Global urinary metabolic profiling procedures using gas chromatography-mass spectrometry

The role of urinary metabolic profiling in systems biology research is expanding. This is because of the use of this technology for clinical diagnostic and mechanistic studies and for the development of new personalized health care and molecular epidemiology (population) studies. The methodologies commonly used for metabolic profiling are NMR spectroscopy, liquid chromatography mass spectrometry (LC/MS) and gas chromatography-mass spectrometry (GC/MS). In this protocol, we describe urine collection and storage, GC/MS and data preprocessing methods, chemometric data analysis and urinary marker metabolite identification. Results obtained using GC/MS are complementary to NMR and LC/MS. Sample preparation for GC/MS analysis involves the depletion of urea via treatment with urease, protein precipitation with methanol, and trimethylsilyl derivatization. The protocol described here facilitates the metabolic profiling of ～400-600 metabolites in 120 urine samples per week.     

Metabolic profiling of a Schistosoma mansoni infection in mouse tissues using magic angle spinning-nuclear magnetic resonance spectroscopy

In order to enhance our understanding of physiological and pathological consequences of a patent Schistosoma mansoni infection in the mouse, we examined the metabolic responses of different tissue samples recovered from the host animal using a metabolic profiling strategy. Ten female NMRI mice were infected with ∼80 S. mansoni cercariae each, and 10 uninfected age- and sex-matched animals served as controls. At day 74 post infection (p.i.), mice were killed and jejunum, ileum, colon, liver, spleen and kidney samples were removed. We employed ¹H magic angle spinning-nuclear magnetic resonance spectroscopy to generate tissue-specific metabolic profiles. The spectral data were analyzed using multivariate modelling methods including an orthogonal signal corrected-projection to latent structure analysis and hierarchical principal component analysis to assess the differences and/or similarities in metabolic responses between infected and non-infected control mice. Most tissues obtained from S. mansoni-infected mice were characterized by high levels of amino acids, such as leucine, isoleucine, lysine, glutamine and asparagine. High levels of membrane phospholipid metabolites, including glycerophosphoryl choline and phosphoryl choline were found in the ileum, colon, liver and spleen of infected mice. Additionally, low levels of energy-related metabolites, including lipids, glucose and glycogen were observed in ileum, spleen and liver samples of infected mice. Energy-related metabolites in the jejunum, liver and renal medulla were found to be positively correlated with S. mansoni worm burden upon dissection. These findings show that a patent S. mansoni infection causes clear disruption of metabolism in a range of tissues at a molecular level, which can be interpreted in relation to the previously reported signature in a biofluid (i.e. urine), giving further evidence of the global effect of the infection.     

Metabolomic profile of hepatitis C virus-infected hepatocytes

Hepatitis C virus (HCV) is capable of disrupting different facets of lipid metabolism and lipids have been shown to play a crucial role in the viral life cycle. The aim of this study was to examine the effect HCV infection has on the hepatocyte metabolome. Huh-7.5 cells were infected using virus produced by the HCV J6/JFH1 cell culture system and cells were harvested 24, 48, and 72-hours following infection. Metabolic profiling was performed using a non-targeted multiple platform methodology combining ultrahigh performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS(2)) and gas chromatography/mass spectrometry (GC/MS). There was a significant increase in a number of metabolites involved in nucleotide synthesis and RNA replication during early HCV infection. NAD levels were also significantly increased along with several amino acids. A number of lipid metabolic pathways were disrupted by HCV infection, resulting in an increase in cholesterol and sphingolipid levels, altered phospholipid metabolism and a possible disruption in mitochondrial fatty acid transport. Fluctuations in 5'-methylthioadenosine levels were also noted, along with alterations in the glutathione synthesis pathway. These results highlight a number of previously unreported metabolic interactions and give a more in depth insight into the effect HCV has on host cell biochemical processes.     

