PubMed

Widening our gaze of red blood storage haze: a role for metabolomics. Transfusion. 2015 Jun;55(6):1139-42 Authors: Zimring JC PMID: 26074173 [PubMed - in process]

A peaklet-based generic strategy for the untargeted analysis of comprehensive two-dimensional gas chromatography mass spectrometry data sets. J Chromatogr A. 2015 Jun 3; Authors: Egert B, Weinert CH, Kulling SE Abstract Comprehensive two-dimensional gas chromatography mass spectrometry (GC×GC-MS) is a well-established key technology in analytical chemistry and increasingly used in the field of untargeted metabolomics. However, automated processing of large GC×GC-MS data sets is still a major bottleneck in untargeted, large-scale metabolomics. For this reason we introduce a novel peaklet-based alignment strategy. The algorithm is capable of an untargeted deterministic alignment exploiting a density based clustering procedure within a time constrained similarity matrix. Exploiting minimal (1)D and (2)D retention time shifts between peak modulations, the alignment is done without the need for peak merging which also eliminates the need for linear or nonlinear retention time correction procedures. The approach is validated in detail using data of urine samples from a large human metabolomics study. The data was acquired by a Shimadzu GCMS-QP2010 Ultra GC×GC-qMS system and consists of 512 runs, including 312 study samples and 178 quality control sample injections, measured within a time period of 22 days. The final result table consisted of 313 analytes, each of these being detectable in at least 75% of the study samples. In summary, we present an automated, reliable and fully transparent workflow for the analysis of large GC×GC-qMS metabolomics data sets. PMID: 26074098 [PubMed - as supplied by publisher]

Influence of HEK293 metabolism on the production of viral vectors and vaccine. Vaccine. 2015 Jun 11; Authors: Petiot E, Cuperlovic-Culf M, Shen CF, Kamen A Abstract Mammalian cell cultures are increasingly used for the production of complex biopharmaceuticals including viral vectors and vaccines. HEK293 is the predominant cell line used for the transient expression of recombinant proteins and a well-established system for the production of viral vectors. Understanding metabolic requirements for high productivity in HEK293 cells remains an important area of investigation. Many authors have presented approaches for increased productivity through optimization of cellular metabolism from two distinct perspectives. One is a non-targeted approach, which is directed to improving feeding strategies by addition of exhausted or critical substrates and eventually removal of toxic metabolites. Alternatively, a targeted approach has attempted to identify specific targets for optimization through better understanding of the cellular metabolism under different operating conditions. This review will present both approaches and their successes with regards to improvement of viral production in HEK293 cells outlining the key relations between HEK293 cell metabolism and viral vector productivity. Also, we will summarize the current knowledge on HEK293 metabolism indicating remaining issues to address and problems to resolve to maximize the productivity of viral vectors in HEK293 cells. PMID: 26073013 [PubMed - as supplied by publisher]

A Metabolomic Approach to Understanding the Metabolic Link between Obesity and Diabetes. Mol Cells. 2015 Jun 15; Authors: Park S, Sadanala KC, Kim EK Abstract Obesity and diabetes arise from an intricate interplay between both genetic and environmental factors. It is well recognized that obesity plays an important role in the development of insulin resistance and diabetes. Yet, the exact mechanism of the connection between obesity and diabetes is still not completely understood. Metabolomics is an analytical approach that aims to detect and quantify small metabolites. Recently, there has been an increased interest in the application of metabolomics to the identification of disease biomarkers, with a number of well-known biomarkers identified. Metabolomics is a potent approach to unravel the intricate relationships between metabolism, obesity and progression to diabetes and, at the same time, has potential as a clinical tool for risk evaluation and monitoring of disease. Moreover, metabolomics applications have revealed alterations in the levels of metabolites related to obesity-associated diabetes. This review focuses on the part that metabolomics has played in elucidating the roles of metabolites in the regulation of systemic metabolism relevant to obesity and diabetes. It also explains the possible metabolic relation and association between the two diseases. The metabolites with altered profiles in individual disorders and those that are specifically and similarly altered in both disorders are classified, categorized and summarized. PMID: 26072981 [PubMed - as supplied by publisher]

