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PubMed1986

Metabolic alterations associated with proliferation of mitogen-activated lymphocytes and of lymphoblastoid cell lines: evaluation of glucose and glutamine metabolism.

In vitro resting, short-term mitogen stimulated, and proliferating rat thymocytes as well as established human T and B lymphoblastoid cell lines were compared in their capacity to metabolize glucose and glutamine as energy source. Furthermore, the pathways of glutamine metabolism in these cells were studied. Compared with resting thymocytes, glucose metabolism of proliferating thymocytes was 36-fold increased during the incubation; 92% of the amount of glucose utilized was converted into trioses mainly lactate, whereas resting cells metabolized only 38% to trioses. However, the latter oxidized 19% of glucose to CO2, as opposed to 1.1% by the proliferating cells. Rates of glucose uptake and degradation to products by the malignant T lymphoblastoid cell line (Jurkat) were nearly identical with those observed with proliferating rat thymocytes, whereas the benign B lymphoblastoid cell lines (DHg-B-1 and LV-B-1) showed significantly higher rates of glucose metabolism. All three transformed lymphoblastoid cell lines, however, metabolized glucose almost completely to lactate as did the proliferating rat thymocytes. Lymphocytes are able to utilize glutamine with glutamate, aspartate and ammonia being the major end-products. A complete recovery of glutamine carbon in the products was obtained with all cells. Glutamine utilization by incubated proliferating rat thymocytes was 8-fold increased as compared to the resting cells. Again the human T lymphoblastoid cell line showed the same rates of glutamine uptake and conversion into products as did the proliferating rat thymocytes, whereas both B lymphoblastoid cell lines had about 2.5-fold enhanced rates as compared to the T cell line. The results indicate that during lymphocyte proliferation caused by mitogen stimulation as well as by permanent transformation into lymphoblastoid cell lines glucose metabolism is altered not only quantitatively but also qualitatively by changing from partly aerobic to almost complete anaerobic glucose breakdown. Glutamine has been found to be a suitable energy source for lymphocytes. About 75% of the amount of glutamate derived from glutamine entered into the citric acid cycle via the aspartate aminotransferase, and the remaining 25% via the glutamate dehydrogenase reaction. The changes in metabolic rates observed in proliferating as well as in transformed or leukemic lymphocytes appear to be reliable parameters to characterize the state of lymphocyte activation or to evaluate the efficacy of lymphokines.

Animals↗
PubMed2003

Cinnamic compound metabolism in human skin and the role metabolism may play in determining relative sensitisation potency.

BACKGROUND: trans-Cinnamaldehyde and trans-cinnamic alcohol cause allergic contact dermatitis (ACD) in humans; cinnamaldehyde is a more potent sensitiser than cinnamic alcohol. These two chemicals are principal constituents of the European Standard 'Fragrance Mix', as used in patch testing diagnostics of sensitisation to fragrances by clinical dermatologists. As contact sensitisers are usually protein reactive compounds, it is hypothesised that cinnamic alcohol (not protein-reactive) is a 'prohapten' that requires metabolic activation, presumably by cutaneous oxidoreductases, to the protein-reactive cinnamaldehyde (a 'hapten'). It is postulated that cinnamaldehyde can be detoxified by aldehyde dehydrogenase (ALDH) to cinnamic acid and/or by alcohol dehydrogenase (ADH) to cinnamic alcohol. Hence, a variety of metabolic pathways may contribute to the relative exposures and hence sensitising potencies of cinnamic alcohol and cinnamaldehyde. OBJECTIVE: To evaluate the extent of cinnamaldehyde and cinnamic alcohol metabolism in human skin and provide evidence for the role of cutaneous ADH and ALDH in such metabolism. METHODS: The extent of cinnamic alcohol and aldehyde metabolism was investigated in human skin homogenates and sub-cellular fractions. A high performance liquid chromatography method was used for analysis of skin sample extracts. Studies were conducted in the presence and absence of the ADH/cytochrome P450 inhibitor 4-methylpyrazole and the cytosolic ALDH inhibitor, disulfiram. RESULTS: Differential metabolism of cinnamic alcohol and cinnamaldehyde was observed in various subcellular fractions: skin cytosol was seen to be the major site of cinnamic compound metabolism. Significant metabolic inhibition was observed using 4-methylpyrazole and disulfiram in whole skin homogenates and cytosolic fractions only. CONCLUSIONS: This study has demonstrated that cutaneous ADH and ALDH activities, located within defined subcellular compartments, play important roles in the activation and detoxification of CAlc and CAld in skin. Such findings are important to the development of computational hazard prediction tools for sensitisation (e.g. the DEREK program) and also to dermatologists in understanding observed interindividual differences, cross-reactivities or co-sensitisation to different cinnamic compounds in the clinic.