GC/MS analysis of the rat urine for metabonomic research

In this paper, an optimized protocol was established and validated for the metabonomic profiling in rat urine using GC/MS. The urine samples were extracted by methanol after treatment with urease to remove excessive urea, then the resulted supernatant was dried, methoximated, trimethylsilylated, and analyzed by GC/MS. Forty-nine endogenous metabolites were separated and identified in GC/MS chromatogram, of which 26 identified compounds were selected for quantitative analysis to evaluate the linearity, precision, and sensitivity of the method. It showed good linearity between mass spectrometry responses and relative concentrations of the 26 endogenous compounds over the range from 0.063 to 1.000 (v/v, urine/urine+water) and satisfactory reproducibility with intra-day and inter-days precision values all below 15%. The metabonomic profiling method based on GC/MS was successfully applied to urine samples from hyperlipidemia model rats. Obviously, separated clustering of model rats and the control rats were shown by principal components analysis (PCA); time-dependent metabonomic modification was detected as well. It was suggested that metabonomic profiling based on GC/MS be a robust method for urine samples.     

Gas chromatography/mass spectrometry in metabolic profiling of biological fluids

One of the objectives of metabonomics is to identify subtle changes in metabolite profiles between biological systems of different physiological or pathological states. Gas chromatography mass spectrometry (GC/MS) is a widely used analytical tool for metabolic profiling in various biofluids, such as urine and blood due to its high sensitivity, peak resolution and reproducibility. The availability of the GC/MS electron impact (EI) spectral library further facilitates the identification of diagnostic biomarkers and aids the subsequent mechanistic elucidation of the biological or pathological variations. With the advent of new comprehensive two dimensional GC (GCxGC) coupled to time-of-flight mass spectrometry (TOFMS), it is possible to detect more than 1200 compounds in a single analytical run. In this review, we discuss the applications of GC/MS in the metabolic profiling of urine and blood, and discuss its advances in methodologies and technologies.     

Measurement of the cortisol production rate in two sisters with 17 alpha-hydroxylase deficiency using [1,2,3,4-13C]cortisol and isotope dilution mass spectrometry

[1,2,3,4-13C]cortisol was i.v. administered to two sisters aged 11 yr (patient I) and 3 yr (patient II) who suffer from 17 alpha-hydroxylase deficiency. This is the first time that the cortisol production rate (CPR) in patients with 17 alpha-hydroxylase deficiency has been measured with a stable labelled tracer using the urinary method. The urine was collected for 3 days. High-performance liquid chromatography (HPLC) of approximately 100 ml urine extracts was carried out to isolate the small amount of cortisol metabolites excreted. The cortisol metabolites were oxidized to 11-oxo-aetiocholanolone. The isotope dilution in the methyl oxime tert-butyldimethylsilyl ether derivatives was measured by selected ion monitoring gas chromatography/mass spectrometry (GC/MS). The CPR calculated from tetrahydrocortisone (THE) and the cortolones was 765 and 536 nmol/day, respectively in patient I. The CPR in patient II was only calculated from THE and was 62 nmol/day. If radioactive labelled cortisol had been used, much larger quantities of urine would have been needed for isolation of sufficient mass of metabolites, even then purification may have been difficult. Steroid profiling of 1 ml urine samples by GC and identification by GC/MS revealed high concentrations of pregnenolone, progesterone, 11 beta-hydroxy progesterone and corticosterone metabolites. Tetrahydrocorticosterone and 5 alpha-tetrahydrocorticosterone were found in urine at elevated excretions of 2.5 and 5.7, 0.9 and 2.0 mumols/24 h, in patients I and II respectively. No cortisol metabolites were detected by routine GC or GC/MS as the low amounts excreted co-eluted with the relatively abundant corticosterone metabolites.     

