Simplest Chemosensor Array for Phosphorylated Saccharides.

We believe that "the simpler we are, the more complete we become" is a key concept of chemical sensing systems. In this work, a "turn-on" fluorescence chemosensor array relying on only two self-assembled molecular chemosensors with ability of both qualitative and quantitative detection of phosphorylated saccharides has been developed. The easy-to-prepare chemosensor array was fabricated by in situ mixing of off-the-shelf reagents (esculetin, 4-methylesculetin, and 3-nitrophenylboronic acid). The fluorescence-based saccharide sensing system was carried out using indicator displacement assay accompanied by photoinduced electron transfer (PeT) under various pH conditions. The simultaneous recognition of 14 types of saccharides including glucose-6-phosphate (G6P) and fructose-6-phosphate (F6P) was achieved with a successful classification rate of 100%. We also succeeded in the quantitative analysis of a mixture of glucose (Glc), as an original substrate, G6P and F6P, as enzymatic products in pseudoglycolysis pathway. Finally, levels of Glc and F6P in human induced pluripotent stem (hiPS) cells were indirectly monitored by using our proposed chemosensor array. Glc and F6P in supernatants of hiPS cells were classified by linear discriminant analysis as a pattern recognition model and the observed clusters represent the activity of hiPS cells. The results show the high accuracy of the proposed chemosensor array in detection of phosphorylated and similarly modified saccharides.

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.

Evaluation of synthase and hemisynthase activities of glucosamine-6-phosphate synthase by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Glucosamine-6-phosphate synthase (GlmS, EC 2.6.1.16) catalyzes the first and rate-limiting step in the hexosamine biosynthetic pathway, leading to the synthesis of uridine-5'-diphospho-N-acetyl-D-glucosamine, the major building block for the edification of peptidoglycan in bacteria, chitin in fungi, and glycoproteins in mammals. This bisubstrate enzyme converts D-fructose-6-phosphate (Fru-6P) and L-glutamine (Gln) into D-glucosamine-6-phosphate (GlcN-6P) and L-glutamate (Glu), respectively. We previously demonstrated that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) allows determination of the kinetic parameters of the synthase activity. We propose here to refine the experimental protocol to quantify Glu and GlcN-6P, allowing determination of both hemisynthase and synthase parameters from a single assay kinetic experiment, while avoiding interferences encountered in other assays. It is the first time that MALDI-MS is used to survey the activity of a bisubstrate enzyme.

Metabolomic profile of diabetic retinopathy: a GC-TOFMS-based approach using vitreous and aqueous humor.

AIM: To identify the potential metabolite markers in diabetic retinopathy (DR) by using gas chromatography coupled with time-of-flight mass spectrometry (GC-TOFMS). METHODS: GC-TOFMS spectra were acquired from vitreous and aqueous humor (AH) samples of patients with DR and non-diabetic participants. Comparative analysis was used to elucidate the distinct metabolites of DR. Metabolic pathway was employed to explicate the metabolic reprogramming pathways involved in DR. Logistic regression and receiver-operating characteristic analyses were carried out to select and validate the biomarker metabolites and establish a therapeutic model. RESULTS: Comparative analysis showed a clear separation between disease and control groups. Eight differentiating metabolites from AH and 15 differentiating metabolites from vitreous were highlighted. Out of these 23 metabolites, 11 novel metabolites have not been detected previously. Pathway analysis identified nine pathways (three in AH and six in vitreous) as the major disturbed pathways associated with DR. The abnormal of gluconeogenesis, ascorbate-aldarate metabolism, valine-leucine-isoleucine biosynthesis, and arginine-proline metabolism might weigh the most in the development of DR. The AUC of the logistic regression model established by D-2,3-Dihydroxypropanoic acid, isocitric acid, fructose 6-phosphate, and L-Lactic acid in AH was 0.965. The AUC established by pyroglutamic acid and pyruvic acid in vitreous was 0.951. CONCLUSIONS: These findings have expanded our understanding of identified metabolites and revealed for the first time some novel metabolites in DR. These results may provide useful information to explore the mechanism and may eventually allow the development of metabolic biomarkers for prognosis and novel therapeutic strategies for the management of DR.