Acrolein↗
PubMed2005

Disturbances of phosphate metabolism: another feature of metabolic syndrome.

BACKGROUND: Despite important recent advances in the understanding of the consequences of metabolic syndrome, its pathophysiological characteristics remain unclear. It has been proposed that disturbances in phosphate metabolism may contribute to the development of this constellation of cardiovascular risk factors. However, there have been insufficient clinical data supporting this hypothesis to date. The aim of our study is to confirm the presence of hypophosphatemia in patients with metabolic syndrome, as well as investigate mechanisms that may underlie the disturbances in phosphate metabolism in this patient group. METHODS: Two hundred fifty-five individuals were enrolled. The diagnosis of metabolic syndrome was based on Adult Treatment Panel III guidelines. Subjects with fewer than 3 criteria served as controls. RESULTS: Patients with metabolic syndrome showed significantly lower phosphate and magnesium levels compared with controls. Because fractional excretion of phosphate was similar in both groups, we assume that hypophosphatemia in patients with metabolic syndrome can be attributed to decreased dietary intake, as well as internal redistribution of this element. Lower magnesium values in the patient group may result from the same mechanisms as lower phosphate levels. In addition, hyperinsulinemia-induced renal magnesium wasting also may be a contributory factor. CONCLUSION: Patients with metabolic syndrome show significantly lower phosphate and magnesium levels compared with healthy individuals. The clinical significance of these disturbances, as well as their importance as targets for preventive or therapeutic interventions, remains to be established.

Adult↗
PubMed2005

Roles of hierarchical and metabolic regulation in the allometric scaling of metabolism in Panamanian orchid bees.

Assessment of the relative importance of variation in enzyme concentration [E] and metabolic regulation in accounting for interspecific variation in metabolic rates is an unrealized area of research. Towards this end, we used metabolic flux rates during hovering and enzymatic flux capacities (V(max) values, equal to [E]xk(cat), where k(cat) is catalytic efficiency) in flight muscles measured in vitro from 14 orchid bee species ranging in body mass from 47 to 1065 mg. Previous studies revealed that, across orchid bee species, wingbeat frequencies and metabolic rates decline in parallel with increasing body mass. V(max) values at some enzymatic steps in pathways of energy metabolism decline with increasing mass while, at most other steps, V(max) values are mass-independent. We quantified the relative importance of ;hierarchical regulation' (alteration in V(max), indicative of alteration in [E]) and ;metabolic regulation' (resulting from variation in substrate, product or modulator concentrations) in accounting for interspecific variation in flux across species. In addition, we applied the method of phylogenetically independent contrasts to remove the potentially confounding effects of phylogenetic relationships among species. In the evolution of orchid bees, hierarchical regulation completely accounts for allometric variation in flux rates at the hexokinase step while, at other reactions, variation in flux is completely accounted for by metabolic regulation. The predominant role played by metabolic regulation is examined at the phosphoglucoisomerase step using the Haldane relationship. We find that extremely small variation in the concentration ratio of [product]/[substrate] is enough to cause the observed interspecific variation in net flux at this reaction in glycolysis.

Animals↗
PubMed2005

Metabolic syndrome and cardiac disease in Japanese men: applicability of the concept of metabolic syndrome defined by the National Cholesterol Education Program-Adult Treatment Panel III to Japanese men--the Tanno and Sobetsu Study.