Pillars article: Downregulation of Th1 cytokine production accompanies induction of Th2 responses by a parasitic helminth, Schistosoma mansoni. J. Exp. Med. 1991. 173: 159-166

In the mouse, infection with Schistosoma mansoni results in an egg-producing infection and associated disease, whereas vaccination with attenuated larval stages produces a substantial and specific immunity in the absence of egg-induced pathology. Preliminary data showing enhanced interleukin-5 (IL-5) production by T cells from infected mice and interferon γ (IFN-γ) synthesis by cells from vaccinated animals (7), suggested differential CD4(+) subset stimulation by the different parasite stimuli. To confirem this hyposthesis, lymphocytes from vaccinated or infected animals were compared for their ability to produce IFN-γ and IL-2 (secreted by Th1 cells) as compared with IL-4 and IL-5 (characteristic Th2 cytokines). After stimulation with specific antigen or mitogen, T cells from vaccinated mice or prepatently infected animals responded primarily with Th1 lymphokines, whereas lymphocytes from patenly infected mice instead produced Th2 cytokines. The Th2 response in infected animals was shown to be induced by schistosome eggs and directed largely against egg antigens, whereas the Th1 reactivity in vaccinated mice was triggered primarily by larval anigens. Interestingly, Th1 responses in mice carrying egg-producing infections were found to be profoundly downregulated. Moreover, the injection of eggs into vaccinated mice resulted in a reduction of antigen and mitogen-stimulated Th1 function accompanied by a coincident expression of Th2 responses. Together, the data suggest that coincident with the induction of Th2 responses, murine schistosome infection results in an inhibition of potentially protective Th1 function. This previously unrecognized downregulation of Th1 cytokine production may be an important immunological consequence of helminth infection related to host adaptation.     

Metabolic alterations in the hamster co-infected with Schistosoma japonicum and Necator americanus

Co-infection with hookworm and schistosomes is a common phenomenon in sub-Saharan Africa, as well as in parts of South America and southeast Asia. As a first step towards understanding the metabolic response of a hookworm-schistosome co-infection in humans, we investigated the metabolic consequences of co-infection in an animal model, using a nuclear magnetic resonance (NMR)-based metabolic profiling technique, combined with multivariate statistical analysis. Urine and serum samples were obtained from hamsters experimentally infected with 250 Necator americanus infective L₃ and 100 Schistosoma japonicum cercariae simultaneously. In the co-infection model, similar worm burdens were observed as reported for single infection models, whereas metabolic profiles of co-infection represented a combination of the altered metabolite profiles induced by single infections with these two parasites. Consistent differences in metabolic profiles between the co-infected and non-infected control hamsters were observed from 4 weeks p.i. onwards. The predominant metabolic alterations in co-infected hamsters consisted of depletion of amino acids, tricarboxylic acid cycle intermediates (e.g. citrate and succinate) and glucose. Moreover, alterations of a series of gut microbial-related metabolites, such as decreased levels of hippurate, 3-hydroxyphenylpropionic acid, 4-hydroxyphenylpropionic acid and trimethylamine-N-oxide, and increased concentrations of 4-cresol glucuronide and phenylacetylglycine were associated with co-infection. Our results provide a first step towards understanding the metabolic response of an animal host to multiple parasitic infections.     

NMR-based metabonomics for detection of Helicobacter pylori infection in gerbils: which is more descriptive

