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PubMed2002

Formate kinetics in methanol poisoning.

OBJECTIVE: We sought to describe the kinetics, dialysis clearance, and laboratory markers of formate (FA), the toxic metabolite of methanol (meOH). METHODS: Data were obtained from a prospective, multicenter study of fomepizole +/- dialysis for methanol poisoning. Inclusion criteria confirmed methanol exposure or suspicion of exposure plus either acidemia or abnormal osmolar gap. Dialysis indications were [meOH] > 50 mg/dL, pH < 7.1, refractory acidosis, or visual toxicity. Serial plasma formate, methanol, pH, and electrolyte measurements were made. Formate was determined by gas chromatography. Endogenous and dialysis elimination half-lives were calculated as t(1/2) = 0.693/Ke, with Ke (elimination constant) derived from the slope of log (FA) vs. time. Half-lives were compared with an unpaired Student's t-test. Dialysis clearance was calculated using the Fick Principle. Pearson correlation analysis compared initial formate with initial pH, serum bicarbonate, and anion gap. RESULTS: Eleven patients were treated in the study. Eight had detectable formate with mean [FA] of 15.1 mmol/L (range 0.5-34.8). Endogenous elimination half-life was 205 +/- 90 minutes. Elimination half-life during dialysis (n = 5) was 150 +/- 37 minutes, which was not different (t = 0.22; NS). The overall dialysis formate clearance rate was 223 +/- 25 mL/min. Correlation coefficients were: pH vs. formate r2 = 0.93; bicarbonate vs. formate r2 = 0.81; and anion gap vs. formate r2 = 0.76 (all p < 0.05). CONCLUSIONS: Although dialysis clears formate, it did not significantly enhance endogenous elimination in our series of patients. Low pH, low bicarbonate, and elevated anion gap correlate independently with formate presence.

Adult↗

PubMed1971

Effects of molybdate and selenite on formate and nitrate metabolism in Escherichia coli.

The effects of adding molybdate and selenite to a glucose-minimal salts medium on the formation of enzymes involved in the anaerobic metabolism of formate and nitrate in Escherichia coli have been studied. When cells were grown anaerobically in the presence of nitrate, molybdate stimulated the formation of nitrate reductase and a b-type cytochrome, resulting in cells that had the capacity for active nitrate reduction in the absence of formate dehydrogenase. Under the same conditions, selenite in addition to molybdate was required for forming the enzyme system which permits formate to serve as an effective electron donor for nitrate reduction. When cells were grown anaerobically on a glucose-minimal salts medium without nitrate, active hydrogen production from formate as well as formate dehydrogenase activity depended on the presence of both selenite and molybdate. The effects of these metals on the formation of formate dehydrogenase was blocked by chloramphenicol, suggesting that protein synthesis is required for the increases observed. It is proposed that the same formate dehydrogenase is involved in nitrate reduction, hydrogen production, and in aerobic formate oxidation.

Chloramphenicol↗

PubMed1996

Inducible cyclo-oxygenase (COX-2) mediates the induction of bone formation by mechanical loading in vivo.

In vivo, indomethacin blockade of bone formation has been used to illustrate the role of prostaglandins. Indomethacin blocks the constitutive (COX-1) and inducible (COX-2) forms of cyclo-oxygenase, and is therefore nonspecific in its action. To test the hypothesis that COX-2 mediates the bone formation response to loading, rats were treated with vehicle, NS-398 (a specific COX-2 inhibitor) or indomethacin at 0.02, 0.2, or 2.0 mg/kg p.o. 3 h before loading the right tibia in four-point bending. Bending or sham loads of 65 N were applied for one bout of 300 cycles and bone formation assessed 5-8 days after loading. Mechanically induced bone formation at the endocortical surface was calculated by subtracting formation indices of the left leg (control) from those of the right (loaded), and woven bone surface and area were measured at the periosteal surface. Endocortical bone formation was significantly increased by bending but not sham loading (p < 0.05). The increase in the endocortical bone formation rate and mineralizing surface caused by bending was only partially inhibited by indomethacin, even at the highest dose, whereas NS-398 completely blocked bone formation at all doses (p < 0.05). The mineral apposition rate was depressed in a dose-response fashion by NS-398 (p < 0.05), but not by indomethacin. Woven bone formation at the periosteal surface was not prevented by treatment with indomethacin nor NS-398, suggesting that its formation is not dependent on prostaglandin production. These data suggest that induction of COX-2 is important for lamellar bone formation elicited by mechanical strain.

