Differences in the fatty acid metabolism of visceral adipose tissue in postmenopausal women.

OBJECTIVE: Visceral fat accumulation and metabolic syndrome incidence among women increase after menopause; therefore, fat metabolic changes and fat redistribution may occur according to menstrual status. The aim of our study was to clarify differences in subcutaneous and visceral adipose tissue metabolism between premenopausal and postmenopausal women, using metabolomics. METHODS: Thirty-nine (16 premenopausal and 23 postmenopausal) women were recruited through elective gynecologic surgery, and both subcutaneous and visceral adipose tissues were collected during surgical operation. Metabolite profiling of adipose tissue was performed by capillary electrophoresis with electrospray ionization time-of-flight mass spectrometry. RESULTS: Sedoheptulose 7-phosphate, a midproduct of the pentose phosphate pathway, was significantly higher (P < 0.05) in visceral adipose tissues of premenopausal women. Dihydroxyacetone phosphate and fructose-1,6-biphosphate, midproducts of glycolysis, were significantly higher (P < 0.05) in subcutaneous adipose tissues of postmenopausal women. The concentrations of fatty acid metabolites-heptanoate (C7:0; premenopausal vs postmenopausal, 4.07 [0.72] vs 2.64 [0.28] nmol/g), octanoate (C8:0; 3.52 [0.29] vs 5.20 [0.29] nmol/g), and pelargonate (C9:0; 8.03 [0.49] vs 10.66 [0.44] nmol/g)-in the visceral fat (but not in subcutaneous fat) of postmenopausal women were significantly higher (P < 0.05) than those in the visceral fat of premenopausal women. CONCLUSIONS: Fatty acid metabolites increase in visceral fat (but not in subcutaneous fat) after menopause. The change in fatty acid metabolism in visceral adipose tissues might be related to metabolic syndrome in postmenopausal women.

Optimization of a blueprint for in vitro glycolysis by metabolic real-time analysis.

Recruiting complex metabolic reaction networks for chemical synthesis has attracted considerable attention but frequently requires optimization of network composition and dynamics to reach sufficient productivity. As a design framework to predict optimal levels for all enzymes in the network is currently not available, state-of-the-art pathway optimization relies on high-throughput phenotype screening. We present here the development and application of a new in vitro real-time analysis method for the comprehensive investigation and rational programming of enzyme networks for synthetic tasks. We used this first to rationally and rapidly derive an optimal blueprint for the production of the fine chemical building block dihydroxyacetone phosphate (DHAP) via Escherichia coli's highly evolved glycolysis. Second, the method guided the three-step genetic implementation of the blueprint, yielding a synthetic operon with the predicted 2.5-fold-increased glycolytic flux toward DHAP. The new analytical setup drastically accelerates rational optimization of synthetic multienzyme networks.

Metabolomics of supragingival plaque and oral bacteria.

Dental caries is initiated by demineralization of the tooth surface through acid production by sugar metabolism of supragingival plaque microflora. To elucidate the sugar metabolic system, we used CE-MS to perform metabolomics of the central carbon metabolism, the EMP pathway, the pentose-phosphate pathway, and the TCA cycle in supra- gingival plaque and representative oral bacteria, Streptococcus and Actinomyces. Supragingival plaque contained all the targeted metabolites in the central carbon metabolism, except erythrose 4-phosphate in the pentose-phosphate pathway. After glucose rinse, glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, dihydroxyacetone phosphate, and pyruvate in the EMP pathway and 6-phosphogluconate, ribulose 5-phosphate, and sedoheptulose 7-phosphate in the pentose-phosphate pathway, and acetyl CoA were increased. Meanwhile, 3-phosphoglycerate and phosphoenolpyruvate in the EMP pathway and succinate, fumarate, and malate in the TCA cycle were decreased. These pathways and changes in metabolites observed in supragingival plaque were similar to the integration of metabolite profiles in Streptococcus and Actinomyces.

