Impact of Up- and Downregulation of Metabolites and Mitochondrial Content on pH and Color of the Longissimus Muscle from Normal-pH and Dark-Cutting Beef.

Limited knowledge is currently available on the biochemical basis for the development of dark-cutting beef. The objective of this research was to determine the metabolite profile and mitochondrial content differences between normal-pH and dark-cutting beef. A gas chromatography-mass spectrometer-based nontargeted metabolomic approach indicated downregulation of glycolytic metabolites, including glucose-1- and 6-phosphate and upregulation of tricarboxylic substrates such as malic and fumaric acids occurred in dark-cutting beef when compared to normal-pH beef. Neurotransmitters such as 4-aminobutyric acid and succinate semialdehyde were upregulated in dark-cutting beef than normal-pH beef. Immunohistochemistry indicated a more oxidative fiber type in dark-cutting beef than normal-pH beef. In support, the mitochondrial protein and DNA content were greater in dark-cutting beef. This increased mitochondrial content, in part, could influence oxygen consumption and myoglobin oxygenation/appearance of dark-cutting beef. The current results demonstrate that the more tricarboxylic metabolites and mitochondrial content in dark-cutting beef impact muscle pH and color.

Quantitation of Glucose-phosphate in Single Cells by Microwell-Based Nanoliter Droplet Microextraction and Mass Spectrometry.

Changes of metabolite concentrations in single cells are significant for exploring the dynamic regulation of important biological processes, such as cell development and differentiation. Accurate quantitation of metabolites is essential for single cell analysis. In this work, we proposed a quantitative method for single-cell metabolites by combining microwell array with droplet microextraction-mass spectrometry. The microwell can confine both single cells and extraction solvent in defined space, avoiding the irregular spread of trace internal standard solution during microextraction, which was the key to improve the precision and accuracy of quantification in extremely small-volume single-cell samples. Glucose-phosphate as a crucial metabolite in glycolysis was detected and quantified in single cells at this work. The calibration curve of glucose-phosphate was obtained with a linear range from amol (10-18 mol) to fmol (10-15 mol), providing the foundation of metabolite quantitation of single cells. We applied this method to investigate the changes of metabolites including glucose-phosphate, 2-deoxy-d-glucose-phosphate, and ribose-phosphate in single K562 cells stimulated by 2-deoxy-d-glucose. With the robust quantitative capabilities, the developed method holds great potential for studying a drugs' mechanism of action and resistance at single cell level.

Online Liquid Chromatography-Sheath-Flow Surface Enhanced Raman Detection of Phosphorylated Carbohydrates.

Online detection and quantification of three phosphorylated carbohydrate molecules: glucose 1-phosphate, glucose 6-phosphate, and fructose 6-phosphate was achieved by coupling sheath-flow surface enhanced Raman spectroscopy (SERS) to liquid chromatography. The presence of an alkanethiol (hexanethiol) self-assembled monolayer adsorbed to a silver SERS-active substrate helps retain and concentrate the analytes of interest at the SERS substrate to improve the detection sensitivity significantly. Mixtures of 2 µM of phosphorylated carbohydrates in pure water as well as in cell culture media were successfully separated by HPLC, with identification using the sheath-flow SERS detector. The quantification of each analyte was achieved using partial least-squares (PLS) regression analysis and acetonitrile in the mobile phases as an internal standard. These results illustrate the utility of sheath-flow SERS for molecular specific detection in complex biological samples appropriate for metabolomics and other applications.

Quantification of Low-Abundant Phosphorylated Carbohydrates Using HILIC-QqQ-MS/MS.

Phosphorylated carbohydrates are central metabolites involved in key plant metabolic pathways, such as glycolysis and central carbon metabolism. Such pathways influence plant growth, development, and stress responses to environmental changes, and ultimately, reflect the plant's energy status. The high polarity of these metabolites, the variety of isomeric structures (e.g., glucose-1-phosphate (G1P)/fructose-6-phosphate (F6P)/mannose-6-phosphate (M6P)/G6P, sucrose-6-phosphate (S6P)/T6P), and rapid metabolic turnover makes their analysis particularly challenging. In this chapter, we describe the use of a set of known phosphorylated carbohydrates to develop and validate a hydrophilic interaction chromatography (HILIC) triple quadrupole (QqQ) tandem mass spectrometry (MS/MS) method in the highly sensitive and selective multiple reaction monitoring (MRM) mode for the target analysis of G1P, F6P, M6P, G6P, S6P, T6P, and the sugar nucleotide uridine 5-diphospho-glucose (UDPG). We present detailed information regarding HILIC column chemistry and practical considerations when coupling it with a QqQ-MS system.