Related Articles Antiandrogenic therapy with finasteride attenuates cardiac hypertrophy and left ventricular dysfunction. Circulation. 2015 Mar 24;131(12):1071-81 Authors: Zwadlo C, Schmidtmann E, Szaroszyk M, Kattih B, Froese N, Hinz H, Schmitto JD, Widder J, Batkai S, Bähre H, Kaever V, Thum T, Bauersachs J, Heineke J Abstract BACKGROUND: In comparison with men, women have a better prognosis when experiencing aortic valve stenosis, hypertrophic cardiomyopathy, or heart failure. Recent data suggest that androgens like testosterone or the more potent dihydrotestosterone contribute to the development of cardiac hypertrophy and failure. Therefore, we analyzed whether antiandrogenic therapy with finasteride, which inhibits the generation of dihydrotestosterone by the enzyme 5-α-reductase, improves pathological ventricular remodeling and heart failure. METHODS AND RESULTS: We found a strongly induced expression of all 3 isoforms of the 5-α-reductase (Srd5a1 to Srd5a3) in human and mouse hearts with pathological hypertrophy, which was associated with increased myocardial accumulation of dihydrotestosterone. Starting 1 week after the induction of pressure overload by transaortic constriction, mice were treated with finasteride for 2 weeks. Cardiac function, hypertrophy, dilation, and fibrosis were markedly improved in response to finasteride treatment in not only male, but also in female mice. In addition, finasteride also very effectively improved cardiac function and mortality after long-term pressure overload and prevented disease progression in cardiomyopathic mice with myocardial Gαq overexpression. Mechanistically, finasteride, by decreasing dihydrotestosterone, potently inhibited hypertrophy and Akt-dependent prohypertrophic signaling in isolated cardiac myocytes, whereas the introduction of constitutively active Akt blunted these effects of finasteride. CONCLUSIONS: Finasteride, which is currently used in patients to treat prostate disease, potently reverses pathological cardiac hypertrophy and dysfunction in mice and might be a therapeutic option for heart failure. PMID: 25632043 [PubMed - indexed for MEDLINE]

Related Articles Comparative metabolite profiling of two rice genotypes with contrasting salt stress tolerance at the seedling stage. PLoS One. 2014;9(9):e108020 Authors: Zhao X, Wang W, Zhang F, Deng J, Li Z, Fu B Abstract BACKGROUND: Rice is sensitive to salt stress, especially at the seedling stage, with rice varieties differing remarkably in salt tolerance (ST). To understand the physiological mechanisms of ST, we investigated salt stress responses at the metabolite level. METHODS: Gas chromatography-mass spectrometry was used to profile metabolite changes in the salt-tolerant line FL478 and the sensitive variety IR64 under a salt-stress time series. Additionally, several physiological traits related to ST were investigated. RESULTS: We characterized 92 primary metabolites in the leaves and roots of the two genotypes under stress and control conditions. The metabolites were temporally, tissue-specifically and genotype-dependently regulated under salt stress. Sugars and amino acids (AAs) increased significantly in the leaves and roots of both genotypes, while organic acids (OAs) increased in roots and decreased in leaves. Compared with IR64, FL478 experienced greater increases in sugars and AAs and more pronounced decreases in OAs in both tissues; additionally, the maximum change in sugars and AAs occurred later, while OAs changed earlier. Moreover, less Na+ and higher relative water content were observed in FL478. Eleven metabolites, including AAs and sugars, were specifically increased in FL478 over the course of the treatment. CONCLUSIONS: Metabolic responses of rice to salt stress are dynamic and involve many metabolites. The greater ST of FL478 is due to different adaptive reactions at different stress times. At early salt-stress stages, FL478 adapts to stress by decreasing OA levels or by quickly depressing growth; during later stages, more metabolites are accumulated, thereby serving as compatible solutes against osmotic challenge induced by salt stress. PMID: 25265195 [PubMed - indexed for MEDLINE]