Analysis of Variance↗

PubMed1994

Mechanism of 3-(glutathion-S-yl)-benzidine formation.

The formation of thioether conjugates is an important mechanism for inactivation of carcinogens. 3-(Glutathion-S-yl)-benzidine (BZ-SG) formation prevents benzidinediimine and peroxidase-mediated benzidine binding to DNA. Benzidinediimine is the two-electron oxidized product of benzidine thought to be the reactive intermediate involved in peroxidase-mediated binding of benzidine to DNA. Diimine interacts with benzidine to form a dimeric complex known as the charge-transfer complex. The latter is in equilibrium with the cation radical. This study evaluated the mechanism by which BZ-SG forms. Benzidinediimine was synthesized and used to study the formation of BZ-SG. With 0.05 mM benzidinediimine, BZ-SG formation was optimum at pH 4.5 and with glutathione at 0.05 to 0.1 mM. By monitoring specific absorption spectra, the reduction of benzidinediimine at pH 4.5 was evaluated. The t1/2 for diimine decay (425 nm) and maximum absorbance of the charge-transfer complex (600 nm) were each at approximately 5 min. Within 10 min, the maximum amount of benzidine had formed from diimine. BZ-SG formation followed the decay of diimine. The relationship between benzidinediimine and benzidine, with respect to BZ-SG formation, was assessed at a fixed concentration of glutathione (0.05 mM) and a fixed total concentration of amine and diimine (0.05 mM). In three separate experiments, each of these three components was radiolabeled independent of the other two components. Experiments with [3H]glutathione indicated that conjugate formation was dependent upon diimine, and not benzidine. With [3H]benzidinediimine or [3H]benzidine, two different calculations were necessary to assess conjugate formation. For [3H]benzidinediimine, the calculation considered that only the radiolabeled diimine formed conjugate, while with [3H]benzidine, a specific activity calculation was necessary to demonstrate that conjugate formation was dependent upon diimine. With 0.05 mM [3H]benzidine, horseradish peroxidase-catalyzed formation of BZ-SG was optimum between 0.05 and 0.0625 mM H2O2. The latter is consistent with conversion of benzidine to diimine before formation of BZ-SG. Specific inhibitors and the absence of oxygen uptake indicated the lack of involvement of cation, thiyl, and carbon-centered radicals. The results are consistent with the existence of the charge-transfer complex and with benzidinediimine reacting with glutathione to form BZ-SG.

Benzidines↗

PubMed1994

Effects of lead on osteoclast-like cell formation in mouse bone marrow cell cultures.

To examine an effect of lead (Pb) on the process of osteoclast-like cell formation from its progenitors, we used a mouse bone marrow culture system in which osteoclast-like multinucleated cells (MNCs) were formed in response to bone-resorbing agents. In a 9-day culture period, Pb dose-dependently stimulated MNC formation over the concentration range 2-10 microM, whereas at 40 microM Pb, MNC formation declined. In an 11-day culture period, MNC formation reached a maximum at 5 microM Pb and decreased with increasing concentration of Pb at 10-40 microM. Pb-stimulated MNC formation was inhibited by both indomethacin and SC19220, an antagonist of prostaglandin E2 (PGE2) receptor. Pb stimulated the production of PGE2 in marrow cell cultures, suggesting that Pb-stimulated MNC formation is dependent on the production of PGE2. 3-Isobutyl-1-methylxanthine potentiated Pb-stimulated MNC formation and 2',5'-dideoxyadenosine, an inhibitor of adenylate cyclase, inhibited it. A calcium ionophore A23187 increased Pb-induced MNC formation and verapamil, a calcium channel blocker, depressed it. It is possible that a PGE2-induced increase in the levels of cyclic adenosine 3',5'-monophosphate (cAMP) and calcium ions in marrow cells is involved in Pb-induced MNC formation. Pb and parathyroid hormone showed a synergistic stimulation on MNC formation. From these results, Pb is thought to induce osteoclast-like cell formation by a mechanism involving PGE2 which increases the intracellular levels of cAMP and calcium ions.

1-Methyl-3-isobutylxanthine↗

PubMed1989

Study of amino acid formation during palmitate oxidation in rat brain mitochondria.