Liquid chromatography/tandem mass spectrometry of glycolytic intermediates: deconvolution of coeluting structural isomers based on unique product ion ratios.

A method has been developed for rapid quantification of nine glycolytic intermediates using ultraperformance liquid chromatography/electrospray-tandem mass spectrometry (UPLC/ESI-MS/MS) to monitor the metabolism of glucose during microbial fermentation. Because comprehensive chromatographic separation is not required, analysis time is significantly less than traditional ion exchange liquid chromatography assays or enzymatic assays. Complete glycolytic intermediate analysis by LC/MS/MS can be achieved in less than 7 min per sample. Quantification is accomplished using isotopically labeled glucose, glucose-6-phosphate, and pyruvate as internal standards. In addition, a method to deconvolute peak areas of coeluting structural isomers based on unique product ion ratios has been developed to allow accurate quantification of the individual isomers 2-phosphoglycerate and 3-phosphoglycerate, as well as glucose-6-phosphate and fructose-6-phosphate. Intrasample precisions for glycolytic intermediate measurements in cell-free extracts using this method vary between 0.9% and 11.8%, averaging 3.5% (RSD). Calibration curves are linear over the range 0.1-100 microg/mL, and detection limits are estimated at 2-49 ng/mL. Spike recoveries in cell extracts vary from 53% to 127% averaging 91%. This method has the potential to demonstrate correlation of glycolytic intermediate flux to microbial production profiles toward acceleration of the bioprocess development cycle.

Identification of fructose 3-phosphate in the lens of diabetic rats.

Fructose 3-phosphate, a novel monosaccharide phosphate, has been identified in the lens of diabetic rats. This compound, which is not present in normal lenses, is a protein glycosylating agent and enzyme inactivator. In addition, because of its structural features, this metabolite is relatively labile and undergoes hydrolysis to yield inorganic phosphate and the potent glycosylating agent, 3-deoxyglucosone. The increase in the concentration of fructose 3-phosphate in the lens of diabetic rats suggests that it and its hydrolysis product, 3-deoxyglucosone, may be responsible in part for the development of some diabetic complications in the lens.

Phenylketonuria: phenylalanine inhibits brain pyruvate kinase in vivo.

The hypothesis that brain damage in phenylketonuria is related to inhibition of pyruvate kinase by phenylalanine was examined in rat brain in vivo. One hour after a single injection of phenylalanine into the rat, the brains were removed and completely frozen in less than a second. The concentration of phenylalanine in the brain was comparable to that found in phenylketonuric patients. Changes in brain glycolytic intermediates were consistent with inhibition of pyruvate kinase in vivo. The inhibition of pyruvate kinase was apparently compensated for by an increase in phosphoenolpyruvate; no decrease in adenosine triphosphate or creatine phosphate was found.

Determination of the cytosolic free NAD/NADH ratio in Saccharomyces cerevisiae under steady-state and highly dynamic conditions.

The coenzyme NAD plays a major role in metabolism as a key redox carrier and signaling molecule but current measurement techniques cannot distinguish between different compartment pools, between free and protein-bound forms and/or between NAD(H) and NADP(H). Local free NAD/NADH ratios can be determined from product/substrate ratios of suitable near-equilibrium redox reactions but the application of this principle is often precluded by uncertainties regarding enzyme activity, localization and coenzyme specificity of dehydrogenases. In Saccharomyces cerevisiae, we circumvented these issues by expressing a bacterial mannitol-1-phosphate 5-dehydrogenase and determining the cytosolic free NAD/NADH ratio from the measured [fructose-6-phosphate]/[mannitol-1-phosphate] ratio. Under aerobic glucose-limited conditions we estimated a cytosolic free NAD/NADH ratio between 101(+/-14) and 320(+/-45), assuming the cytosolic pH is between 7.0 and 6.5, respectively. These values are more than 10-fold higher than the measured whole-cell total NAD/NADH ratio of 7.5(+/-2.5). Using a thermodynamic analysis of central glycolysis we demonstrate that the former are thermodynamically feasible, while the latter is not. Furthermore, we applied this novel system to study the short-term metabolic responses to perturbations. We found that the cytosolic free NAD-NADH couple became more reduced rapidly (timescale of seconds) upon a pulse of glucose (electron-donor) and that this could be reversed by the addition of acetaldehyde (electron-acceptor). In addition, these dynamics occurred without significant changes in whole-cell total NAD and NADH. This approach provides a new experimental tool for quantitative physiology and opens new possibilities in the study of energy and redox metabolism in S. cerevisiae. The same strategy should also be applicable to other microorganisms.