Related Articles A multienzyme complex channels substrates and electrons through acetyl-CoA and methane biosynthesis pathways in Methanosarcina. PLoS One. 2014;9(9):e107563 Authors: Lieber DJ, Catlett J, Madayiputhiya N, Nandakumar R, Lopez MM, Metcalf WW, Buan NR Abstract Multienzyme complexes catalyze important metabolic reactions in many organisms, but little is known about the complexes involved in biological methane production (methanogenesis). A crosslinking-mass spectrometry (XL-MS) strategy was employed to identify proteins associated with coenzyme M-coenzyme B heterodisulfide reductase (Hdr), an essential enzyme in all methane-producing archaea (methanogens). In Methanosarcina acetivorans, Hdr forms a multienzyme complex with acetyl-CoA decarbonylase synthase (ACDS), and F420-dependent methylene-H4MPT reductase (Mer). ACDS is essential for production of acetyl-CoA during growth on methanol, or for methanogenesis from acetate, whereas Mer is essential for methanogenesis from all substrates. Existence of a Hdr:ACDS:Mer complex is consistent with growth phenotypes of ACDS and Mer mutant strains in which the complex samples the redox status of electron carriers and directs carbon flux to acetyl-CoA or methanogenesis. We propose the Hdr:ACDS:Mer complex comprises a special class of multienzyme redox complex which functions as a "biological router" that physically links methanogenesis and acetyl-CoA biosynthesis pathways. PMID: 25232733 [PubMed - indexed for MEDLINE]

Related Articles Allele compensation in tip60+/- mice rescues white adipose tissue function in vivo. PLoS One. 2014;9(5):e98343 Authors: Gao Y, Hamers N, Rakhshandehroo M, Berger R, Lough J, Kalkhoven E Abstract Adipose tissue is a key regulator of energy homestasis. The amount of adipose tissue is largely determined by adipocyte differentiation (adipogenesis), a process that is regulated by the concerted actions of multiple transcription factors and cofactors. Based on in vitro studies in murine 3T3-L1 preadipocytes and human primary preadipocytes, the transcriptional cofactor and acetyltransferase Tip60 was recently identified as an essential adipogenic factor. We therefore investigated the role of Tip60 on adipocyte differentiation and function, and possible consequences on energy homeostasis, in vivo. Because homozygous inactivation results in early embryonic lethality, Tip60+/- mice were used. Heterozygous inactivation of Tip60 had no effect on body weight, despite slightly higher food intake by Tip60+/- mice. No major effects of heterozygous inactivation of Tip60 were observed on adipose tissue and liver, and Tip60+/- displayed normal glucose tolerance, both on a low fat and a high fat diet. While Tip60 mRNA was reduced to 50% in adipose tissue, the protein levels were unaltered, suggesting compensation by the intact allele. These findings indicate that the in vivo role of Tip60 in adipocyte differentiation and function cannot be properly addressed in Tip60+/- mice, but requires the generation of adipose tissue-specific knock out animals or specific knock-in mice. PMID: 24870614 [PubMed - indexed for MEDLINE]