The interrelation of palmitate oxidation with amino acid formation in rat brain mitochondria has been investigated in purified mitochondria of nonsynaptic origin by measuring the formation of aspartate, alpha-ketoglutarate, and glutamate during palmitate oxidation, and also by assaying 14C-products of [1-14C]palmitate oxidation. Oxidation of palmitate (or [1-14C]palmitate) resulted in the formation of aspartate (or 14C-aspartate), and the oxidation was inhibited by aminooxyacetate (an inhibitor of transaminase). Palmitate oxidation also resulted in alpha-ketoglutarate formation, which was sensitive to the effect of aminooxyacetate. Addition of NH4Cl was found to increase 14C-products and formation of alpha-ketoglutarate, whereas glutamate formation was not increased unless the rate of palmitate oxidation was reduced by 50% by aminooxyacetate or alpha-ketoglutarate was added exogenously. Exogenous alpha-ketoglutarate was found to decrease 14C-products, but not aspartate formation. These results indicated that palmitate oxidation was closely related to aspartate formation via aspartate aminotransferase. During palmitate oxidation without aminooxyacetate or added alpha-ketoglutarate, however, alpha-ketoglutarate was not available for glutamate formation via glutamate dehydrogenase. We discuss the possibility that this was because (a) oxidative decarboxylation of alpha-ketoglutarate to form succinyl-CoA was favored over glutamate formation for the competition for alpha-ketoglutarate in the same pool, and (b) the pool of alpha-ketoglutarate produced in the aspartate aminotransferase reaction did not serve as substrate for glutamate formation.

Aminooxyacetic Acid↗

PubMed1993

Adipose glycerolipid formation: effect of nutritional and hormonal states.

The potential of glycerolipid formation from sn-glycerol-3-phosphate (GP) and 2-monoacylglycerol (MG) was studied in adipose microsomal fractions under various nutritional and hormonal states. Glycerolipid formation from GP was followed in the presence of [14C]glycerol-3-phosphate and palmitoyl-CoA and was assayed by measuring the formation of butanol-soluble product, consisting mainly of [14C]phosphatidate. Glycerolipid formation from MG was determined in the presence of 2-mono-oleyl glycerol and [14C]palmitoyl-CoA, and was estimated by the formation of both [14C]di- and triacylglycerol. Glycerolipid formation from GP was decreased significantly during food deprivation, in experimental diabetes, in the presence of lipolytic hormone, and during aging. Glycerolipid formation from MG did not change under these conditions and continued at the same rate as observed in control animals. The rate of glycerolipid formation from GP was 7-20 times greater than from MG in the various fat depots. Measurement of the adipose monoacylglycerol concentration did not show any correlation with the glycerolipid formation from MG. The studies suggest that glycerolipid formation from MG is active in various fat depots, and is substantial when glycerolipid formation via GP is impaired.

Adipose Tissue↗

PubMed1999

The analgesic NSAID lornoxicam inhibits cyclooxygenase (COX)-1/-2, inducible nitric oxide synthase (iNOS), and the formation of interleukin (IL)-6 in vitro.

OBJECTIVE: To investigate anti-inflammatory effects of lornoxicam in vitro on COX-1/COX-2, on NO formation from iNOS and on the formation of the pro-inflammatory cytokines TNF-alpha, IL-1beta, IL-6, and IL-8. MATERIALS AND METHODS: COX-1 inhibition in intact cells was assessed employing two systems: measurement of aggregation in human washed platelets and assessment of TXB2 formation in HEL cells. COX-2 inhibition was assessed by measuring 6-keto-PGF1alpha in supernatants of intact cells of LPS-stimulated J774.2 cells (murine) and of Mono Mac 6 cells (human). In whole blood inhibition of COX-1 was performed by measuring TXB2 formation after clotting, and COX-2 inhibition was examined in LPS-stimulated whole blood cultures. The reduction of NO levels as a measure of the inhibition of cellular NO formation was assayed in supernatants of LPS-stimulated RAW 264.7 cells using the Griess reaction. Compound influence on the formation ofTNF-alpha, IL-1beta, IL-6, and IL-8 was examined using LPS-stimulated monocytic cells (THP-1) and measurement of cytokine concentrations by specific ELISAs. RESULTS: In intact human cells, lornoxicam showed a balanced inhibition of COX-1/-2 exhibiting the lowest IC50 (0.005 microM/0.008 microM) of the large panel of NSAIDs tested. Similar results were obtained in the whole blood for COX-1/-2. NO formation was dose-dependently inhibited by lornoxicam (IC50 of 65 microM) whereas piroxicam, diclofenac, ibuprofen, ketorolac and naproxen inhibited the NO formation markedly less. Indomethacin was approximately equipotent with lornoxicam. In stimulated monocytic cells (THP-1), lornoxicam showed a marked inhibition of IL-6 formation (IC50 54 microM) while the formation ofTNF-alpha, IL-1beta and IL-8 was only moderately affected. CONCLUSIONS: Of the panel of NSAIDs tested, lornoxicam was found to be the most potent balanced inhibitor of human COX-1/-2. The equipotent COX-isoenzyme inhibition by lornoxicam is complemented by a marked inhibition of IL-6 production and of iNOS-derived NO formation. The in vitro activities described support the marked anti-inflammatory and analgesic activities of lornoxicam found in animal models as well as in clinical studies.