Metabolic alterations in the human erythrocyte produced by increases in glucose concentration. The role of the polyol pathway.

Human erythrocytes incubated in medium containing 50 mM glucose have increased intracellular sorbitol and fructose concentrations as compared with samples incubated with 5 mM glucose. Increased medium glucose concentration did not significantly alter total glucose consumption or lactate production. However, the intracellular lactate:pyruvate ratio rose, the concentrations of fructose diphosphate, and triose phosphates increased, and the 2,3-diphosphoglycerate concentration fell. [(14)C]O(2) production from glucose-1-(14)C also increased with increased medium glucose concentration. These changes are believed to reflect changes in the redox states of the diphosphopyridine nucleotide/reduced form of diphosphopyridine nucleotide (NAD/NADH) and nicotinamide-adenine dinucleotide phosphate/reduced form of nicotinamide-adenine dinucleotide phosphate (NADP/NADPH) couples resulting from increased activity of the polyol pathway. Addition of pyruvate to the incubation media prevented these changes. These studies illustrate that an increase in the red cell's normal substrate, glucose, can produce changes in red cell metabolism.

Fructose metabolizing enzymes from mouse liver: influence of age and caloric restriction.

The influence of caloric restriction (CR) on the activities of liver fructose metabolizing enzymes and metabolite levels were studied in young (3 months) and old (30 months) mice. Fructokinase activity was increased (P<0.05) in both young and old CR mice when compared to controls while triokinase activity was increased (P<0.05) only in old CR versus control mice. Aldolase was not altered by CR in either old or young mice. No age-related differences in activities were observed in controls although a trend towards an increase was observed for triokinase, while significant age-related increases were observed for fructokinase and triokinase, but not aldolase, in CR mice. Both young and old mice on CR showed significant decreases in fructose and fructose-1-phosphate, however, no age-related changes in metabolite levels were observed for either control or CR mice. A fructose-1-phosphate kinase activity was also measured and found to be unchanged in both young and old mice on CR, but the activity was significantly lower in the old mice compared with young. We show here that the enzymes involved in fructose metabolism are influenced by CR and that this could contribute to alterations in gluconeogenesis and glycolysis observed with CR.

Effects of glucose on insulin release and on intermediary metabolism of isolated perifused pancreatic islets from fed and fasted rats.

We examined the relationship between glucose-induced insulin release and the intermediary metabolism of islets from fed and fasted rats. Isolated islets were perifused and insulin release measured in the effluent. At various times after switching islets from 2.4 to 8.6 or 14.5 mM glucose or from 2.4 to 14.5 and back to 2.4 mM glucose, islets were quickly frozen, freeze dried, and subsequently analyzed for tissue content of glucose-6-P, fructose-1,6-P2 plus triose-P, Pi, ATP, ADP, 5'-AMP, NADH, NADPH, total NAD, and total NADP using enzymatic fluorometric procedures. When islets from fed rats were exposed to high glucose, there were concomitant increases of insulin release and islet content of glucose-6-P, fructose-1,6-P2 plus triose-P, NADH, and NADPH. During stimulation Pi and 5'-AMP content fell markedly. The total adenine nucleotide content remained constant. Similar secretory and metabolic changes occurred when 1.5 mM Pi was added to the perifusion fluid. When glucose-stimulated islets were switched back to low glucose for 10 min, all substances but fructose-1,6-P2 plus triose-P, 5'-AMP, NADPH, and possibly ATP returned to the prestimulatory level. Starvation of rats for 3 days blocked the secretory response to 8.6 mM glucose. Fructose-1,6-P2 plus triose-P rose but it did not attain the level existing in islets from fed rats. The ratios (ATP)/(5'-AMP) and (ATP)/(Pi)(adp) increased to the values observed in glucose-stimulated islets of fed rats. The metabolic changes in islets from fed rats exposed to high glucose are consistent with an activation of glycolysis occurring concomitantly with stimulated rates of insulin release. This occurs despite the decrease of important activators of glycolysis--Pi and 5'-AMP. The enhanced glycolysis possibly results from P-fructokinase activation by increased fructose-6-P levels. Activation of glycolysis with 8.6 mM glucose was not as pronounced in islets from starved rats. Despite the different secretory response of islets from fet and fasted rats, the changes of phosphorylation state in the islets, in particular, Pi and 5'-AMP levels, were similar.