Background:  Helicobacter pylori , the human pathogenic gram-negative microaerophilic bacterium, causes chronic gastric infection in more than half of the human population regardless of race. The infection of microbe is not yet controllable to pose a substantial public health impact and a growing social burden. The management of H. pylori infection primarily necessitates accurate and timely diagnosis at case level, on-demand supervision of pathologic progression, and reliable evaluation of the impact of pharmacologic interventions on the patients' population.
Methods:  The characterization of H. pylori infection on gerbils model was performed by metabolic profiling, employing ¹H NMR spectroscopy compounding multivariate pattern recognition strategies. In the same manner, urine samples were individually collected from 10 gerbils infected with H. pylori GS13, and from 10 uninfected control animals equally accessible to feed and water.
Results:  The resultant metabolic profiles indicate that H. pylori infection disturbs carbohydrate metabolism to elevate the levels of α- and β-glucose, and cis -aconitate (a TCA cycle intermediate). In addition to the energy metabolism alteration, the colonization of H. pylori in gerbil stomach generates a remarkable deviation of amino acid metabolism as indicated by depletion of taurine and arginine, and elevation of proline and glutamine in the animal urine. Moreover, the H. pylori infection modifies the gut microbiota as highlighted by a range of microbial-related metabolites such as indoxyl sulfate and hippurate.
Conclusions:  These findings demonstrate that the ¹H NMR-based urine metabolic profiling is a promising technique capable of providing an accurate, noninvasive, and rapid diagnosis of H. pylori infection.     

Evidence of altered secondary lymphoid-tissue chemokine responsiveness in Balb/c mice infected with Schistosoma mansoni

To determine the extent to which splenic T cells were affected by Schistosoma mansoni infection, we investigated the ability of the T cells to produce interferon (IFN)-gamma, as well as their chemotactic ability 7 wk PI. In this study, we report that splenic T cells from Balb/c mice with S. mansoni infections were capable of producing levels of IFN-gamma comparable with splenic T cells from naive mice. However, the T cells exhibited altered chemotactic activity, as evidenced by an inability to respond to secondary lymphoid-tissue chemokine (SLC/CCL21). Although no difference in chemokine expression was found between the spleens of infected versus control mice, chemokine production was greater in the livers of infected versus control mice. Collectively, these data indicate that Balb/c mice with 7-wk S. mansoni infection possess splenic T cells with altered chemotactic activity and that the alterations may be a consequence of the granulomatous response in the liver.     

Quantitative metabolomic profiling of serum and urine in DSS-induced ulcerative colitis of mice by (1)H NMR spectroscopy

Quantitative profiling of a large number of metabolic compounds is a promising method to detect biomarkers in inflammatory bowel diseases (IBD), such as ulcerative colitis (UC). We induced an experimental form of UC in mice by treatment with dextran sulfate sodium (DSS) and characterized 53 serum and 69 urine metabolites by use of (1)H NMR spectroscopy and quantitative ("targeted") analysis to distinguish between diseased and healthy animals. Hierarchical multivariate orthogonal partial least-squares (OPLS) models were developed to detect and predict separation of control and DSS-treated mice. DSS treatment resulted in weight loss, colonic inflammation, and increase in myeloperoxidase activity. Metabolomic patterns generated from the OPLS data clearly separated DSS-treated from control mice with a slightly higher predictive power (Q(2)) for serum (0.73) than urine (0.71). During DSS colitis, creatine, carnitine, and methylamines increased in urine while in serum, maximal increases were observed for ketone bodies, hypoxanthine, and tryptophan. Antioxidant metabolites decreased in urine whereas in serum, glucose and Krebs cycle intermediates decreased strongly. Quantitative metabolic profiling of serum and urine thus discriminates between healthy and DSS-treated mice. Analysis of serum or urine seems to be equally powerful for detecting experimental colitis, and a combined analysis offers only a minor improvement.     

Toxoplasma gondii and Schistosoma mansoni synergize to promote hepatocyte dysfunction associated with high levels of plasma TNF-alpha and early death in C57BL/6 mice.