Control analysis of the role of triosephosphate isomerase in glucose metabolism in Lactococcus lactis.

Triosephosphate isomerase (TPI), which catalyses the conversion of dihydroxyacetone phosphate (DHAP) to glyceraldehyde-3-phosphate (G3P), was studied for its control on glycolysis and mixed acid production in L. lactis subspecies lactis IL1403 and L. lactis subspecies cremoris MG1363. Strains in which the TPI activity was modulated from 3%-225% (IL1403) or 13%-103% (MG1363) of the wild-type level were constructed by changing the expression of the tpiA gene. The enzyme was found to be present in high excess in the wild-type cells and 10% TPI activity still supported more than 70% of the wild-type glycolytic flux in both strains. Homolactic product formation was preserved throughout the range of TPI activities studied, although a slight increase in the amount of acetate and formate production was observed in the strains with strongly reduced TPI activity for both IL1403 and MG1363. The upstream metabolites glucose-6-phosphate, fructose-1,6-bisphosphate and DHAP in the IL1403 derivatives were essentially unchanged for TPI activities from 26% to 225%. At a TPI activity of 3%, the level of DHAP increased four times. The finding that an increased level of DHAP coincides with an increase in formate production is surprising and indicates that pyruvate formate lyase is not inhibited by DHAP under these conditions.

Substrate product equilibrium on a reversible enzyme, triosephosphate isomerase.

The highly efficient glycolytic enzyme, triosephosphate isomerase, is expected to differentially stabilize the proposed stable reaction species: ketone, aldehyde, and enediol(ate). The identity and steady-state populations of the chemical entities bound to triosephosphate isomerase have been probed by using solid- and solution-state NMR. The 13C-enriched ketone substrate, dihydroxyacetone phosphate, was bound to the enzyme and characterized at steady state over a range of sample conditions. The ketone substrate was observed to be the major species over a temperature range from -60 degrees C to 15 degrees C. Thus, there is no suggestion that the enzyme preferentially stabilizes the reactive intermediate or the product. The predominance of dihydroxyacetone phosphate on the enzyme would support a mechanism in which the initial proton abstraction in the reaction from dihydroxyacetone phosphate to D-glyceraldehyde 3-phosphate is significantly slower than the subsequent chemical steps.

A potential role of the cytoskeleton of Saccharomyces cerevisiae in a functional organization of glycolytic enzymes.

Numerous individual enzymes participate in a given synthetic or degradative pathway in which the product of one reaction becomes the substrate for the subsequent enzyme. This raises the question of whether the product of one 'soluble' enzyme diffuses freely through the available cell volume, where it accidentally collides with the subsequent 'soluble' enzyme. Alternatively, enzymes acting in a given pathway may be organized in ordered structures, metabolons. Certain glycolytic enzymes have been shown to co-localize with the cytoskeleton in mammalian cells. We deleted genes coding for proteins associated with the cytoskeleton of Saccharomyces cerevisiae: TPM1 coding for tropomyosin, SAC6 for fimbrin and CIN1 for a microtubule-associated protein. Single deletions or deletions of two such genes had no effect on the specific activities of glycolytic enzymes, or on the rates of glucose consumption and ethanol production. However, the concentrations of glycolytic metabolites during a switch from a gluconeogenic mode of metabolism, growth on an ethanol medium, to glycolysis after glucose addition showed transient deviations from the normal change in metabolite concentrations, as observed in wild type cells. However, all metabolites in mutant strains reached wild-type levels within 2-4 h after the shift. Only ATP levels remained low in all but the tmp1-Delta-sac6-Delta double mutant strains. These observations can be interpreted to mean that metabolic reorganization from a gluconeogenic to a glycolytic metabolism is facilitated by an intact cytoskeleton in yeast.

Analysis of glycolysis metabolites by capillary zone electrophoresis with indirect UV detection.

