanisms were involved oxidative stress, energy metabolism, lipid metabolism, amino acid metabolism, and inflammation. Correlation analysis were then performed to further confirm the metabolites involved in oxidative stress. Correlation analysis showed that six plasma metabolites had high correlations with plasma LDH, MDA, and SOD. This study provides evidence that an NMR-based metabonomics approach integrated with biochemical assessment can help to better understand the underlying mechanisms as well as the holistic effect of multiple compounds from TCM.