To address the question of how the murine host responds to a prototypic type 1 cytokine inducer while concurrently undergoing a helminth-induced type 2 cytokine response, C57BL/6 strain animals with patent schistosomiasis mansoni were orally infected with the cystogenic Toxoplasma gondii strain ME49. Schistosoma mansoni infection resulted in a significantly higher mortality rate when mice were subsequently orally infected with ME49, and these animals displayed a defective IFN-gamma and NO response relative to animals infected with T. gondii alone. Plasma levels of TNF-alpha and aspartate transaminase in double-infected mice were greatly elevated relative to mice infected with either parasite alone. Consistent with the latter observation, these animals exhibited severe liver pathology, with regions of coagulative necrosis and hepatocyte vacuolization unapparent in mice carrying either infection alone. Interestingly, mean egg granuloma size was approximately 50% of that in mice with S. mansoni infection alone. The exacerbated liver pathology in coinfected mice did not appear to be a result of uncontrolled tachyzoite replication, because both parasite-specific RT-PCR analysis and immunohistochemical staining demonstrated a low number of tachyzoites in the liver. We hypothesize that mortality in these animals results from the high level of systemic TNF-alpha, which mediates a severe liver pathology culminating in death of the animal.     

A contribution to the study of acute schistosomiasis (an experimental trial)

In an attempt to establish an experimental model of acute schistosomiasis, sequential histological changes were investigated in the skin, lung, liver and spleen of mice infected with 30 or 100 cercariae of Schistosoma mansoni according to four sets of experiments: single infection, repeated infections, unisexual infection and infection in mice born from infected mothers. Animals were killed every other day from exposure up to 50 days after infection. Only mild, isolated, focal inflammatory changes were found before the appearance of mature eggs in the liver, even when repeated infections were made. Severe changes of reactive hepatitis and splenitis appeared suddenly when the first mature eggs were deposited, around the 37th to 42nd day after infection. The mature eggs induced lytic and coagulative necrosis of hepatocytes around them which was soon followed by dense infiltration of eosinophils. So, mature egg-induced lesions appeared as the major factors in the pathogenesis of acute schistosomiasis in mice. Mice born from infected mothers were apparently able to rapidly modulate the egg-lesions, forming early fibrotic granulomas. The murine model of acute schistosomiasis appeared adequate for the study of pathology and pathogenesis of acute schistosomiasis.     

Metabonomic and metallomic profiling in the amniotic fluid of malnourished pregnant rats

Epidemiology and studies in animal models have revealed that prenatal malnutrition is highly correlated with abnormal fetal neurodevelopment. We present here a combined metabonomic and metallomic profiling technique to associate the metabolic and trace-elemental composition variations of rat amniotic fluid (AF) in malnourished pregnant rats with the retardation of fetal rat neurodevelopment. The AF samples from three groups of pregnant Sprague-Dawley rats, which were fed either a normal diet, a low-protein diet, or "a famine diet", were subjected to GC/MS and ICP/MS combined with multivariate data analysis (MVDA). PCA scores plot of both GC/MS and ICP/MS data showed similar and unique metabolic signatures of AF in response to the different diets. Rats in the famine group released increased amounts of glycine, inositol, putrescine, and rubidium and decreased amounts of methionine, dopa, tryptophan, glutamine, zinc, cobalt, and selenium in the AF. These discriminable variations in the AF may indicate the abnormality of a number of metabolic pathways in fetal rats including the folate cycle and methionine pathway, the monoamine pathway, and tri-iodothyronine (T3) metabolism. The abnormalities may be the result of metabolites or elemental differences or a combination of both. This study demonstrates the potential of combining profiling of small-molecule metabolites and trace elements to broaden the understanding of biological variations associated with fetal neurodevelopment induced by environmental perturbation.     

Apolipoprotein A-I mimetic peptide inhibits atherosclerosis by altering plasma metabolites in hypercholesterolemia