The glycolysis metabolites glucose 6-phosphate (G6-P), fructose 6-phosphate (F6-P), fructose 1,6-bisphosphate (F1,6-BP), fructose 2,6-bisphosphate (F2,6-BP), glyceraldehyde phosphate (GAP), dihydroxyacetone phosphate (DHAP), phosphoenolpyruvate (PEP), pyruvate, and lactate were analyzed by capillary zone electrophoresis (CZE) with indirect UV detection. The chromophores phthalic acid, sorbic acid, and 4-hydroxybenzoic acid were studied as background electrolytes. Both detection sensitivity and resolution were found to depend on the pH and the concentration of the carrier electrolyte. Optimum separation and detection of the phosphate compounds were accomplished upon reversal of electroendosmotic flow (EOF) with OFM Anion-BT (Waters Corp., Milford, MA) at a concentration of 4-6 mM 4-hydroxybenzoic acid, pH 11.6-12.0, with the detection wave-length set at 280 nm. The highly alkaline pH allowed the successful separation of the isomers F6-P and G6-P, as well as F1,6-BP and F2,6-BP, respectively. The effect of sample ionic strength on the detection limits of G6-P, F6-P, F1,6-BP, and F2,6-BP was also investigated: These limits ranged from 1 to 3 microM in both low- and high-ionic-strength samples. However, high Mg2+ concentrations in the sample led to a progressive loss of resolution between F1,6-BP and F2,6-BP, unless the inlet reservoir was replenished with fresh electrolyte after every injection. Linearity of detection was observed over one to two orders of magnitude.

Enzymatic determination of carbon (14C)-labeled glycerol in biological samples.

A method for determination of glycerol-specific-radioactivity in biological samples is presented. It is based on the following steps: (a) enzymatic conversion of glycerol to dihydroxyacetone-phosphate, (b) quantitative trapping of dihydroxyacetone-phosphate in SPE amino (NH2) columns, (c) eluation with HCl 0.5 N of dihydroxyacetone-phosphate followed by radioactivity counting and (d) estimation of the radioactivity thus trapped compared with that of enzymatically untreated aliquots of the same samples. No interferences from other 14C-labeled materials tested such as D-glucose, L-alanine, L-glutamine and D-beta-hydroxybutyrate were observed. This inexpensive and high-speed method can be applied in routine multiple estimations of glycerol-specific-radioactivity in biological samples in tracer metabolic studies.

Critical micellar concentrations of palmitoyl dehydroxyacetone phosphate and 1-palmitoyl-rac-glycerol 3-phosphate.

By fluorescent titration method, the critical micellar concentrations of palmitoyl CoA, palmitoyl dihydroxyacetone-P, and 1-palmitoyl-rac-glycerol-3-P in 75 mM Tris-HCl, pH 7.5, were determined to be 41, 58, and 70 microM, respectively.

A sensitive multienzymatic assay for the measurement of pyruvate, dihydroxyacetone phosphate, oxaloacetate, and acetoacetate in clear extracts from biological samples.

A multienzymatic method for the measurement of pyruvate, dihydroxyacetone phosphate, oxaloacetate, and acetoacetate is presented. The determination procedure is considered suitable because it is simple, sensitive, and its advantages could be demonstrated by comparison with the original methods.

[Changes in enzyme systems and lipogenesis metabolites in experimental tuberculosis]. / Izmenenie nekotorykh fermentnykh sistem i metabolitov lipogeneza pri éksperimental'nom tuberkuleze.