Results of a 6-year follow-up study were used to determine whether the concept of and the criteria for metabolic syndrome as defined by the National Cholesterol Education Program-Adult Treatment Panel III (NCEP-ATP III) can be applied to Japanese men for prediction of the occurrence of cardiac disease. The subjects were 808 men who underwent mass health check-ups in 1993 and who were not on medication for hypertension, diabetes or hyperlipidemia. Individuals who had hypertriglyceridemia, hypo-high density lipoprotein (HDL) cholesterolemia, high blood pressure, and/or high fasting plasma glucose levels were identified on the basis of the NCEP-ATP III criteria. Not in conformity with the NCEP-ATP Ill, however, a cut-off value of 85 cm was used for waist girth as an indicator of abdominal obesity. The subjects who had 3 or more risk factors were judged as having metabolic syndrome. The proportion of subjects having metabolic syndrome was 25.3%. In the 6-year follow-up study, cardiac disease occurred in 11.7% of the subjects in the metabolic syndrome group and in 6.7% of the subjects in the non-metabolic syndrome group. Results of regression analysis using Cox's proportional hazards model showed that subjects in the metabolic syndrome group had a 2.2-times greater risk of developing cardiac disease than did subjects in the non-metabolic syndrome group. The concept of metabolic syndrome as defined in the NCEP-ATP III was therefore considered to be useful for predicting the occurrence of cardiac disease in Japanese men.

Aged↗
PubMed2005

A prospective study on the prevalence of metabolic syndrome among healthy french families: two cardiovascular risk factors (HDL cholesterol and tumor necrosis factor-alpha) are revealed in the offspring of parents with metabolic syndrome.

OBJECTIVE: The purpose of this study was to estimate the longitudinal variation of prevalence of metabolic syndrome within French families and to observe biological parameters involved in cardiovascular disease among their offspring. RESEARCH DESIGN AND METHODS: Three hundred seventy-one apparently healthy families (1,366 individuals) taken from the STANISLAS cohort were studied. The subjects were examined at two time points with a 5-year interval (t(0) and t(+5)). The crude prevalence of metabolic syndrome was assessed among parents according to the National Cholesterol Education Program Adult Treatment Panel III (NCEP ATP) definition. RESULTS: The prevalence of metabolic syndrome was 5.9% in men and 2.1% in women at t(0), rising to 7.2 and 5.4% in men and women, respectively, at t(+5). Children of parents having metabolic syndrome showed higher levels of tumor necrosis factor-alpha (TNF-alpha), whereas their HDL cholesterol and apolipoprotein (apo) E concentrations were lower compared with those of age- and sex-matched control subjects (P </= 0.05). When applying NCEP ATP definitions that included either antihypertensive drugs only or all the drugs involved in metabolic syndrome, we found that the three parameters shared by the three different versions of the definition were TNF-alpha, HDL cholesterol, and an interaction between alcohol consumption and parental metabolic syndrome on HDL cholesterol concentration. CONCLUSIONS: Metabolic syndrome increases with age in supposedly healthy families from the STANISLAS cohort. In offspring of affected people, it seems to be predictive of higher values of TNF-alpha and low HDL cholesterol levels, which are two major cardiovascular factors. Therefore, in terms of prevention, it is important to identify and follow subjects with metabolic syndrome as well as their offspring, even in apparently healthy populations, to enable early disease management.

Adolescent↗
PubMed1975

[The lipid metabolism of the small intestine and its correlation to the lipid and lipoprotein metabolism of the total organism].

