These are scientific publications for Discovery Metabolomics using an Agilent GC/MS system. Please note that the links below will take you to outside of Agilent’s website to PubMed.
1. Integrated analysis of serum and liver metabonome in liver transplanted rats by gas chromatography coupled with mass spectrometry.
Wang Y, Tao Y, Lin Y, Liang L, Wu Y, Qu H, Liang T, Cheng Y.
Anal Chim Acta. 2009 Feb 2;633(1):65-70. Epub 2008 Nov 25.
In this paper, we present a metabonomic method for the investigation of abnormal metabolic process in both serum and liver tissue of liver transplanted rats. Syngeneic transplantation was performed on male Lewis rats. The serum and grafted liver on day 1, 3, and 7 post-transplant were collected to analyze endogenous metabolites using gas chromatography coupled with mass spectrometry (GC-MS). The method was validated with acceptable linearity, precision, and repeatability. Thirty-four metabolites in serum and 29 metabolites in liver were identified. Results of correlation analysis illustrated metabolites with similar function exhibited similar variations in liver and serum. The data processed by principle component analysis (PCA) showed time-dependent biochemical variations. As a consequence, the present study may offer specific putative pathways in the pathophysiological mechanism of orthotopic liver transplantation.
2. Hydroxylated metabolites of polybrominated diphenyl ethers in human blood samples from the United States.
Qiu X, Bigsby RM, Hites RA.
Environ Health Perspect. 2009 Jan;117(1):93-8. Epub 2008 Aug 1.
BACKGROUND: A previous study from our laboratory showed that polybrominated diphenyl ethers (PBDEs) were metabolized to hydroxylated PBDEs (HO-PBDEs) in mice and that para-HO-PBDEs were the most abundant and, potentially, the most toxic metabolites. OBJECTIVE: The goal of this study was to determine the concentrations of HO-PBDEs in blood from pregnant women, who had not been intentionally or occupationally exposed to these flame retardants, and from their newborn babies. METHODS: Twenty human blood samples were obtained from a hospital in Indianapolis, Indiana, and analyzed for both PBDEs and HO-PBDEs using electron-capture negative-ionization gas chromatographic mass spectrometry.
RESULTS: The metabolite pattern of HO-PBDEs in human blood was quite different from that found in mice; 5-HO-BDE-47 and 6 HO-BDE-47 were the most abundant metabolites of BDE-47, and 5'-HO-BDE-99 and 6'-HO-BDE-99 were the most abundant metabolites of BDE-99. The relative concentrations between precursor and corresponding metabolites indicated that BDE-99 was more likely to be metabolized than BDE-47 and BDE-100. In addition, three bromophenols were also detected as products of the cleavage of the diphenyl ether bond. The ratio of total hydroxylated metabolites relative to their PBDE precursors ranged from 0.10 to 2.8, indicating that hydroxylated metabolites of PBDEs were accumulated in human blood. CONCLUSIONS: The quite different PBDE metabolite pattern observed in humans versus mice indicates that different enzymes might be involved in the metabolic process. Although the levels of HO-PBDE metabolites found in human blood were low, these metabolites seemed to be accumulating.
3. A serum metabolomic investigation on hepatocellular carcinoma patients by chemical derivatization followed by gas chromatography/mass spectrometry.
Xue R, Lin Z, Deng C, Dong L, Liu T, Wang J, Shen X.
Rapid Commun Mass Spectrom. 2008 Oct;22(19):3061-8.
The purpose of this study was to investigate the serum metabolic difference between hepatocellular carcinoma (HCC, n = 20) male patients and normal male subjects (n = 20). Serum metabolome was detected through chemical derivatization followed by gas chromatography/mass spectrometry (GC/MS). The acquired GC/MS data was analyzed by stepwise discriminant analysis (SDA) and support vector machine (SVM). The metabolites including butanoic acid, ethanimidic acid, glycerol, L-isoleucine, L-valine, aminomalonic acid, D-erythrose, hexadecanoic acid, octadecanoic acid, and 9,12-octadecadienoic acid in combination with each other gave the strongest segregation between the two groups. By applying these variables, our method provided a diagnostic model that could well discriminate between HCC patients and normal subjects. More importantly, the error count estimate for each group was 0%. The total classifying accuracy of the discriminant function tested by SVM 20-fold cross validation was 75%. This technique is different from traditional ones and appears to be a useful tool in the area of HCC diagnosis.
4. Arabidopsis 10-formyl tetrahydrofolate deformylases are essential for photorespiration.
Collakova E, Goyer A, Naponelli V, Krassovskaya I, Gregory JF 3rd, Hanson AD, Shachar-Hill Y.
Plant Cell. 2008 Jul; 20(7):1818-32. Epub 2008 Jul 15.