Lung tissue phospholipid metabolism has been studied under the conditions of experimental tuberculosis. These investigations were combined with the simultaneous studying of the qualitative and quantitative composition of individual phospholipids, of the changes in the content of glycerophosphate and dioxiacetonphosphate, as well as, of the abnormalities in the activity of glycerokinase and glycerophosphatedehydrogenase, which play an important role in the catalysis of the initial processes of phosphatidogenesis. Our data obtained have shown that formation of tuberculosis process is accompanied by the pronounced increase of glycerokinase and glycerophosphatedehydrogenase activities, which leads to the significant accumulation of the dioxiacetonphosphate and the free glycerol concentrations. It was established by us, that under the conditions of the pathology studied, the significant decrease of the quantity of phosphatidylcholines, phosphatidylethanolamines and phosphatidylserines took place, while the level of sphingomyelins was increased. The results of our investigations shed the light on the up-to-date understanding of the role of different fractions of phospholipids, as well as, of corresponding enzymatic systems in the pathogenesis of lung tuberculosis and in the formation of focus of necrosis in lung tissue.

Estimation of acyldihydroxyacetone phosphate and lysophosphatidate in animal tissues.

Chemical and enzymatic methods have been developed to measure small quantities (10(-8) - 10(-10) mol) of acyldihydroxyacetone phosphate in animal tissues. Lipids extracted from tissue samples with acidic CHCl3/methanol were subjected to solvent partitioning at two different pH values for partial purification of this keto-lipid from other lipids. This lipid was then estimated radiometrically either by chemical reduction with NaB3H4 or by enzymatic reduction with [4B-3H]NADPH using a partially purified acyldihydroxyacetone-phosphate reductase (EC 1.1.1.101). Thin-layer chromatography revealed the presence of a number of 3H-labeled lipids in the NaB3H4-reduced product and further purification of the product was necessary to estimate the amount of acyl[2-3H]glycerol 3-phosphate formed. The enzymatic reduction was very specific for acyl/alkyldihydroxyacetone phosphate. The amounts (nmol/g) of these keto-lipids estimated in different tissues by the enzymatic method were 10.06 +/- 0.64 (guinea pig liver), 4.3 +/- 0.15 (rat liver), 2.1 (rat testis), 1.5 (rad kidney) and 1.2 (rat brain). Monoacylglycerol 3-phosphate, i.e., lysophosphatidic acid, which was co-purified with acyldihydroxyacetone phosphate, was found to be present in relatively larger amounts in tissues. The amounts (nmol/g) of this lipid, estimated by enzymatically measuring the amounts of sn-glycerol 3-phosphate released after alkaline methanolysis of the partially purified lipid extracts, were 143 (guinea pig liver), 58 (rat liver), 53 (rat kidney) and 92 (rat brain). Stearic acid (18:0) was found to be the major (65%) fatty acid present in the lysophosphatidate purified from guinea pig liver.

Inhibition of glyceraldehyde 3-phosphate dehydrogenase by adenine nucleotides in pachytene primary spermatocytes from rat testes.

The effect of adenine nucleotides on the catalytic activity of spermatocyte glyceraldehyde 3-phosphate dehydrogenase (GA3PDH) was studied for its significance to metabolic regulation. In the presence of glucose (10 mM), the ATP level declined whereas the ADP and AMP levels remained unchanged. During incubation with glucose, fructose 1,6-bisphosphate (fruc 1,6-P2) and dihydroxyacetone phosphate (DHAP) were accumulated markedly. GA3PDH was inhibited by ATP (Ki = 2.27 mM), ADP (Ki = 1.21 mM) and AMP (Ki = 0.73 mM) competitively with NAD (Km = 0.24 mM). The results suggest that glycolysis in spermatocytes is regulated by GA3PDH.

Reagent for the enzymatic determination of serum total triglycerides with improved lipolytic efficiency.

A fully enzymatic assay is described for the determination of triglycerides. The coupled activities of triacylglycerol acylhydrolase and glycerol kinase result in the formation of glycerol-3-phosphate. The system also contains L-alpha-glycerol-phosphate oxidase, which produces hydrogen peroxide from glycerol-3-phosphate, and a sensitive chromogenic indicator system, consisting of peroxidase, 4-chlorophenol and 4-aminophenazone. We evaluated this method with respect to kinetics, linearity, blank rates, precision, accuracy, reagent stability and interfering substances. The accuracy of the triglyceride assay demands that each enzymatic reaction step be complete and homogeneous. We therefore developed HPTLC-1) and HPLC-2) methods to monitor the course and completeness of each step.