It is attempted to discuss the intestinal lipid metabolism particularly as it relates to systemic lipid and lipoprotein metabolism. In an introductory review of recent concepts of intestinal fat absorption special reference is given to the significance of the mixed lipid micelle during the intraluminal phase of fat absorption, to special morphological and functional features of the lumen-absorptive cell interface and to the proposed role of a fatty acid binding protein in the intracellular transport of long chain fatty acids. Furthermore the metabolic fate of absorbed fatty acids within the absorptive epithelial cell is reviewed and pointed out, that certain proteins (f.i. apoprotein B) are of crucial significance for the export of intestinal lipids into lymph. Absorption of dietary fat, however, is not the only function of the intestine in lipid metabolism. Intestinal mucosa is also capable of de novo synthesis of lipids, a function which is elaborated upon in the discussion of intestinal biosynthesis of cholesterol. Since in addition biosynthesis of cholesterol in the liver has been shown to be under sensitive control of cholesterol in intestinal lymph lipoproteins and the intestine represents the only organ, in which serum cholesterol can be excreted (after conversion to bile salts in the liver), the intestine occupies the central role in cholesterol metabolism. The intensive interrelation of intestinal functions and systemic lipoprotein metabolism is underscored by the more recent finding, that the intestine also synthesizes endogenous lipoproteins, which are secreted constantly into intestinal lymph even in the fasting state, thus contributing to plasma very low density lipoproteins. The intestine is the only organ, in which extrahepatic production of endogenous lipoproteins has been demonstrated. In the special section of this paper own original work is presented, elaborating on intestinal metabolism of plasma free fatty acids (FFA) and dealing with the important question, whether plasma FFA are incorporated into intestinal lymph lipoproteins. Studies were conducted in rats initially, which were given a rapid i.v. injection of labelled fatty acids. Mucosal radioactivity of the small intestine was greatest 2 minutes after i.v. 14C-palmitate, and accounted for 1% of administered isotope. Of mucosal 14C, 42% were present in water soluble metabolites, including CO2 and ketoacids, 28% in phospholipids and only 16% in triglycerides. The specific activity of mucosal triglyceride fatty acids (TGFA) was 11 times that of serum TGFA, confirming triglyceride synthesis by intestinal mucosa. The unexpectedly low percent incorporation of 14C-palmitate into triglycerides and its preferential conversion to water soluble metabolites, reflecting oxidation, were confirmed in double label experiments (3H-palmitate intraluminally, 14C-palmitate i.v.), which showed marked differences in the metabolism of fatty acids entering mucosa simultaneously from the two sources. While i.v...

Adult↗
PubMed2025

Hierarchical metabolic engineering for rewiring cellular metabolism.

Metabolic engineering is a key enabling technology for rewiring cellular metabolism to enhance production of chemicals, biofuels, and materials from renewable resources. However, how to make cells into efficient factories is still challenging due to its robust metabolic networks. To open this door, metabolic engineering has realized great breakthroughs through three waves of technological research and innovations, especially the third wave. To understand the third wave of metabolic engineering better, we discuss its mainstream strategies and examples of its application at five hierarchies, including part, pathway, network, genome, and cell level, and provide insights as to how to rewire cellular metabolism in the context of maximizing product titer, yield, and productivity. Finally, we highlight future perspectives on metabolic engineering for the successful development of cell factories.

Metabolic Engineering↗
PubMed1999

Correlating cell cycle with metabolism in single cells: combination of image and metabolic cytometry.

BACKGROUND: We coin two terms: First, chemical cytometry describes the use of high-sensitivity chemical analysis techniques to study single cells. Second, metabolic cytometry is a form of chemical cytometry that monitors a cascade of biosynthetic and biodegradation products generated in a single cell. In this paper, we describe the combination of metabolic cytometry with image cytometry to correlate oligosaccharide metabolic activity with cell cycle. We use this technique to measure DNA ploidy, the uptake of a fluorescent disaccharide, and the amount of metabolic products in a single cell. METHODS: A colon adenocarcinoma cell line (HT29) was incubated with a fluorescent disaccharide, which was taken up by the cells and converted into a series of biosynthetic and biodegradation products. The cells were also treated with YOYO-3 and Hoechst 33342. The YOYO-3 signal was used as a live-dead assay, while the Hoechst 33342 signal was used to estimate the ploidy of live cells by fluorescence image cytometry. After ploidy analysis, a cell was injected into a fused-silica capillary, where the cell was lysed. Fluorescent metabolic products were then separated by capillary electrophoresis and detected by laser-induced fluorescence. RESULTS: Substrate uptake measured with metabolic cytometry gave rise to results similar to those measured by use of laser scanning confocal microscopy. The DNA ploidy histogram obtained with our simple image cytometry technique was similar to that obtained using flow cytometry. The cells in the G(1) phase did not show any biosynthetic activity in respect to the substrate. Several groups of cells with unique biosynthetic patterns were distinguished within G(2)/M cells. CONCLUSIONS: This is the first report that combined metabolic and image cytometry to correlate formation of metabolic products with cell cycle. A complete enzymatic cascade is monitored on a cell-by-cell basis and correlated with cell cycle.