Meningococcal X polysaccharide quantification by high-performance anion-exchange chromatography using synthetic N-acetylglucosamine-4-phosphate as standard.

A method for meningococcal X (MenX) polysaccharide quantification by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) is described. The polysaccharide is hydrolyzed by strong acidic treatment, and the peak of glucosamine-4-phosphate (4P-GlcN) is detected and measured after chromatography. In the selected conditions of hydrolysis, 4P-GlcN is the prevalent species formed, with GlcN detected for less than 5% in moles. As standard for the analysis, the monomeric unit of MenX polysaccharide, N-acetylglucosamine-4-phosphate (4P-GlcNAc), was used. This method for MenX quantification is highly selective and sensitive, and it constitutes an important analytical tool for the development of a conjugate vaccine against MenX.

Purification, characterization, and gene cloning of glucose-1-phosphatase from Citrobacter braakii.

Citrobacter braakii produced an intracellular acid glucose phosphatase (AgpC) which was purified 986 fold to homogeneity with the specific activity of 286 units/mg. AgpC hydrolyzed a wide variety of phosphorylated compounds with high activity for glucose-1-phosphate and glucose-6-phosphate. The optimum pH and temperature for the enzyme activity was pH 5.0 and 45 degrees C, respectively. The Km value for glucose-1-phosphate was 5.12 mM with a Vmax 27.8 U mg(-1). Its molecular weight was 46 kDa by SDS-PAGE gel and the sequence of N-terminal amino acid residues identified was Gln-Thr-Ala-Pro-Glu-Gly-Tyr-Gln-Leu-Gln. The glucose-1-phosphatase gene (agpC) was cloned from the C. braakii genomic library. This gene comprised 1,242 nucleotides and encoded a polypeptide of 413 amino acids. The result of its BLAST search showed a significant similarity with glucose-1-phosphatase from enterobacteria such as E. coli, Enterobacter, Shigella, and Salmonella.

Lithium-mediated suppression of morphogenesis and growth in Candida albicans.

Hyphal development in Candida albicans contributes to virulence, and inhibition of filamentation is a target for the development of antifungal agents. Lithium is known to impair Saccharomyces cerevisiae growth in galactose-containing media by inhibition of phosphoglucomutase, which is essential for galactose metabolism. Lithium-mediated phosphoglucomutase inhibition is reverted by Mg(2+). In this study we have assessed the effect of lithium upon C. albicans and found that growth is inhibited preferentially in galactose-containing media. No accumulation of glucose-1-phosphate or galactose-1-phosphate was detected when yeasts were grown in the presence of galactose and 15 mM LiCl, though we observed that in vitro lithium-mediated phosphoglucomutase inhibition takes place with an IC(50) of 2 mM. Furthermore, growth inhibition by lithium was not reverted by Mg(2+). These results show that lithium-mediated inhibition of growth in a galactose-containing medium is not due to inhibition of galactose conversion to glucose-6-phosphate but is probably due to inhibition of a signaling pathway. Deletion of the Ser-Thr protein phosphatase SIT4 and treatment with rapamycin have been shown to inhibit filamentous differentiation. We observed that C. albicans filamentation was inhibited by lithium in solid medium containing either galactose as the sole carbon source or 10% fetal bovine serum. These results suggest that suppression of hyphal outgrowth by lithium could be related to inhibition of the target of rapamycin (TOR) pathway.

Revised procedures for yeast metabolites extraction: application to a glucose pulse to carbon-limited yeast cultures, which reveals a transient activation of the purine salvage pathway.

In this study we have revised our original procedure of yeast metabolites extraction. We showed that: (a) less than 5% of intracellular metabolites leaks out during the step of rapid arrest of cellular metabolism by quenching yeast cells into a 60% methanol solution kept at -40 degrees C; and (b) with a few exception, the stability of metabolites were not altered during the 3 min boiling procedure in a buffered ethanol solution. However, there was a loss of external added metabolites of 5-30%, depending on the type of metabolites. This was mainly attributable to their retention on cellular debris after ethanol treatment, which prevented centrifugation of the cellular extracts before evaporation of ethanol. We further simplified our previous high-performance ionic chromatography (HPIC) techniques for easier, more reliable and robust quantitative measurements of organic acids, sugar phosphates and sugar nucleotides, and extended these techniques to purine and pyrimidine bases, using a variable wavelength detector set at 220 and 260 nm in tandem with a pulsed electrochemical or suppressed conductivity detector. These protocols were successfully applied to a glucose pulse to carbon-limited yeast cultures on purines metabolism. This study showed that glucose induced a fast activation of the purine salvage pathway, as indicated by a transient drop of ATP and ADP with a concomitant rise of IMP and inosine. This metabolic perturbation was accompanied by a rapid increase in the activity of the ISN1-encoded specific IMP-5'-nucleotidase. The mechanism of this activation remains to be determined.