In prokaryotes, PurU (10-formyl tetrahydrofolate [THF] deformylase) metabolizes10-formyl THF to formate and THF for purine and Gly biosyntheses. The Arabidopsis thaliana genome contains two putative purU genes, At4g17360 and At5g47435. Knocking out these genes simultaneously results in plants that are smaller and paler than the wild type. These double knockout (dKO) mutant plants show a 70-fold increase in Gly levels and accumulate elevated levels of 5- and 10-formyl THF. Embryo development in dKO mutants arrests between heart and early bent cotyledon stages. Mature seeds are shriveled, accumulate low amounts of lipids, and fail to germinate. However, the dKO mutant is only conditionally lethal and is rescued by growth under nonphotorespiratory conditions. In addition, culturing dKO siliques in the presence of sucrose restores normal embryo development and seed viability, suggesting that the seed and embryo development phenotypes are a result of a maternal effect. Our findings are consistent with the involvement of At4g17360 and At5g47435 proteins in photorespiration, which is to prevent excessive accumulation of 5-formyl THF, a potent inhibitor of the Gly decarboxylase/Ser hydroxymethyltransferase complex. Supporting this role, deletion of the At2g38660 gene that encodes the bifunctional 5,10-methylene THF dehydrogenase/5,10-methenyl THF cyclohydrolase that acts upstream of 5-formyl THF formation restored the wild-type phenotype in dKO plants.
5. Application of dissimilarity indices, principal coordinates analysis, and rank tests to peak tables in metabolomics of the gas chromatography/mass spectrometry of human sweat.
Xu Y, Gong F, Dixon SJ, Brereton RG, Soini HA, Novotny MV, Oberzaucher E, Grammer K, Penn DJ.
Anal Chem. 2007 Aug 1; 79(15):5633-41. Epub 2007 Jun 30.
The majority of works in metabolomics employ approaches based on principal components analysis (PCA) and partial least-squares, primarily to determine whether samples fall within large groups. However, analytical chemists rarely tackle the problem of individual fingerprinting, and in order to do this effectively, it is necessary to study a large number of small groups rather than a small number of large groups and different approaches are required, as described in this paper. Furthermore, many metabolomic studies on mammals and humans involve analyzing compounds (or peaks) that are present in only a certain portion of samples, and conventional approaches of PCA do not cope well with sparse matrices where there may be many 0s. There is, however, a large number of qualitative similarity measures available for this purpose that can be exploited via principal coordinates analysis (PCO). It can be shown that PCA scores are a specific case of PCO scores, using a quantitative similarity measure. A large-scale study of human sweat consisting of nearly 1000 gas chromatography/mass spectrometry analyses from the sweat of an isolated population of 200 individuals in Carinthia (Southern Austria) sampled once per fortnight over 10 weeks was employed in this study and grouped into families. The first step was to produce a peak table requiring peak detection, alignment, and integration. Peaks were reduced from 5080 to 373 that occurred in at least 1 individual over 4 out of 5 fortnights. Both qualitative (presence/absence) and quantitative (equivalent to PCA) similarity measures can be computed. PCO and the Kolomorogov-Smirnoff (KS) rank test are applied to these similarity matrices. It is shown that for this data set there is a reproducible individual fingerprint, which is best represented using the qualitative similarity measure as assessed both by the Hotelling t2 statistic as applied to PCO scores and the probabilities associated with the KS rank test.
6. Systematic identification of conserved metabolites in GC/MS data for metabolomics and biomarker discovery.
Styczynski MP, Moxley JF, Tong LV, Walther JL, Jensen KL, Stephanopoulos GN.
Anal Chem. 2007 Feb 1;79(3):966-73.
Analysis of metabolomic profiling data from gas chromatography-mass spectrometry (GC/MS) measurements usually relies upon reference libraries of metabolite mass spectra to structurally identify and track metabolites. In general, techniques to enumerate and track unidentified metabolites are nonsystematic and require manual curation. We present a method and software implementation, freely available at http://spectconnect.mit.edu, that can systematically detect components that are conserved across samples without the need for a reference library or manual curation. We validate this approach by correctly identifying the components in a known mixture and the discriminating components in a spiked mixture. Finally, we demonstrate an application of this approach with a brief analysis of the Escherichia coli metabolome. By systematically cataloguing conserved metabolite peaks prior to data analysis methods, our approach broadens the scope of metabolomics and facilitates biomarker discovery.
7. Metabolite profiling studies in Saccharomyces cerevisiae: an assisting tool to prioritize host targets for antiviral drug screening.
Schneider K, Krömer JO, Wittmann C, Alves-Rodrigues I, Meyerhans A, Diez J,Heinzle E.
Microb Cell Fact. 2009 Jan 30;8:12.