Biodegradation, Environmental↗
PubMed1999

Cryopreserved human hepatocytes: characterization of drug-metabolizing enzyme activities and applications in higher throughput screening assays for hepatotoxicity, metabolic stability, and drug-drug interaction potential.

Cryopreserved human hepatocytes were extensively characterized in our laboratory. The post-thaw viability, measured via dye exclusion, ranged from 55 to 83%, for hepatocytes cryopreserved from 17 donors. Post-thaw viability and yield (viable cells per vial) were found to be stable up to the longest storage duration evaluated of 120 days. Drug-metabolizing enzyme activities of the cryopreserved hepatocytes (mean of ten donors) as percentages of the freshly isolated cells were: 97%, for cytochrome P450 isoform (CYP) 1A2, 78% for CYP2A6, 96% for CYP2C9. 86% for CYP2Cl9, 90% for CYP2D6, 164% for CYP3A4, 76% for UDP-glucuronidase, and 88% for umbelliferone sulfotransferase. Known species-differences in 7-ethoxycoumarin (7-EC) metabolism were reproduced by cryopreserved hepatocytes from human, rat, rabbit, dog, and monkey, illustrating the utility of cryopreserved hepatocytes from multiple animal species in the evaluation of species-differences in drug metabolism. Higher throughput screening (HTS) assays were developed using cryopreserved human hepatocytes for hepatotoxicity, metabolic stability, and inhibitory drug-drug interactions. Dose-dependent cytotoxicity, measured using MTT metabolism as an endpoint, was observed for the known hepatotoxic chemicals tamoxifen, clozapine, cadmium chloride, diclofenac, amiodarone, tranylcypromine, precocene II, but not for 2-thiouracil. Cell density- and time-dependent metabolism of 7-EC and dextromethorphan were observed in the HTS assay for metabolic stability. Known CYP isoform-specific inhibitors were evaluated in the HTS assay for inhibitory drug-drug interactions. Furafylline, sulfaphenazole, quinidine, and ketoconazole were found to be specific inhibitors of CYP1A2, CYP2C9, CYP2D6, and CYP3A4, respectively. Tranylcypromine and diethyldithiocarbamate were found to be less specific, with inhibitory effects towards several CYP isoforms, including CYP2A6, CYP2C9, CYP2C19, and CYP2E1. These results suggest that cryopreserved human hepatocytes represent a useful experimental tool for the evaluation of drug metabolism, toxicity, and inhibitory drug-drug interaction potential.

Adult↗
PubMed1990

Validation of the tolbutamide metabolic ratio for population screening with use of sulfaphenazole to produce model phenotypic poor metabolizers.

The present study has validated kinetically a convenient method to measure tolbutamide hydroxylation capacity in human beings by use of urinary metabolic ratios. The known in vivo and in vitro inhibitory properties of sulfaphenazole were used to convert control phase subjects to phenotypically "poor" metabolizers of tolbutamide. Six healthy subjects were given a single 500 mg oral dose of tolbutamide with and without sulfaphenazole, 500 mg every 12 hours. Tolbutamide, hydroxytolbutamide, and carboxytolbutamide in urine were determined by newly developed HPLC procedures. Plasma tolbutamide clearance and half-life were measured, as were the metabolic ratio (hydroxytolbutamide + carboxytolbutamide/tolbutamide) in successive 6-hour urine collections. The mean tolbutamide plasma clearance decreased from 0.196 +/- 0.026 ml/min/kg without sulfaphenazole to 0.039 +/- 0.009 ml/min kg with sulfaphenazole, and the mean half-life of tolbutamide increased from 7.28 +/- 0.89 hours to 38.76 +/- 13.30 hours. The metabolic ratio determined in the 6 to 12 hour urine collection period decreased from 794.0 +/- 86.6 to 126.0 +/- 79.3, and this collection period also gave the best separation of subjects between phases. There was a good correlation between tolbutamide plasma clearance and metabolic ratio (rs = 0.853, p less than 0.01, n = 12) and between the percentage decrease in plasma tolbutamide clearance and the percentage decrease in metabolic ratio (r = 0.932, p less than 0.01, n = 6). The tolbutamide urinary metabolic ratio therefore effectively distinguishes tolbutamide hydroxylase activity in "normal" subjects and in those converted to model phenotypically "poor" metabolizers by sulfaphenazole.