Relationship between glucose transport and metabolism in isolated bovine mammary epithelial cells.

Glucose transport by isolated bovine mammary epithelial cells involves translocation across the cell membrane into a compartment that exchanges slowly with the bulk cytosol. The significance to glucose metabolism of this compartmentalization was examined by generation, modeling, and analysis of transport and metabolism data. Net uptake of 5 mM 3-O-methyl-d-glucose by isolated bovine mammary epithelial cells was measured at 37 degrees C. Time-course curves were better fitted by a double exponential equation than a single exponential equation and were subjected to compartmental analysis to obtain glucose transport model parameters. Lactose synthesis and glucose oxidation rates and cellular concentrations of intermediary metabolites, glucose-6-phosphate and glucose-1-phosphate, were measured at varied media glucose concentrations. A model that integrates both glucose transport and metabolism under-predicted the rates of lactose synthesis and glucose oxidation by a factor of 3. To account for the observed glucose use rates, glucose must be available for phosphorylation once translocated across the cell membrane (intermediate compartmentalization of translocated glucose does not exclude access to hexokinase). Metabolic control analysis indicated that, at physiological glucose concentrations, phosphorylation by hexokinase exerts 80% of the control of glucose metabolism to lactose and CO(2), and transport exerts the remaining 20%.

Kinetic measurements of phosphoglucomutase by direct analysis of glucose-1-phosphate and glucose-6-phosphate using ion/molecule reactions and Fourier transform ion cyclotron resonance mass spectrometry.

A method for the direct determination of kinetic constants for phosphoglucomutase and its phosphorylated products is described. Fourier transform ion cyclotron resonance gas-phase ion/molecule reactions between trimethyl borate and glucose phosphate, phosphorylated at either the 1 or the 6 position, generate mass spectra distinguishable with regard to product ion distribution. A multicomponent quantification method is utilized to determine the composition of a binary mixture of the two positional isomers. Using this method, the conversion between glucose-1-phosphate and glucose-6-phosphate can be directly monitored without the use of coupling enzymes. The values of K(m) for glucose-1-phosphate and glucose-6-phosphate were determined using the substrate-velocity plot and the Haldane relationship, respectively. Values of V(max) for both the forward and the reverse directions were measured, and the equilibrium constant for the reversible reaction was determined using this methodology. Kinetic parameters measured correlate well with those obtained using traditional methods. The assay was demonstrated to be accurate and particularly convenient to determine kinetic constants for enzymatic systems that involve the interconversion of phosphorylated positional isomers.

Investigation of ion/molecule reactions as a quantification method for phosphorylated positional isomers. an FT-ICR approach.

A rapid and accurate method of quantifying positional isomeric mixtures of phosphorylated hexose and N-acetylhexosamine monosacchrides by using gas-phase ion/molecule reactions coupled with FT-ICR mass spectrometry is described. Trimethyl borate, the reagent gas, reacts readily with the singly charged negative ions of phosphorylated monosaccharides to form two stable product ions corresponding to the loss of one or two neutral molecules of methanol from the original adduct. Product distribution in the ion/molecule reaction spectra differs significantly for isomers phosphorylated in either the 1- or the 6-position. As a result, the percents of total ion current of these product ions for a mixture of the two isomers vary with its composition. In order to determine the percentage of each isomer in an unknown mixture, a multicomponent quantification method is utilized in which the percents of total ion current of the two product ions for each pure monosaccharide phosphate and the mixture are used in a two-equation, two-unknown system. The applicability of this method is demonstrated by successfully quantifying mock mixtures of four different isomeric pairs: Glucose-1-phosphate and glucose-6-phosphate; mannose-1-phosphate and mannose-6-phosphate; galactose-1-phosphate and galactose-6-phosphate; N-acetylglucosamine-1-phosphate and N-acetylglucosamine-6-phosphate. The effects of mixture concentrations and ion/molecule reaction conditions on the quantification are also discussed. Our results demonstrate that this assay is a fast, sensitive, and robust method to quantify isomeric mixtures of phosphorylated monosaccharides.

Thermal decomposition of specifically phosphorylated D-glucoses and their role in the control of the Maillard reaction.