The cellular proteins Pat1p, Lsm1p, and Dhh1p are required for the replication of some positive-strand viruses and therefore are potential targets for new antiviral drugs. To prioritize host targets for antiviral drug screening a comparative metabolome analysis in Saccharomyces cerevisiae reference strain BY4742 Matalpha his3Delta1 leu2Delta0 lys2Delta0 ura3Delta0 and deletion strains pat1Delta, lsm1Delta and dhh1Delta was performed. RESULTS: GC/MS analysis permitted the quantification of 47 polar metabolites and the identification of 41 of them. Metabolites with significant variation between the strains were identified using partial least squares to latent structures discriminate analysis (PLS-DA). The analysis revealed least differences of pat1Delta to the reference strain as characterized by Euclidian distance of normalized peak areas. The growth rate and specific production rates of ethanol and glycerol were also most similar with this strain. CONCLUSION: From these results we hypothesize that the human analog of yeast Pat1p is most likely the best drug target candidate.
8. Urinary markers of adrenarche: reference values in healthy subjects, aged 3-18 years.
Remer T, Boye KR, Hartmann MF, Wudy SA.
J Clin Endocrinol Metab. 2005 Apr;90(4):2015-21. Epub 2005 Jan 25.
Information on the urinary excretion of dehydroepiandrosterone (DHEA) and its direct metabolites is scarce for healthy subjects during growth. We used gas chromatography-mass spectrometry urinary steroid profiling to noninvasively study adrenarchal metabolome in 400 healthy subjects, aged 3-18 yr. Urinary 24-h excretion rates of DHEA did not increase significantly before age 7-8 yr. However, DHEA together with its 16alpha-hydroxylated downstream metabolites, 16alpha-hydroxy-DHEA and 3beta,16 alpha,17beta-androstenetriol (DHEA&M), as well as the DHEA metabolite, 5-androstene-3beta,17beta-diol (ADIOL), and the sum of major urinary androgen metabolites (C19) rose consistently from the youngest to the oldest age group. The significant increases (P < 0.01) observed for 24-h excretion rates of C19, ADIOL, and DHEA&M were 2- to 4-fold in boys and girls between age 3 and 8 yr. DHEA&M, for example, rose from about 20 to 80 microg/d (P < 0.0001) during this period. Until the age of 16 yr, DHEA&M excretion also increased to nearly 1000 microg/d. Patterns of steroidogenic enzyme activities were assessed (from definite ratios of urinary steroid metabolites) for 21-hydroxylase, 3beta-hydroxysteroid dehydrogenase, 17beta-hydroxysteroid dehydrogenase, and 5alpha-reductase. Our results indicate for healthy boys and girls that adrenarche is a gradual process starting much earlier than hitherto believed. Efficient metabolism of DHEA, especially to 16-hydroxylated steroids, may explain the almost constant levels seen for this steroid until age 7-8 yr. The established reference values for DHEA, DHEA&M, ADIOL, C19 (including androsterone and etiocholanolone), and urinary parameters of steroidogenic enzyme activities could be useful to identify nutritional, environmental, and pathophysiological interrelations with the progressive maturational process of adrenarche. Our data may also be used as reference data for the diagnosis of steroid-related disorders.
9. Identification of 19 new metabolites induced by abnormal amino acid conjugation in isovaleric acidemia.
Loots DT, Erasmus E, Mienie LJ.
Clin Chem. 2005 Aug;51(8):1510-2.
10. Metabolic profiling of the sink-to-source transition in developing leaves of quaking aspen.
Jeong ML, Jiang H, Chen HS, Tsai CJ, Harding SA.
Plant Physiol. 2004 Oct; 136(2):3364-75. Epub 2004 Sep 24.
Profiles of small polar metabolites from aspen (Populus tremuloides Michx.) leaves spanning the sink-to-source transition zone were compared. Approximately 25% of 250 to 300 routinely resolved peaks were identified, with carbohydrates, organic acids, and amino acids being most abundant. Two-thirds of identified metabolites exhibited greater than 4-fold changes in abundance during leaf ontogeny. In the context of photosynthetic and respiratory measurements, profile data yielded information consistent with expected developmental trends in carbon-heterotrophic and carbon-autotrophic metabolism. Suc concentration increased throughout leaf expansion, while hexose sugar concentrations peaked at mid-expansion and decreased sharply thereafter. Amino acid contents generally decreased during leaf expansion, but an early increase in Phe and a later one in Gly and Ser reflected growing commitments to secondary metabolism and photorespiration, respectively. The assimilation of nitrate and utilization of stored Asn appeared to be marked by sequential changes in malate concentration and Asn transaminase activity. Principal component and hierarchical clustering analysis facilitated the grouping of cell wall maturation (pectins, hemicelluloses, and oxalate) and membrane biogenesis markers in relation to developmental changes in carbon and nitrogen assimilation. Metabolite profiling will facilitate investigation of nitrogen use and cellular development in Populus sp. varying widely in their growth and pattern of carbon allocation during sink-to-source development and in response to stress.