Related Articles The potential biomarker panels for identification of Major Depressive Disorder (MDD) patients with and without early life stress (ELS) by metabonomic analysis. PLoS One. 2014;9(5):e97479 Authors: Ding X, Yang S, Li W, Liu Y, Li Z, Zhang Y, Li L, Liu S Abstract OBJECTIVE: The lack of the disease biomarker to support objective laboratory tests still constitutes a bottleneck in the clinical diagnosis and evaluation of major depressive disorder (MDD) and its subtypes. We used metabonomic techniques to screen the diagnostic biomarker panels from the plasma of MDD patients with and without early life stress (ELS) experience. METHODS: Plasma samples were collected from 25 healthy adults and 46 patients with MDD, including 23 patients with ELS and 23 patients without ELS. Furthermore, gas chromatography/mass spectrometry (GC/MS) coupled with multivariate statistical analysis was used to identify the differences in global plasma metabolites among the 3 groups. RESULTS: The distinctive metabolic profiles exist either between healthy subjects and MDD patients or between the MDD patients with ELS experience (ELS/MDD patients) and the MDD patients without it (non-ELS/MDD patients), and some diagnostic panels of feature metabolites' combination have higher predictive potential than the diagnostic panels of differential metabolites. CONCLUSIONS: These findings in this study have high potential of being used as novel laboratory diagnostic tool for MDD patients and it with ELS or not in clinical application. PMID: 24870353 [PubMed - indexed for MEDLINE]

Related Articles Combined systems approaches reveal highly plastic responses to antimicrobial peptide challenge in Escherichia coli. PLoS Pathog. 2014 May;10(5):e1004104 Authors: Kozlowska J, Vermeer LS, Rogers GB, Rehnnuma N, Amos SB, Koller G, McArthur M, Bruce KD, Mason AJ Abstract Obtaining an in-depth understanding of the arms races between peptides comprising the innate immune response and bacterial pathogens is of fundamental interest and will inform the development of new antibacterial therapeutics. We investigated whether a whole organism view of antimicrobial peptide (AMP) challenge on Escherichia coli would provide a suitably sophisticated bacterial perspective on AMP mechanism of action. Selecting structurally and physically related AMPs but with expected differences in bactericidal strategy, we monitored changes in bacterial metabolomes, morphological features and gene expression following AMP challenge at sub-lethal concentrations. For each technique, the vast majority of changes were specific to each AMP, with such a plastic response indicating E. coli is highly capable of discriminating between specific antibiotic challenges. Analysis of the ontological profiles generated from the transcriptomic analyses suggests this approach can accurately predict the antibacterial mode of action, providing a fresh, novel perspective for previous functional and biophysical studies. PMID: 24789011 [PubMed - indexed for MEDLINE]

Related Articles Systems medicine: a new approach to clinical practice. Arch Bronconeumol. 2014 Oct;50(10):444-51 Authors: Cardinal-Fernández P, Nin N, Ruíz-Cabello J, Lorente JA Abstract Most respiratory diseases are considered complex diseases as their susceptibility and outcomes are determined by the interaction between host-dependent factors (genetic factors, comorbidities, etc.) and environmental factors (exposure to microorganisms or allergens, treatments received, etc.) The reductionist approach in the study of diseases has been of fundamental importance for the understanding of the different components of a system. Systems biology or systems medicine is a complementary approach aimed at analyzing the interactions between the different components within one organizational level (genome, transcriptome, proteome), and then between the different levels. Systems medicine is currently used for the interpretation and understanding of the pathogenesis and pathophysiology of different diseases, biomarker discovery, design of innovative therapeutic targets, and the drawing up of computational models for different biological processes. In this review we discuss the most relevant concepts of the theory underlying systems medicine, as well as its applications in the various biological processes in humans. PMID: 24397963 [PubMed - indexed for MEDLINE]