Mechanisms of hyperglycemia-induced insulin resistance in whole body and skeletal muscle of type I diabetic patients.

To examine the mechanisms of hyperglycemia-induced insulin resistance, eight insulin-dependent (type I) diabetic men were studied twice, after 24 h of hyperglycemia (mean blood glucose 20.0 +/- 0.3 mM, i.v. glucose) and after 24 h of normoglycemia (7.1 +/- 0.4 mM, saline) while receiving identical diets and insulin doses. Whole-body and forearm glucose uptake were determined during a 300-min insulin infusion (serum free insulin 359 +/- 22 and 373 +/- 29 pM, after hyper- and normoglycemia, respectively). Muscle biopsies were taken before and at the end of the 300-min insulin infusion. Plasma glucose levels were maintained constant during the 300-min period by keeping glucose for 150 min at 16.7 +/- 0.1 mM after 24-h hyperglycemia and increasing it to 16.5 +/- 0.1 mM after normoglycemia and by allowing it thereafter to decrease in both studies to normoglycemia. During the normoglycemic period (240-300 min), total glucose uptake (25.0 +/- 2.8 vs. 33.8 +/- 3.9 mumol.kg-1 body wt.min-1, P less than 0.05) was 26% lower, forearm glucose uptake (11 +/- 4 vs. 18 +/- 3 mumol.kg-1 forearm.min-1, P less than 0.05) was 35% lower, and nonoxidative glucose disposal (8.9 +/- 2.2 vs. 19.4 +/- 3.3 mumol.kg-1 body wt-1min-1, P less than 0.01) was 54% lower after 24 h of hyper- and normoglycemia, respectively. Glucose oxidation rates were similar. Basal muscle glycogen content was similar after 24 h of hyperglycemia (234 +/- 23 mmol/kg dry muscle) and normoglycemia (238 +/- 22 mmol/kg dry muscle). Insulin increased muscle glycogen to 273 +/- 22 mmol/kg dry muscle after 24 h of hyperglycemia and to 296 +/- 33 mmol/kg dry muscle after normoglycemia (P less than 0.05 vs. 0 min for both). Muscle ATP, free glucose, glucose-6-phosphate, and fructose-6-phosphate concentrations were similar after both 24-h treatment periods and did not change in response to insulin. We conclude that a marked decrease in whole-body, muscle, and nonoxidative glucose disposal can be induced by hyperglycemia alone.

Effects of brazilin on the production of fructose-2,6-bisphosphate in rat hepatocytes.

Increased hepatic glucose output is one of the major mechanisms of hyperglycemia in diabetic patients. Fructose-2,6-bisphosphate (F-2,6-BP), a gluconeogenic intermediate, plays a critical role in hepatic glucose output by regulating gluconeogenesis and glycolysis in the liver. Brazilin, an active component of sappan wood (Caesalpinia sappan), decreases blood glucose in diabetic animals. In this study, the effect of brazilin on gluconeogenic intermediate production and enzyme activity were examined to investigate the hypoglycemic mechanism of brazilin. Brazilin increased the production of F-2,6-BP in hepatocytes by elevating intracellular levels of fructose-6-phosphate (F-6-P) and hexose-6-phosphate (H-6-P). Brazilin was also found to significantly increase the activity of 6-phosphofructo-2-kinase (PFK-2) and pyruvate kinase in glucagon-treated hepatocytes. However, glucose-6-phosphatase activity was not affected by brazilin. This data suggests that brazilin inhibits hepatic gluconeogenesis by elevating the F-2,6-BP level in hepatocytes, possibly by elevating cellular F-6-P/H-6-P levels and PFK-2 activity. Increased pyruvate kinase activity may also play a role in the anti-gluconeogenic action of brazilin.