An apolipoprotein A-I mimetic peptide, D-4F, has been shown to improve vasodilation and inhibit atherosclerosis in hypercholesterolemic low-density lipoprotein receptor-null (LDLr(-/-)) mice. To study the metabolic variations of D-4F ininhibiting atherosclerosis, metabonomics, a novel system biological strategy to investigate the pathogenesis, was developed. Female LDLr(-/-) mice were fed a Western diet and injected with or without D-4F intraperitoneally. Atherosclerotic lesion formation was measured, whereas plasma metabolic profiling was obtained on the basis of ultra-high-performance liquid chromatography in tandem with time-of-flight mass spectrometry operating in both positive and negative ion modes. Data were processed by multivariate statistical analysis to graphically demonstrate metabolic changes. The partial least-squares discriminate analysis model was validated with cross-validation and permutation tests to ensure the model's reliability. D-4F significantly inhibited the formation of atherosclerosis in a time-dependent manner. The metabolic profiling was altered dramatically in hypercholesterolemic LDLr(-/-) mice, and a significant metabolic profiling change in response to D-4F treatment was observed in both positive and negative ion modes. Thirty-six significantly changed metabolites were identified as potential biomarkers. A series of phospholipid metabolites, including lysophosphatidylcholine (LysoPC), lysophosphatidylethanolamine (LysoPE), phosphatidylcholine (PC), phatidylethanolamine (PE), sphingomyelin (SM), and diacylglycerol (DG), particularly the long-chain LysoPC, was elevated dramatically in hypercholesterolemic LDLr(-/-) mice but reduced by D-4F in a time-dependent manner. Quantitative analysis of LysoPC, LysoPE, PC, and DG using HPLC was chosen to validate the variation of these potential biomarkers, and the results were consistent with the metabonomics findings. Our findings demonstrated that D-4F may inhibit atherosclerosis by regulating phospholipid metabolites specifically by decreasing plasma long-chain LysoPC.     

Schistosome fecundity: influence of host genotype and intensity of infection

The host genetic influence on the fecundity of Schistosoma mansoni was studied by measuring egg excretion and accumulation of eggs in the tissues of two inbred strains of mice. The two strains, NIH/Ola and CBA/Ca, differed in both parameters. Egg excretion after infection in the NIH/Ola reached a maximum and declined earlier than was the case for the CBA/Ca mice. More eggs accumulated in the gut and lungs of CBA/Ca, while the NIH/Ola had more eggs in the liver by 100 days post-infection. Statistical analysis of both tissue eggs and faecal eggs, using a robust, non-parametric method, indicated that there is significant evidence for a density dependent reduction in fecundity of worms in more heavily infected animals. We conclude that both the genetic constitution of the murine host and the intensity of infection affect the fecundity of Schistosoma mansoni worms.     

1H NMR-based metabolomic study on resistance to diet-induced obesity in AHNAK knock-out mice

AHNAK is a giant protein of approximately 700 kDa identified in human neuroblastomas and skin epithelial cells. Recently, we found that AHNAK knock-out (AHNAK(-/-)) mice have a strong resistance to high-fat diet-induced obesity. In this study, we applied (1)H NMR-based metabolomics with multivariate statistical analysis to compare the altered metabolic patterns detected in urine from high-fat diet (HFD) fed wild-type and AHNAK(-/-) mice and investigate the mechanisms underlying the resistance to high-fat diet-induced obesity in AHNAK(-/-) mice. In global profiling, principal components analysis showed a clear separation between the chow diet and HFD groups; wild-type and AHNAK(-/-) mice were more distinctly separated in the HFD group compared to the chow diet group. Based on target profiling, the urinary metabolites of HFD-fed AHNAK(-/-) mice gave higher levels of methionine, putrescine, tartrate, urocanate, sucrose, glucose, threonine, and 3-hydroxyisovalerate. Furthermore, two-way ANOVAs indicated that diet type, genetic type, and their interaction (gene × diet) affect the metabolite changes differently. Most metabolites were affected by diet type, and putrescine, threonine, urocanate, and tartrate were also affected by genetic type. In addition, cis-aconitate, succinate, glycine, histidine, methylamine (MA), phenylacetylglycine (PAG), methionine, putrescine, uroconate, and tartrate showed interaction effects. Through the pattern changes in urinary metabolites of HFD-fed AHNAK(-/-) mice, our data suggest that the strong resistance to HFD-induced obesity in AHNAK(-/-) mice comes from perturbations of amino acids, such as methionine, putrescine, threonine, and histidine, which are related to fat metabolism. The changes in metabolites affected by microflora such as PAG and MA were also observed. In addition, resistance to obesity in HFD-fed AHNAK(-/-) mice was not related to an activated tricarboxylic acid cycle. These findings demonstrate that (1)H NMR-based metabolic profiling of urine is suitable for elucidating possible biological pathways perturbed by functional loss of AHNAK on HFD feeding and could elucidate the mechanism underlying the resistance to high-fat diet-induced obesity in AHNAK(-/-) mice.     