Gluconeogenesis and phosphate reabsorption in isolated lactate- or pyruvate-perfused rat kidneys.

Recent experiments suggest that cytoplasmic free NAD inhibits renal phosphate reabsorption and that gluconeogenesis, by increasing NAD concentration, inhibits phosphate reabsorption. To examine these relationships further we measured phosphate reabsorption by isolated kidneys perfused with either lactate or pyruvate. Lactate perfusion increased the ratio of the cytoplasmic redox couple (glycerol-3-phosphate/dihydroxyacetone phosphate) in snap-frozen kidneys 3-fold relative to the ratio with pyruvate perfusion (p less than 0.001). Inulin clearance and fractional phosphate reabsorption were lower with lactate than with pyruvate perfusion. Phosphate reabsorption decreased throughout the lactate perfusion but was constant for 1 h of pyruvate perfusion. Basal and norepinephrine (NE)-stimulated gluconeogenesis were lower with lactate than with pyruvate perfusion. With lactate and pyruvate perfusions, NE induced equal increases in fractional sodium reabsorption, although only with pyruvate perfusion was there increased phosphate reabsorption. In these experiments, oxidative phosphorylation, not cytoplasmic free NAD concentrations, influenced phosphate reabsorption, and NE-stimulated gluconeogenesis was associated with increased phosphate reabsorption.

Intrauterine malnutrition in the rat: alterations of fetal glycerol metabolism.

This investigation intended to clarify the effects of malnutrition in utero on enzymes of glycerol metabolism and the stores of phosphorylated glycerol intermediates in liver, striated muscle, and brain in the rat. Pregnant Wistar rats were restricted to an intake of 50% (M) of ad libitum fed controls (C) from the seventh day of gestation onward. Fetuses were removed 2 days (-2), or 1 day (-1), before term, or at the day of birth (DOB) The M fetuses and newborn rats were stunted. Their hepatic alpha-glycerophosphate oxidase (GPO) levels were lower than those of C in utero (mean +/- SEM: M = 23.1 +/- 1.5, 15.8 +/- 1.1, and 31.6 +/- 4.5; C = 34.8 +/- 4.9, 39.8 +/- 7.0, and 23.6 +/- 5.0 nmol/min X cm at -2, -1, and DOB, respectively; F = 7.29 [1,57], P less than .01). In muscle, this enzyme, as well as liver and brain alpha-glycerophosphate dehydrogenase (GPD), alpha-glycerophosphate (GP), and dihydroxyacetone phosphate (DHAP), only varied with the developmental stage. Although the latter was a significant differential factor in all the determinations, maternal diet only affected brain DHAP stores (M = 1.85 +/- 0.36, 1.03 +/- 0.16, 0.74 +/- 0.10; C = 2.33 +/- 0.46, 1.87 +/- 0.21, 1.13 +/- 0.18 mumol/g at -2, -1, and DOB, respectively; F = 9.03 [1,53], P less than .01). These findings support the contention that intrauterine malnutrition can alter normal ontogenesis of glycerol metabolism enzymes in certain organs and become a negative factor disturbing normal gluconeogenesis and glycogenolysis, with potential disruption of energy homeostasis immediately after birth.

[Contents of glycogen, glycolytic activity and contents of metabolites in the atrioventricular system of bovine hearts (author's transl)]. / Glykogengehalte, glykolytische Aktivität und Metabolitgehalte im Atrioventricularsystem des Rinderherzens.