Administration, Oral↗
PubMed2007

CYP2C76-mediated species difference in drug metabolism: a comparison of pitavastatin metabolism between monkeys and humans.

The monkey is often used to predict metabolism of drugs in humans since it generally shows a metabolic pattern similar to humans. However, metabolic profiles different from humans are occasionally seen in monkeys for some drugs including pitavastatin. Recently, we have successfully identified a monkey-specific cytochrome P450 (CYP) 2C76, which possibly accounts for a species difference between monkeys and humans because of its sequence and functional uniqueness. The present study on the role of CYP2C76 and other monkey CYP2Cs in pitavastatin metabolism, as an example, has revealed that CYP2C76 is important for the metabolism of the lactone form, indicating a major role of CYP2C76 for the difference in the metabolism of pitavastatin and possibly other drugs between monkeys and humans. The current investigation on the involvement of CYP2C76 in the metabolism of other drugs is expected to reveal further the further importance of this monkey-specific drug-metabolizing enzyme.

Animals↗
PubMed1984

The role of secondary metabolism in the metabolic activation of 7,12-dimethylbenz[a]anthracene by rat liver microsomes.

The effect of induction by either phenobarbital (PB) or 3-methylcholanthrene (MC) on the kinetics of both primary and secondary metabolism of 7,12-dimethylbenz[a]anthracene (DMBA) have been studied. PB and MC induction stimulate the initial rate of total DMBA metabolism 4- and 8-fold, respectively. Individual metabolites exhibited distinct time courses dependent on the inducer. With both induced and uninduced microsomes, formation of DMBA 5,6-dihydrodiol exhibited a 2-5 min time lag before reaching a maximum rate while, in contrast, formation of 7-hydroxymethyl-12-methylbenz[a]anthracene (7HOMMBA) declined substantially from linearity at a very early point in the reaction. This was most apparent after 2 min and total levels decreased after 5 min. Regioselectivity for both DMBA and 7HOMMBA were measured based on the initial rates of mono-oxygenation derived from these kinetics. In each set of microsomes, prior 7-hydroxylation redirected metabolism towards DMBA 3,4-dihydrodiol formation and away from 5,6-dihydrodiol. Large differences were noted for the effectiveness and regioselectivity of secondary metabolism via 7HOMMBA. MC-induced liver microsomes exhibited a stronger preference for secondary metabolism of 7HOMMBA than PB-induced and control microsomes. In all cases, 7HOMMBA dihydrodiols, formed from DMBA, appeared in the same ratio as when formed directly from 7HOMMBA, suggesting formation from 7HOMMBA rather than from 7-hydroxylation of primary dihydrodiols. The extent of secondary metabolism of primary products was calculated as the difference between the calculated formation (determined from the appropriate regioselectivity and DMBA disappearance) and the quantitated level at a particular reaction time. However, for MC-induced microsomes, quantitation of 7HOMMBA mono-oxygenated metabolites accounted for less than 50% of the calculated 7HOMMBA secondary metabolism. This discrepancy results from selective further oxidation of 7HOMMBA phenols. For control and PB-induced liver microsomes, quantitation of identifiable 7HOMMBA mono-oxygenated metabolites agreed well with calculated secondary 7HOMMBA metabolism. Based on the results presented here, factors that favor hydroxylation of PAH prior to bay-region diol-epoxide formation are discussed.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1979

Effects of Light and Inhibitors on Glutamate Metabolism in Leaf Discs of Vicia faba L: Sources of ATP for Glutamine Synthesis and Photoregulation of Tricarboxylic Acid Cycle Metabolism.