Metabolome-scale de novo pathway reconstruction using regioisomer-sensitive graph alignments. Bioinformatics. 2015 Jun 15;31(12):i161-i170 Authors: Yamanishi Y, Tabei Y, Kotera M Abstract MOTIVATION: Recent advances in mass spectrometry and related metabolomics technologies have enabled the rapid and comprehensive analysis of numerous metabolites. However, biosynthetic and biodegradation pathways are only known for a small portion of metabolites, with most metabolic pathways remaining uncharacterized. RESULTS: In this study, we developed a novel method for supervised de novo metabolic pathway reconstruction with an improved graph alignment-based approach in the reaction-filling framework. We proposed a novel chemical graph alignment algorithm, which we called PACHA (Pairwise Chemical Aligner), to detect the regioisomer-sensitive connectivities between the aligned substructures of two compounds. Unlike other existing graph alignment methods, PACHA can efficiently detect only one common subgraph between two compounds. Our results show that the proposed method outperforms previous descriptor-based methods or existing graph alignment-based methods in the enzymatic reaction-likeness prediction for isomer-enriched reactions. It is also useful for reaction annotation that assigns potential reaction characteristics such as EC (Enzyme Commission) numbers and PIERO (Enzymatic Reaction Ontology for Partial Information) terms to substrate-product pairs. Finally, we conducted a comprehensive enzymatic reaction-likeness prediction for all possible uncharacterized compound pairs, suggesting potential metabolic pathways for newly predicted substrate-product pairs. CONTACT: maskot@bio.titech.ac.jp. PMID: 26072478 [PubMed - as supplied by publisher]

The Caenorhabditis elegans lipidome A primer for lipid analysis in Caenorhabditis elegans. Arch Biochem Biophys. 2015 Jun 10; Authors: Witting M, Schmitt-Kopplin P Abstract Lipids play important roles in biology, ranging from building blocks of membranes to signaling lipids. The nematode and model organism Caenorhabditis elegans has been used to explore lipid metabolism and several techniques for their analysis have been employed. These techniques include different possibilities ranging from visualization of lipid droplets, analysis of total fatty acids to analysis of complex lipids using lipidomics approaches. Lipidomics evolved from metabolomics, the latest off-spring of the "omics"-technologies and aims to characterize the lipid content of a given organism or system. Although being an extensively studied model organism, only a few applications of lipidomics to C. elegans have been reported to far, but the number is steadily increasing with more applications expected in the near future. This review gives an overview on the C. elegans lipidome, lipid classes it contains and ways to analyze them. It serves as primer for scientists interested in studying lipids in this model organism and list methods used so far and what information can be derived from them. Lastly, challenges and future (methodological) research directions, together with new methods potentially useful for C. elegans lipid research are discussed. PMID: 26072113 [PubMed - as supplied by publisher]

Related Articles Multi-level systems biology modeling characterized the atheroprotective efficiencies of modified dairy fats in a hamster model. Am J Physiol Heart Circ Physiol. 2015 Jun 12;:ajpheart.00032.2015 Authors: Martin JC, Berton A, Ginies C, Bott R, Scheercousse P, Saddi A, Gripois D, Landrier JF, Dalemans D, Alessi MC, Delplanque B Abstract We assessed the atheroprotective efficiency of modified dairy fats in hyperlipidemic hamsters. A systems biology approach was implemented to reveal and quantify the dietary fat-related components of the disease. Three modified dairy fats (40% energy) were prepared from regular butter by mixing with a plant oil mixture, by removing cholesterol alone or by removing cholesterol in combination with reducing saturated fatty acids. A plant oil mixture with regular butter was used as the control diet. The atherosclerosis severity (aortic cholesteryl-ester level) was higher in the regular butter-fed hamsters than in the other four groups (P < 0.05). Eighty-seven of the 1666 variables measured from multi-platform analysis were found to be strongly associated with the disease. When aggregated into 10 biological clusters combined into a multivariate predictive equation, these 87 variables explained 81% of the disease variability. The biological cluster "regulation of lipid transport and metabolism" appeared central to atherogenic development relative to diets. The "vitamin E metabolism" cluster was the main driver of atheroprotection with the best performing transformed dairy fat. Under conditions that promote atherosclerosis, the impact of dairy fats on atherogenesis could be greatly ameliorated by technological modifications. Our modeling approach allowed for identifying and quantifying the contribution of complex factors to atherogenic development in each dietary setup. PMID: 26071539 [PubMed - as supplied by publisher]