Production of fructose and fructose-3-phosphate in maturing rat lenses.

PURPOSE: A large increase in glycation of crystallins between 1 and 8 months has been demonstrated in lenses obtained from aging rats. The objective of this study was to investigate if an age-associated increase in the levels of any of the phosphorylated and nonphosphorylated sugars in the aging rat lenses could be correlated with this increase. METHODS: Lenses were obtained from Sprague-Dawley rats ranging in age from 2 to 20 months. Trichloroacetic extracts of these tissues were analyzed by using 31P-NMR for sugar phosphates and high-pressure liquid chromatography equipped with an electrochemical detector for sugars and polyols. RESULTS: Although no elevation in the lenticular glucose levels was observed, an age-associated increase in the concentrations of polyol pathway-associated metabolites--sorbitol, fructose, sorbitol-3-phosphate, and fructose-3-phosphate--was detected. In contrast, no significant changes were observed in glycolytic or pentose shunt metabolites. CONCLUSION: Aging lenses accumulate increased concentrations of fructose and fructose-3-phosphate. Because fructose-3-phosphate is a potent glycating agent and a potential in vivo source of 3-deoxyglucosone, its accumulation in the lens, along with fructose, may be a contributing factor in the age-associated increase of nonenzymatic glycation in rat lenses.

31P nuclear magnetic resonance study on perfused brain slices of guinea pig.

31P-NMR measurements of brain slices of guinea pig were successfully carried out for the first time with a new perfusion and spinning technique. The 31P-NMR spectrum showed resonance lines of phosphocreatine, fructose-6-phosphate, ATP, and inorganic phosphate. In the time course of the 31P-NMR spectra, the concentrations of phosphocreatine and fructose-6-phosphate, which were estimated from the initial spectrum, were 4.3 and 1.1 mumol/g of fresh tissue, respectively, and the estimates from the following ones showed that the concentration of phosphocreatine decreased to 2.7 mumol/g of fresh tissue while that of fructose-6-phosphate did not change significantly throughout the measurements.

Relationship between the concentrations of glycolytic intermediates and expression of the L-type pyruvate kinase gene in cultured hepatocytes.

Previous studies have suggested that some glycolytic intermediates are involved in the regulation of L-type pyruvate kinase gene expression by carbohydrates such as glucose and fructose. To find such intermediates, we examined the relationship between the levels of L-type pyruvate kinase mRNA and glycolytic metabolites in hepatocytes cultured under various conditions. Of the metabolites, the levels of 3-phosphoglycerate and phosphoenol-pyruvate only increased significantly under conditions under which the expression of the L-type pyruvate kinase gene was stimulated. The level of glucose 6-phosphate, which was reported to be involved in dietary stimulation of this gene expression, was not correlated with the mRNA level since marked accumulation of deoxyglucose 6-phosphate occurring on the addition of deoxyglucose, a nonmetabolizable glucose analog, was not accompanied by an increase in the L-type pyruvate kinase mRNA level. In addition, we found that fructose at a low concentration in the presence of glucose failed to increase the mRNA and metabolite levels in contrast to other reports that the promoter activity of the L-type pyruvate kinase gene is stimulated by this treatment. Thus we propose that 3-phosphoglycerate and/or phosphoenolpyruvate are involved in the carbohydrate regulation of L-type pyruvate kinase gene expression.

Fructose-3-phosphate production and polyol pathway metabolism in diabetic rat hearts.