Metabolism of [(14)C]glutamate was studied in leaf discs of Vicia faba L. in light and in darkness. In white light glutamine was the main labeled product. In the dark label was principally in compounds closely associated with tricarboxylic acid cycle metabolism, predominantly aspartate. Entry of label from glutamate into tricarboxylic acid metabolism appeared to be at least partially by decarboxylation of glutamate to gamma-amino butyric acid, followed by conversion to succinate. 3-(3,4-dichlorophenyl)-1, 1-Dimethylurea inhibited light-enhanced synthesis of glutamine and caused reversion toward the dark pattern of metabolism. Methionine sulfoximine severely inhibited glutamine synthesis and caused accumulation of labeled malate.Monochromatic 650 nanometer light gave similar results to white light. Monochromatic light of 710 nanometers had a much smaller effect on glutamine synthesis but did significantly raise the ratio of labeled malate to aspartate. gamma-Amino [(14)C]butyric acid was metabolized entirely via tricarboxylic acid cycle metabolism in light or dark, and in the light the ratio of labeled malate to aspartate was raised.These results suggest that illuminated leaves metabolize glutamate to glutamine mainly in the chloroplasts. When chloroplastic glutamine synthesis fails to take place, either in darkness or in the presence of inhibitors, glutamate is apparently metabolized outside the chloroplast. Light lowers the NAD(+) to NADH ratio outside the chloroplast, consequently altering the equilibrium of the malate dehydrogenase reaction. Alteration of the malate to aspartate ratio by 710 nanometer light suggests that ATP generated by photosystem I-dependent cyclic photophosphorylation may affect extrachloroplastic NAD(+) to NADH ratios.

Journal Article↗
PubMed1988

NMR study of rat diaphragm exposed to metabolic and compensated metabolic acidosis.

When exposed to hypercapnia, several muscles deteriorate with respect to their mechanical performance. Exposure to metabolic acidosis and, perhaps surprisingly, to compensated metabolic acidosis has the same effect on the diaphragm. The mechanisms involved in these effects remain unclear. If the diaphragmatic intracellular pH (pHi) is assumed to decrease with hypercapnia, to remain unchanged during metabolic acidosis, and to increase during compensated metabolic acidosis, it would appear that different mechanisms must be responsible for the depreciation in the diaphragm's mechanical performance. The present experiments using 31P nuclear magnetic resonance (31P-NMR) spectroscopy were undertaken to determine the effect of metabolic acidosis and compensated metabolic acidosis on pHi and on high-energy phosphate metabolites in the resting rat diaphragm. A whole diaphragm was slightly stretched while being stitched onto a fiberglass mesh. The area approximated that at functional residual capacity. It was superfused in the NMR sample tube with a phosphate-free Krebs-Ringer bicarbonate solution [( HCO3-] = 6 meqO equilibrated with either 95% O2-5% CO2 or 98.75% O2-1.25% CO2). Spectra were acquired during 15-min intervals for control (30 min of normal Krebs-Ringer bicarbonate superfusate, equilibrated with 95% O2-5% CO2), for 120 min of exposure to either form of acidosis and for 60 min of recovery with normal superfusate. The pHi decreased rapidly during metabolic acidosis but did not change significantly during compensated metabolic acidosis. In both forms of acidosis, phosphocreatine declined gradually but not significantly, whereas ATP and inorganic phosphate did not change at all. The results suggest that HCO3- passes freely through the diaphragmatic sarcolemma, very much like the cardiac sarcolemma.(ABSTRACT TRUNCATED AT 250 WORDS)

Acidosis↗
PubMed2001

Metabolic and endocrine effects of metabolic acidosis in humans.