Animals↗

PubMed1988

Normal and warfarin-resistant rat hepatocyte metabolism of vitamin K 2,3-epoxide: evidence for multiple pathways of hydroxyvitamin K formation.

Vitamin K and 3- (and/or 2)-hydroxy-2,3-dihydro-2-methyl-3-phytyl-1,4-naphthoquinone (hydroxyvitamin K) have been identified as metabolites of vitamin K 2,3-epoxide incubated with hepatocytes isolated from normal and warfarin-resistant rats. Dithiothreitol added to the extracellular medium differentially enhanced the formation of both metabolites: hydroxyvitamin K formation, almost undetectable in the absence of dithiothreitol, was particularly affected. Addition of the vitamin K 2,3-epoxide reductase inhibitors warfarin (5 to 100 microM) and brodifacoum (1 to 5 microM) to normal rat hepatocyte cultures produced a slight increase in hydroxyvitamin K formation and a marked inhibition of vitamin K formation. Brodifacoum was a weak inhibitor of hydroxyvitamin K formation at higher concentrations. Hepatocytes from warfarin-resistant rats catalyzed hydroxyvitamin K formation 1.5 to 2 times faster and vitamin K formation 1.5 to 2 times slower than did normal rat hepatocytes. The addition of warfarin to these cultures had no effect on epoxide metabolism to hydroxyvitamin K and only partially diminished metabolism to vitamin K. In contrast, brodifacoum (1 microM) addition produced 50% inhibition of hydroxyvitamin K formation and almost complete inhibition of vitamin K formation. These data suggest that in resistant, but not in normal rat hepatocytes, the vitamin K 2,3-epoxide reductase makes a significant contribution to hydroxyvitamin K formation. A second sulfhydryl-dependent pathway, present in both strains, is also involved in the formation of this metabolite. They also suggest that in resistant rats, warfarin inhibition of the vitamin K 2,3-epoxide reductase, and presumably the sulfhydryl-dependent vitamin K reductase, is incomplete and independent of concentration.

Animals↗

PubMed1989

Leukotriene formation by human polymorphonuclear leukocytes from endogenous arachidonate. Physiological triggers and modulation by prostanoids.

Human polymorphonuclear leukocytes (PMN) were isolated from freshly drawn venous blood by Dextran sedimentation and discontinuous Percoll gradient centrifugation. The effects of several putative triggers of the leukotriene formation such as C5a, PAF, FMLP, C3a, PMA, LTC4, LTD4, LTB4 or arachidonate were studied by RP-HPLC analysis. 280 nM C5a, 100 nM FMLP, 1 microM PAF or 20 microM arachidonate induced a marginal formation of 1.5-18 ng of LTB4 plus LTB4 metabolites/2 x 10(7) PMN. 560 nM C3a, 100 nM PMA, 1 microM LTC4, 1 microM LTD4 and 1 microM LTB4 each failed to induce any formation of 5-lipoxygenase products. Pretreatment of the cells with 40 microM ethylmercurithiosalicylate (merthiolate) enhanced the leukotriene formation by 100 nM FMLP about 40-fold, by 280 nM C3a about 120-fold and by 1 microM PAF about 14-fold. Merthiolate itself induced no leukotriene formation from human PMN and reduced the leukotriene formation by 20 microM arachidonate. The FMLP/merthiolate-induced activation of the PMN was concentration-dependent in respect to both FMLP and merthiolate. 1 microM LTC4, 1 microM LTD4 or 1 microM LTB4 also failed to trigger any LTB4 formation of merthiolate-treated PMN. 560 nM C3a or 100 nM PMA in combination with 40 microM merthiolate induced a slight formation of 28 ng and 10 ng of LTB4 plus LTB4 metabolites, respectively. The FMLP/merthiolate-induced leukotriene formation was modulated by prostanoids. PGE2, PGE1, PGD2 and 6-keto-PGE1 each evoked a concentration-dependent inhibition of the leukotriene formation with IC50 values of 0.07 microM, 0.18 microM, 0.27 microM and 6 microM respectively. In addition, significant inhibitory effects by PGI2, Iloprost (a carbacyclin analogue of prostacyclin), PGF2a or 6-keto-PGF1a were achieved; the corresponding IC50 values, however, amounted to 19-59 microM. Thus these compounds were about 500-fold less potent in comparison with PGE2 in inhibiting LTB4 formation by human PMN.