Related Articles Phenolic amides are potent inhibitors of de novo nucleotide biosynthesis. Appl Environ Microbiol. 2015 Jun 12; Authors: Pisithkul T, Jacobson TB, O'Brien TJ, Stevenson DM, Amador-Noguez D Abstract An outstanding challenge towards efficient production of biofuels and value-added chemicals from plant biomass is the impact that lignocellulose-derived inhibitors have on microbial fermentations. Elucidating the mechanisms that underlie their toxicity is critical for developing strategies to overcome them. Here, using E. coli as a model system, we investigated the metabolic effects and toxicity mechanisms of feruloyl and coumaroyl amide, the predominant phenolic compounds in ammonia-pretreated biomass hydrolysates. Using metabolomics, isotope tracers, and biochemical assays, we show that these two phenolic amides act as potent and fast-acting inhibitors of purine and pyrimidine biosynthetic pathways. Feruloyl or coumaroyl amide exposure leads to: 1) rapid buildup of 5-phosphoribosylpyrophosphate (PRPP), a key precursor in nucleotide biosynthesis; 2) rapid decrease in the levels of pyrimidine biosynthetic intermediates; and 3) long-term generalized decrease in nucleotide and deoxynucleotide levels. Tracer experiments using (13)C-sugars and (15)N-ammonia demonstrated that carbon and nitrogen flux into nucleotides and deoxynucleotides are inhibited by these phenolic amides. We found that these effects are mediated via direct inhibition of glutamine amidotransferases that participate in nucleotide biosynthetic pathways. In particular, feruloyl amide is a competitive inhibitor of glutamine PRPP amidotransferase (PurF), which catalyzes the first committed step in de novo purine biosynthesis. Finally, external nucleoside supplementation prevents phenolic amide-mediated growth inhibition by allowing nucleotide biosynthesis via salvage pathways. The results presented here will help develop strategies to overcome toxicity of phenolic compounds and facilitate engineering of more efficient microbial producers of biofuels and chemicals. PMID: 26070680 [PubMed - as supplied by publisher]

Related Articles Compartmentation of metabolism of the C12-, C9-, and C5-n-dicarboxylates in rat liver, investigated by mass isotopomer analysis: Anaplerosis from dodecanedioate. J Biol Chem. 2015 Jun 12; Authors: Jin Z, Bian F, Tomcik K, Kelleher JK, Zhang GF, Brunengraber H Abstract We investigated the compartmentation of the catabolism of dodecanedioate (DODA), azelate and glutarate in perfused rat livers, using a combination of metabolomics and mass isotopomer analyses. Livers were perfused with recirculating or nonrecirculating buffer containing one fully (13)C-labeled dicarboxylate. Information on the peroxisomal vs mitochondrial catabolism was gathered from the labeling patterns of acetyl-CoA proxies, i.e., total acetyl-CoA, the acetyl moiety of citrate, C-1+2 of β-hydroxybutyrate, malonyl-CoA and acetylcarnitine. Additional information was obtained from the labeling patterns of citric acid cycle intermediates and related compounds. The data characterize the partial oxidation of DODA and azelate in peroxisomes, with terminal oxidation in mitochondria. We did not find evidence of peroxisomal oxidation of glutarate. Unexpectedly, DODA contributes a substantial fraction to anaplerosis of the citric acid cycle. This opens the possibility to use water-soluble DODA in nutritional or pharmacological anaplerotic therapy when other anaplerotic substrates are impractical or contraindicated, e.g., in propionic acidemia and methylmalonic acidemia. PMID: 26070565 [PubMed - as supplied by publisher]

17 new pubmed citations were retrieved for your search. Click on the search hyperlink below to display the complete search results: metabolomics These pubmed results were generated on 2015/06/13PubMed comprises more than 24 million citations for biomedical literature from MEDLINE, life science journals, and online books. Citations may include links to full-text content from PubMed Central and publisher web sites.