Metabolic acidosis is an important acid-base disturbance in humans. It is characterised by a primary decrease in body bicarbonate stores and is known to induce multiple endocrine and metabolic alterations. Metabolic acidosis induces nitrogen wasting and, in humans, depresses protein metabolism. The acidosis-induced alterations in various endocrine systems include decreases in IGF-1 levels due to peripheral growth hormone insensitivity, a mild form of primary hypothyroidism and hyperglucocorticoidism. Metabolic acidosis induces a negative calcium balance (resorption from bone) with hypercalciuria and a propensity to develop kidney stones. Metabolic acidosis also results in hypophosphataemia due to renal phosphate wasting. Negative calcium balance and phosphate depletion combine to induce a metabolic bone disease that exhibits features of both osteoporosis and osteomalacia. In humans at least, 1,25-(OH)2 vitamin D levels increase, probably through phosphate depletion-induced stimulation of 1-alpha hydroxylase. The production rate of 1,25-(OH)2 vitamin D is thus stimulated, and parathyroid hormone decreases secondarily. There is experimental evidence to support the notion that even mild degrees of acidosis, such as that occurring by ingestion of a high animal protein diet, induces some of these metabolic and endocrine effects. The possible role of diet-induced acid loads in nephrolithiasis, age-related loss of lean body mass and osteoporosis is discussed.

Acidosis↗
PubMed2001

Metabolism of dimethyl-4,4'-dimethoxy-5,6,5',6'-dimethylene dioxybiphenyl-2,2'-dicarboxylate (DDB) by human liver microsomes: characterization of metabolic pathways and of cytochrome P450 isoforms involved.

Metabolic fate of DDB and identification of P450 isozymes involved in the metabolism of DDB were investigated in human liver microsomes. DDB was rapidly metabolized to five different metabolites, and the structures of each metabolite were characterized based on UV, mass, and NMR spectral analyses. The major metabolic pathways of DDB in human liver microsomes were identified as O-demethylation of the carboxymethyl moiety (M4) and demethylenation of the methylenedioxyphenyl group (M2). The intramolecular lactonization between the hydroxyl group at the C6 and carboxymethyl group at the C2' of M2 resulted in the generation of M5, which was either hydrolyzed to its hydrolyzed derivative (M1) or further metabolized to the O-demethylated derivative (M3). The interconversion of M1, M2, and M5 took place nonenzymatically depending on the solvent condition. M5 was predominantly detected at the acidic condition, whereas M1 was preferentially detected at the basic environment. Cytochrome P450 (P450) isoform(s) involved in the metabolism of DDB was identified using several in vitro approaches. Chemical inhibition using isoform-selective P450 inhibitors, correlation of DDB metabolites formation with several isoform-specific P450 activities in a panel of liver microsomes, metabolism by microsomes derived from P450 cDNA-expressed B-lymphoblastoid cells, and immunoinhibition by isoform-specific anti-P450 antibodies collectively indicated that CYP1A2, CYP2C9, and CYP3A4 are responsible for the metabolism of DDB. O-Dealkylation of the carboxymethyl group was preferentially catalyzed by CYP1A2, whereas demethylenation of the methylenedioxyphenyl moiety was catalyzed by CYP3A4 and CYP2C9.

Antibodies↗
PubMed2003

MetaFluxNet, a program package for metabolic pathway construction and analysis, and its use in large-scale metabolic flux analysis of Escherichia coli.

We have developed MetaFluxNet which is a stand-alone program package for the management of metabolic reaction information and quantitative metabolic flux analysis. It allows users to interpret and examine metabolic behavior in response to genetic and/or environmental modifications. As a result, quantitative in silico simulations of metabolic pathways can be carried out to understand the metabolic status and to design the metabolic engineering strategies. The main features of the program include a well-developed model construction environment, user-friendly interface for metabolic flux analysis (MFA), comparative MFA of strains having different genotypes under various environmental conditions, and automated pathway layout creation. The usefulness and functionality of the program are demonstrated by applying to metabolic pathways in E. coli. First, a large-scale in silico E. coli model is constructed using MetaFluxNet, and then the effects of carbon sources on intracellular flux distributions and succinic acid production were investigated on the basis of the uptake and secretion rates of the relevant metabolites. The results indicated that among three carbon sources available, the most reduced substrate is sorbitol which yields efficient succinic acid production. The software can be downloaded from http://mbel.kaist.ac.kr/.

Computer Simulation↗