One of the main shortcomings of the information available on the Maillard reaction is the lack of knowledge to control the different pathways, especially when it is desired to direct the reaction away from the formation of carcinogenic and other toxic substances to more aroma and color generation. The use of specifically phosphorylated sugars may impart some elements of control over the aroma profile generated by the Maillard reaction. Thermal decomposition of 1- and 6-phosphorylated glucoses was studied in the presence and absence of ammonia and selected amino acids through pyrolysis/gas chromatography/mass spectrometry using nonpolar PLOT and medium polar DB-1 columns. The analysis of the data has indicated that glucose-1-phosphate relative to glucose undergoes more extensive phosphate-catalyzed ring opening followed by formation of sugar-derived reactive intermediates as was indicated by a 9-fold increase in the amount of trimethylpyrazine and a 5-fold increase in the amount of 2,3-dimethylpyrazine, when pyrolyzed in the presence of glycine. In addition, glucose-1-phosphate alone generated a 6-fold excess of acetol as compared to glucose. On the other hand, glucose-6-phosphate enhanced retro-aldol reactions initiated from a C-6 hydroxyl group and increased the subsequent formation of furfural and 4-cyclopentene-1,3-dione. Furthermore, it also stabilized 1- and 3-deoxyglucosone intermediates and enhanced the formation of six carbon atom-containing Maillard products derived directly from them through elimination reactions such as 1,6-dimethyl-2,4-dihydroxy-3-(2H)-furanone (acetylformoin), 2-acetylpyrrole, 5-methylfurfural, 5-hydroxymethylfurfural, and 4-hydroxy-2,5-dimethyl-3-(2H)-furanone (Furaneol), due to the enhanced leaving group ability of the phosphate moiety at the C-6 carbon. However, Maillard products generated through the nucleophilic action of the C-6 hydroxyl group such as 2-acetylfuran and 2,3-dihydro-3,5-dihydroxy-4H-pyran-4-one were retarded, due to the blocked nucleophilic atom at C-6.

Analysis of phosphate position in hexose monosaccharides using ion-molecule reactions and SORI-CID on an FT-ICR mass spectrometer.

Through the use of ion-molecule reactions and SORI-CID, the phosphate position in hexose phosphate monosaccharides has been determined in the negative ion mode. Trimethyl borate was used as a reagent gas and was found to react readily with the phosphorylated hexose monosaccharides. After reaction of the reagent gas with the hexose phosphate, ion activation of the precursor by SORI-CID yielded different MS/MS spectra. Different diagnostic ions were generated for the two isomers, thus enabling differentiation and linkage position determination of the phosphate moiety.

High-performance thin-layer chromatography method for inositol phosphate analysis.

A simple and inexpensive high-performance thin-layer chromatography (HPTLC) method for the analysis of inositol mono- to hexakisphosphates on cellulose precoated plates is described. Plates were developed in 1-propanol-25% ammonia solution-water (5:4:1) and substance quantities as low as 100-200 pmol were detected by molybdate staining. Chromatographic mobilities of nucleotides and phosphorylated carbohydrates were also characterized. Charcoal treatment was employed to separate nucleotides from inositol phosphates with similar R(F) values prior to HPTLC analysis. Practical application of the HPTLC system is demonstrated by analysis of grain extracts from wild type and low-phytate mutant barley as well as phytate degradation products resulting from barley phytase activity.

Simultaneous determination of glucosamine and glucosamine 4-phosphate in Lipid A with high-performance anion-exchange chromatography (HPAEC).

A method for simultaneous determination of glucosamine (GlcN) and glucosamine 4-phosphate (GlcN-4-P) in Lipid A with high-performance anion-exchange chromatography (HPAEC) is described. Lipid A is hydrolyzed with 4 M HCl for 16 h at 100 degrees C, and the peaks of glucosamine and glucosamine 4-phosphate were measured. The true GlcN value can be computed from the GlcN value after correction for the incomplete hydrolysis of GlcN-4-P, or by the combined yield of GlcN and GlcN-4-P.

Growth dependence of alpha-glucan phosphorylase activity in Thermus thermophilus.

Glycogen from the thermophilic eubacterium Thermus thermophilus has been characterized by enzymatic, chemical and spectroscopic analysis. With an average chain length of seven glucose units, the glycogen from T. thermophilus is one of the most highly branched glycogens known. In contrast to other bacterial species, in T. thermophilus, accumulation of glycogen appears not be affected by low nitrogen concentration. For the first time, alpha-glucan phosphorylase activity and glycogen content were measured throughout the growth cycle of T. thermophilus in order to gain insight into glycogen metabolism. In contrast to the situation that prevails in Escherichia coli, additional carbon sources had no effect on alpha-glucan phosphorylase activity in T. thermophilus. Maximal activity of the thermophilic enzyme was found in the early logarithmic phase of growth, suggesting a function of the alpha-glucan phosphorylase in T. thermophilus as an outgrowth-specific enzyme.