Related Articles p53 attenuates AKT signaling by modulating membrane phospholipid composition. Oncotarget. 2015 Jun 3; Authors: Rueda-Rincon N, Bloch K, Derua R, Vyas R, Harms A, Hankemeier T, Khan NA, Dehairs J, Bagadi M, Binda MM, Waelkens E, Marine JC, Swinnen JV Abstract The p53 tumor suppressor is the central component of a complex network of signaling pathways that protect organisms against the propagation of cells carrying oncogenic mutations. Here we report a previously unrecognized role of p53 in membrane phospholipids composition. By repressing the expression of stearoyl-CoA desaturase 1, SCD, the enzyme that converts saturated to mono-unsaturated fatty acids, p53 causes a shift in the content of phospholipids with mono-unsaturated acyl chains towards more saturated phospholipid species, particularly of the phosphatidylinositol headgroup class. This shift affects levels of phosphatidylinositol phosphates, attenuates the oncogenic AKT pathway, and contributes to the p53-mediated control of cell survival. These findings expand the p53 network to phospholipid metabolism and uncover a new molecular pathway connecting p53 to AKT signaling. PMID: 26061814 [PubMed - as supplied by publisher]

Related Articles Pyrimidine Metabolism: Dynamic and Versatile Pathways in Pathogens and Cellular Development. J Genet Genomics. 2015 May 20;42(5):195-205 Authors: Garavito MF, Narváez-Ortiz HY, Zimmermann BH Abstract The importance of pyrimidines lies in the fact that they are structural components of a broad spectrum of key molecules that participate in diverse cellular functions, such as synthesis of DNA, RNA, lipids, and carbohydrates. Pyrimidine metabolism encompasses all enzymes involved in the synthesis, degradation, salvage, interconversion and transport of these molecules. In this review, we summarize recent publications that document how pyrimidine metabolism changes under a variety of conditions, including, when possible, those studies based on techniques of genomics, transcriptomics, proteomics, and metabolomics. First, we briefly look at the dynamics of pyrimidine metabolism during nonpathogenic cellular events. We then focus on changes that pathogen infections cause in the pyrimidine metabolism of their host. Next, we discuss the effects of antimetabolites and inhibitors, and finally we consider the consequences of genetic manipulations, such as knock-downs, knock-outs, and knock-ins, of pyrimidine enzymes on pyrimidine metabolism in the cell. PMID: 26059768 [PubMed - as supplied by publisher]

Related Articles Determination of the in vivo NAD/NADH ratio in S. cerevisiae under anaerobic conditions using alcohol dehydrogenase as sensor reaction. Yeast. 2015 Jun 9; Authors: Bekers KM, Heijnen JJ, van Gulik WM Abstract With the current quantitative metabolomics techniques only whole cell concentrations of NAD and NADH can be quantified. These measurements cannot provide information on the in vivo redox state of the cells, which is determined by the ratio of the free forms only. In this work we quantified the free NAD/NADH ratio in yeast under anaerobic conditions using alcohol dehydrogenase (ADH) and the lumped reaction of glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase as sensor reactions. We show that with an alternative accurate acetaldehyde determination method, based on rapid sampling, instantaneous derivatization with 2,4 diaminophenol hydrazine (DNPH) and quantification with HPLC, the ADH catalyzed oxidation of ethanol to acetaldehyde can be applied as a relatively fast and simple sensor reaction to quantify the free NAD/NADH ratio under anaerobic conditions. We evaluated the applicability of ADH as a sensor reaction in the yeast Saccharomyces cerevisiae grown in anaerobic glucose limited chemostats during steady state and dynamic conditions. The results found in this study show that the cytosolic redox status (NAD/NADH ratio) of yeast is at least one order of magnitude lower, and thus much more reduced, under anaerobic conditions compared to aerobic glucose-limited steady state conditions. The more reduced state of the cytosol under anaerobic conditions has major implications for (central) metabolism. Accurate determination of the free NAD/NADH ratio is therefore of importance for the unravelling of in vivo enzyme kinetics and to judge accurately the thermodynamic reversibility of each redox reaction. PMID: 26059529 [PubMed - as supplied by publisher]