Arachidonic Acids↗

PubMedDate not supplied

Estrogen induces bone formation on non-resorptive surfaces in the rat.

We have recently found that 17 beta-estradiol (E2) stimulates bone formation in rat cancellous bone, and that this bone formation is suppressed by (3-amino-1-hydroxypropylidene)-1-bisphosphonate (AHPrBP). To analyse the relationship between bone resorption and bone formation in the action of E2, we injected 13-week-old female rats sequentially with three fluorochromes (calcein, tetracycline and xylenol orange) at 7-day intervals. E2 (40 micrograms/kg) or vehicle was injected daily for 15 days, starting 24 hrs after the first fluorochrome. A third group was injected with AHPrBP (0.3 mg/kg) 24 hrs after the first two fluorochromes. The rats were killed 48 hrs after the third fluorochrome. We found that the perimeter of all three fluorochrome labels was increased by E2. The entire perimeter of the first label was non-crenated. Since the first label was given before E2-administration, this suggests that label that would otherwise have been eluted from the bone surface had been fixed in bone by E2-induced bone formation, which might have occurred either through prolongation of pre-existing bone formation, or induction of bone formation on quiescent surfaces. In either case, our results suggest that resorption did not precede formation at the site of bone formation. Since induction of bone formation by E2 is suppressed by inhibition of bone resorption, this suggests that the coupling of E2-induced formation to resorption in the rat does not necessarily require that formation occurs at the same site as bone resorption.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2004

Biochemical markers of bone formation reflect endosteal bone loss in elderly men--MINOS study.

In the skeleton of elderly men, two opposite activities occur: bone loss at the endosteal envelope, which increases bone fragility, and periosteal apposition, which improves bending strength of bone. Both may contribute to serum bone formation markers although they have an opposite effect on bone fragility. The aim of this study was to determine if circulating bone formation markers reflect periosteal bone formation and endosteal bone remodelling in 640 men aged 55-85 years belonging to the MINOS cohort. We measured biochemical markers of bone formation (osteocalcin, bone alkaline phosphatase, N-terminal extension propeptide of type I collagen) and bone resorption (urinary and serum beta-isomerised C-terminal telopeptide of collagen type I, total and free deoxypyridinoline). Parameters of bone size (cross-sectional surface of third lumbar vertebral body measured by X-ray, projected areas of total hip, femoral neck, radius and ulna measured by dual-energy X-ray absorptiometry) increased with age (r = 0.20-0.32, P < 0.0001). In contrast, parameters related to bone loss (areal bone mineral density [aBMD], volumetric bone mineral density [vBMD] and cortical thickness) and determined mainly by bone resorption, decreased with ageing (r = -0.14 to -0.23, P < 0.005-0.0001). Men in the highest quartile of bone resorption markers had lower aBMD (3.8-10.2%, P < 0.05-0.0001), lower vBMD (3.9-13.0%, P < 0.05-0.0001), and lower cortical thickness (1.5-9.6%, P < 0.05-0.0001) than men in the lowest quartile. Markers of bone resorption were not significantly associated with estimates of bone size at any skeletal site. Markers of bone formation were not associated with estimates of periosteal formation after adjustment for covariates. In contrast, men in the highest quartile of the bone formation markers had significantly lower aBMD (4.0-11.7%, P < 0.05-0.0001), lower vBMD (4.2-16.3, P < 0.05-0.0001) and lower cortical thickness (4.0-7.4%, P < 0.05-0.0001) than men in the lowest quartile. In summary, serum levels of bone formation markers are negatively correlated with the estimates of endosteal bone loss. In contrast, they disclose no association with parameters reflecting periosteal apposition. Thus, in elderly men, bone formation markers reflect endosteal bone remodelling, probably because of the coupling between resorption and formation activities. In contrast, they do not reflect the periosteal bone formation, probably because the periosteal surface is smaller and has a slower remodelling rate than the endosteal surface.