Previous studies have suggested that polyol-pathway and nonenzymatic glycation may be involved in the development of cardiac myopathy, a well-known manifestation of diabetes. Although the exact etiology of this complication is not fully understood, it is likely to be multifactorial. In this study, we investigated the metabolic consequences of diabetes and the effect of aldose reductase inhibitor (ARI) treatment on cardiac tissues of Sprague-Dawley rats. Perchloric acid (PCA) extracts of hearts from the animals were examined using 31P-nuclear magnetic resonance (NMR), gas chromatography/mass spectrometry (GC/MS), and high-performance liquid chromatography (HPLC). In 31P-NMR spectra of diabetic animals, a peak resonating at the chemical shift of 5.8 ppm with a coupling constant of 10 Hz was identified as fructose-3-phosphate (F3P). Undetectable in controls (< approximately 20 nmol/g), this metabolite was present at a concentration of 81.3 +/- 16.3 nmol/g wet weight (n = 4) in diabetic rat hearts. GC/MS analysis of these extracts from diabetics also identified a decomposition product of F3P, 3-deoxyglucosone (3DG), at a concentration of 9.4 +/- 3.5 nmol/g (n = 3), compared with 0.98 +/- 0.43 nmol/g (n = 3) in controls. No evidence was found for the expected detoxification products of 3-DG, 3-deoxyfructose and 2-keto 3-deoxygluconate. Concomitant with the elevation of F3P and 3DG, fructose and sorbitol levels were also elevated in diabetic animals. Surprisingly, ARI treatment was found to have no effect on the levels of these metabolites. These data suggest that either the heart may be unique in its production of fructose or it may not readily transport the ARI sorbinil. Production of the potent glycating agents F3P and 3DG in diabetics suggests that these compounds may be contributing factors in the glycation of cardiac proteins in the diabetic rat heart.

Effects of fructose ingestion on sorbitol and fructose 3-phosphate contents of erythrocytes from healthy men.

To investigate the effect of fructose ingestion on sorbitol and fructose 3-phosphate (F3P) in erythrocytes, we administered 50 g fructose with and without treatment with an aldose reductase inhibitor, epalrestat, to seven healthy, normal-glucose-tolerant, male volunteers aged 20-43 years. The same subjects were given 50 g glucose on another day. The sorbitol and F3P contents in their erythrocytes increased significantly, reaching peak levels at 60 min and 180 min, respectively, following fructose in gestion. On the other hand, glucose ingestion did not cause any statistically significant change in sorbitol content in their erythrocytes, although it significantly elevated their F3P content. Treatment with epalrestat had no significant effect on incremental changes in erythrocyte sorbitol and F3P content following fructose ingestion. This suggests that oral fructose may be converted directly to sorbitol and F3P in erythrocytes instead of being converted via glucose. Thus, the dietary intake of fructose may affect the concentrations of sorbitol and F3P in erythrocytes in normal men.

Metabolism of fructose-1,6-bisphosphate by mature boar spermatozoa.

Mature epididymal boar spermatozoa converted glucose and fructose to carbon dioxide and lactate and maintained high concentrations of ATP. In the presence of (S)-alpha-chlorohydrin these processes were inhibited and there was an accumulation of fructose-1,6-bisphosphate and dihydroxyacetone phosphate. With fructose-1,6-bisphosphate as the substrate, the concentration of ATP was maintained, carbon dioxide was evolved and dihydroxyacetone phosphate accumulated. Cells pre-incubated with (S)-alpha-chlorohydrin did not maintain ATP levels, evolved less carbon dioxide and produced dihydroxyacetone phosphate. Assays of incubates in which fructose-1,6-bisphosphate was used as the substrate showed the presence of equilibrium quantities of fructose-6-phosphate and glucose-6-phosphate which were not detected when either fructose or glucose were used as substrates. [14C]Fructose and [14C]glucose were not produced from [14C]fructose-1,6-bisphosphate in spermatozoal incubates which had or had not been pre-incubated with (S)-alpha-chlorohydrin. Evidence is presented that a high concentration of fructose-1,6-bisphosphate leads to the formation of fructose-6-phosphate and glucose-6-phosphate but not of fructose and/or glucose.