Luminometric measurement of subnanomole amounts of key metabolites in extracts from isolated heart muscle cells.

In principle, luminometry allows very sensitive metabolite measurements as shown with standards in aqueous solutions (e.g., buffers). However, components of complex biological samples may largely interfere with luminometric reactions. We now describe a procedure by which subnanomole amounts of intermediary metabolites (malate, glucose 6-phosphate) can be measured by luminometry in extracts from isolated mammalian cells, namely rat heart muscle cells. Basically, measurements occur in two steps: (i) Enzymatically catalyzed reactions involving the metabolite to be measured lead to the stoichiometric production of NAD(P)H; (ii) the oxidation of this NAD(P)H in a luciferase/reductase system results in light production which is proportional to the original concentration of the metabolite. The reaction scheme is thus as follows: (1) Metabolite (malate, glucose 6-phosphate) + NAD(P)+ --> X + NAD(P)H + H+; (2) NAD(P)H + O2 + RCOH --> NAD(P)+ + RCOOH + H2O + hnu. The cardiomyocytes used are previously subjected to an ethanolic extraction in which the cellular NAD(P)H is destroyed by acidification. Subsequent evaporation of the extracts allows to neutralize and to concentrate the samples. This contributes, along with other experimental maneuvers, to increasing the sensitivity of the method. With this procedure, we were able to detect amounts of approximately 70 pmol of malate and approximately 90 pmol of glucose 6-phosphate in cardiomyocyte samples. In addition, the calculated cellular concentrations of malate and glucose 6-phosphate (101.1 +/- 4.5, and 202.8 +/- 26.1 microM, respectively, in the absence of exogenous substrate) correspond to values previously reported for heart tissue. In principle, the procedure described could be applied to the measurement of any ethanol-extractable metabolite that can be converted in reactions involving NAD(P)+.

Transcriptional glucose signaling through the glucose response element is mediated by the pentose phosphate pathway.

Glucose catabolism induces the expression of the L-type pyruvate kinase (L-PK) gene through the glucose response element (GIRE). The metabolic pathway used by glucose after its phosphorylation to glucose 6-phosphate by glucokinase to induce L-PK gene expression in hepatocytes remains unknown. The sugar alcohol xylitol is metabolized to xylulose 5-phosphate, an intermediate of the nonoxidative branch of the pentose phosphate pathway. In this study, we demonstrated that xylitol at low concentration (O.5 mM) induced the expression of the L-PK/CAT construct in glucose-responsive mhAT3F hepatoma cells at the same level as 20 mM glucose, while it did not affect intracellular concentration of glucose 6-phosphate significantly. The effect of xylitol on the induction of the L-PK gene expression was noncumulative with that of glucose since 20 mM glucose plus 5 mM xylitol induced the expression of the L-PK/CAT construct similarly to 20 mM glucose alone. In hepatocytes in primary culture, 5 mM xylitol induced accumulation of the L-PK mRNA even in the absence of insulin. Furthermore, the response to xylitol as well as glucose required the presence of a functional GIRE. It can be assumed from these results that glucose induces the expression of the L-PK gene through the nonoxidative branch of the pentose phosphate pathway. The effect of xylitol at low concentration suggests that the glucose signal to the transcriptional machinery is mediated by xylulose 5-phosphate.

Phosphorylating enzymes involved in glucose fermentation of Actinomyces naeslundii.

Enzymatic activities involved in glucose fermentation of Actinomyces naeslundii were studied with glucose-grown cells from batch cultures. Glucose could be phosphorylated to glucose 6-phosphate by a glucokinase that utilized polyphosphate and GTP instead of ATP as a phosphoryl donor. Glucose 6-phosphate was further metabolized to the end products lactate, formate, acetate, and succinate through the Embden-Meyerhof-Parnas pathway. The phosphoryl donor for phosphofructokinase was only PPi. Phosphoglycerate kinase, pyruvate kinase, and acetate kinase coupled GDP as well as ADP, but P(i) compounds were not their phosphoryl acceptor. Cell extracts showed GDP-dependent activity of phosphoenolpyruvate carboxykinase, which assimilates bicarbonate and phosphoenolpyruvate into oxaloacetate, a precursor of succinate. Considerable amounts of GTP, polyphosphate, and PPi were found in glucose-fermenting cells, indicating that these compounds may serve as phosphoryl donors or acceptors in Actinomyces cells. PPi could be generated from UTP and glucose 1-phosphate through catalysis of UDP-glucose synthase, which provides UDP-glucose, a precursor of glycogen.