In the atrioventricular system (AVS) consisting of the compact node, the penetrating bundle and the branching bundles of about 250 bovine hearts there were made several studies: 1. In quickly removed and fixed specimens (distal AV-node, penetrating bundle) determination of a metabolic state with respect to glycogen, glucose, lactate, ATP, ADP, AMP, creatinephosphate, total creatine, gluc-6-phosphate, fructose-6-phosphate, fructose-1,6-diphosphate, dihydroxyacetonphosphate and pyruvate. 2. Determination of glycogen contents and glygolytic activity in AVS and its parts for ischemic times up to three hours. 3. The determination of metabolic contents in samples of connective tissue in atrium and ventricle of bovine hearts. The AV-nodes are poor in glycogen comparable with glycogen content of central nervous system and other ganglia. Penetrating bundles of Hiss and branching bundle belong after liver to the glycogen richest parenchyma of animal tissues. Even after ischemia of 3 h only a part of glycogen was recovered as lactate. The greater part of glycogen must be considered as a structural element of Hiss bundle and branching bundles of the ventricles.

Substrate form of D-frutose 1,6-bisphosphate utilized by fructose 1,6-bisphosphatase.

Rapid quench kinetic experiments on fructose 1,6-bisphosphatase demonstrate a stereospecificity for the alpha anomer of fructose 1,6-bisphosphate relative to the beta configuration. The beta anomer is only utilized after mutarotation to the alpha form in a process that is not enzyme catalyzed. Studies employing analogues of the acyclic keto configuration indicate that the keto form is utilized at a rate less than 5% that of the alpha anomer, a finding also confirmed by computer simulation of the rapid quench data. Chemical trapping experiments of the keto analogue, xylulose 1,5-bisphosphate, and the normal substrate suggest that interconversion of the acyclic and anomeric configurations is retarded by their binding to the enzyme. A hypothesis is advanced attributing substrate inhibition of fructose 1,6-bisphosphatase to possible binding of the keto species.

Molar absorptivities of beta-NADH and beta-NAD at 260 nm.

To determine the molar absorptivities of beta-NADH and beta-NAD at 260 nm, we purified the reduced form of the coenzyme by repeated anion-exchange chromatography. A NADH preparation was so obtained for which the 260 nm/340 nm absorbance ratio was 2.265. When calculated with epsilon 340 beta-NADH = 6.22 times 10-3 for beta-NADH at 260 nm and 25 degrees C, a molar absorptivity of 14.1 times 10-3 liter - mol minus 1 - cm minus 1 resulted from this quotient. By use of the lactate dehydrogenase or glycerol-3-phosphate dehydrogenase assay, respectively, and referring to the new absorption coefficient for NADH at 260 nm, the molar absorptivity of beta-NAD at 260 nm and 25 degrees C was established to be 17.4 times 10-3 liter - mol minus 1 - cm minus 1. The values observed are lower than those reported thus far.

[Studies on the effect of radiation on electrolyte changes and metabolism of the myocardium. V. Changes in enzyme activities and glycolysis metabloites due to radiation]. / Untersuchungen über den Einfluss von Röntgenstrahlen auf Elektrolytver-schiebungen und Stoffwechsel des Herzmuskels

The behavior of enzyme activities, substrates and metabolites of glycosis as well as of the pentose phosphate shunt following local irradiation (250 to 6000 R surface dose) is biochemically investigated in the guinea-pig's myocardium. During irradiation, an activation of phosphorylase-a is going on while the total phosphorylase content remains unchanged. Enzyme activities of hexokinase and phosphofructokinase are increased in dependence on dosage as well as time. The glycogen content is being reduced; tissular concentration of the metabolites glucose-1-phosphate, glucose-6-phosphate, glyceraldehyde-3-phosphate, glycerol-3-phosphate, and pyruvate increases following irradiation; the content of fructose-1,6-diphosphate, dihydroxyacetonephosphate, and lactate is decreased. The activity of glucose-6-phosphate dehydrogenas is slightly inhibited, whereas 6-phosphogluconate-dehydrogenase remains unaffected.