Metabolism of the designer drug 4-bromo-2,5-dimethoxyphenethylamine (2C-B) in mice, after acute administration

4-Bromo-2,5-dimethoxyphenethylamine (2C-B) is a psychoactive drug of abuse often sold under the general street name "Ecstasy". Recent reports on the abuse of 2C-B and analogues denote the lack of knowledge on this drug metabolism. In the present study, we investigated the metabolic profile of 2C-B in the mouse and found unchanged 2C-B and several metabolites, which could be identified by GC/MS in the mice urine. The identification of 2C-B metabolites may give important clues for the biological and toxicological effects of this drug of abuse and provides new important data for forensic analysis on samples taken from 2C-B abusers.     

Urine metabolomics analysis for adrenal incidentaloma activity detection and biomarker discovery

This study describes the development of a method suitable for the analysis of nineteen major urinary steroid metabolites in human urine. The analytes of interest were isolated from urine using solid phase extraction, subjected to enzymatic hydrolysis and again extracted applying solid phase extraction. After derivatization, methyloxime-trimethylsilyl ether derivatives of steroid hormones were identified by gas chromatography-mass spectrometry (GC/MS) and quantified by gas chromatography with flame ionization detector (GC/FID). The quantification method was validated for linearity, trueness, precision and selectivity. The limits of detection were between 6.2 and 7.2 ng/mL and limits of quantification were between 12.3 and 14.8 ng/mL. The established method was applied to analyze 28 urine samples from patients diagnosed with non-functioning adrenal incidentalomas (AIs) and 30 healthy subjects. Hierarchical cluster analysis (HCA) and principal component analysis (PCA) were employed to visualize the differences between metabolic profiles of patients and the control group and to determine possible markers of AIs activity. Both multivariate methods separated seven patients from the rest of the examined individuals. Five urinary metabolites including α-cortol, tetrahydrocorticosterone, tetrahydrocortisol, allo-tetrahydrocortisol and etiocholanolone were identified as potential biomarkers of pathological adrenal function. The altered metabolites reflected pathological metabolism mainly of cortisol and cortisone. This research proved that metabolomics is a suitable tool for disease research.     

A metabolite profiling approach to follow the sprouting process of mung beans (Vigna radiata)

A metabolite profiling methodology based on capillary gas chromatography/mass spectrometry (GC/MS) was employed to investigate time-dependent metabolic changes in the course of the sprouting of mung beans (Vigna radiata). Intact mung beans and sprout samples taken during the germination process were subjected to an extraction and fractionation procedure covering a broad spectrum of lipophilic (e.g. fatty acid methyl esters, hydrocarbons, fatty alcohols, sterols) and hydrophilic (e.g. sugars, acids, amino acids, amines) low molecular weight constituents. Investigation of the obtained fractions by GC resulted in the detection of more than 450 distinct peaks of which 146 were identified by means of MS. Statistical assessment of the metabolite profiling data via principal component analysis demonstrated that the metabolic changes during the sprouting of mung beans are reflected by time-dependent shifts of the scores which were comparable for two spouting processes independently conducted under the same conditions. Analysis of the loadings showed that polar metabolites were major contributors to the separation along the first principal component. The dynamic changes of single metabolites revealed significantly increased levels of monosaccharides, organic acids and amino acids and a decrease in fatty acid methyl esters in mung bean sprouts.