Absorptiometry, Photon↗

PubMed1993

Inhibitory effect of interleukin-4 on osteoclast-like cell formation in mouse bone marrow culture.

Recently, interleukin 4 (IL-4) was reported to inhibit bone resorption in mouse long bone culture. To test the effect of IL-4 on the formation of osteoclast-like cells, we used a mouse bone marrow culture system that formed mononuclear and multinucleated cells with osteoclast characteristics. Recombinant mouse IL-4 dose-dependently inhibited the formation of tartrate-resistant acid phosphatase-positive multinucleated cells [TRAP(+)MNC] induced by 1,25(OH)2D3, PTH(1-34) or Interleukin-1 alpha (IL-1 alpha). The minimum effective inhibitory concentration was 0.01 ng/ml, and 1 ng/ml IL-4 completely inhibited TRAP(+)MNC formation. IL-4 also dose-dependently inhibited the formation of tartrate-resistant acid phosphatase-positive mononuclear cells. Treatment of cultures with IL-4 for the first or last 48 h of an 8-day culture period inhibited TRAP(+)MNC formation to the same extent, whereas IFN-gamma and calcitonin suppressed TRAP(+)MNC formation mainly at the early and the late phase, respectively. IL-4, as a macrophage fusion factor, at higher concentrations (0.1-10 ng/ml), increased formation of tartrate-resistant acid phosphatase-negative multinucleated cells [TRAP(-)MNC] with giant macrophage characteristics. The half-maximal concentrations inhibiting TRAP(+)MNC formation and stimulating TRAP(-)MNC formation were 0.05 ng/ml and 2 ng/ml, respectively. These results demonstrate that IL-4 inhibits bone resorption by inhibiting the recruitment of osteoclast precursor and formation of multinucleated osteoclast-like cells, and by stimulating the formation of macrophage polykaryons.

Acid Phosphatase↗

PubMed2003

The effects of nitric oxide and peroxynitrite on the formation of prostaglandin and arachidonoyl-CoA formed from arachidonic acid in rabbit kidney medulla microsomes.

Under physiological conditions, small amounts of free arachidonic acid (AA) are released from membrane phospholipids, and cyclooxygenase (COX) and acyl-CoA synthetase (ACS) competitively act on this fatty acid to form prostaglandins (PGs) and arachidonoyl-CoA (AA-CoA). To clarify factors deciding the metabolic fate of free AA into these two pathways, we investigated the effects of a nitric oxide (NO) donor 1-hydroxyl-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene (NOC7), and peroxynitrite (ONOO(-)) on the formation of PG and AA-CoA from high and low concentrations of AA (60 and 5 micro M) in rabbit kidney medulla microsomes. The kidney medulla microsomes were incubated with 60 or 5 micro M [14C]-AA in 0.1M Tris/HCl buffer (pH 8.0) containing cofactors of COX (reduced GSH and hydroquinone) and cofactors of ACS (ATP, MgCl(2) and CoA). After incubation, PG (as total PGs) and AA-CoA were separated by selective extraction using petroleum ether and ethyl acetate. When 60 micro M AA was used as the substrate concentration, NOC7 stimulated the PG formation at 0.5 micro M, and inhibited it at 50 and 100 micro M, without affecting the AA-CoA formation. When 5 micro M AA was used as the substrate concentration, NOC7 showed no effect on the PG and AA-CoA formation up to 10 micro M or below, but enhanced the AA-CoA formation with a coincident decrease in the PG formation at 50 micro M or over. Experiments utilizing a NO antidote, carboxy-2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide, revealed that the observed effects of NOC7 using 60 and 5 micro M AA are caused by NO. On the other hand, ONOO(-) stimulated the PG formation from 60 micro M AA, with no alteration in the AA-CoA formation at a concentration of 100 micro M, but when 5 micro M AA was used as the substrate concentration, it was without effect on the PG and AA-CoA formation. These findings indicate that actions of NO and ONOO(-) on the PG and AA-CoA formation by the kidney medulla microsomes may change depending on the substrate concentration. The effects of NO using 5 micro M AA were reversed by the addition of the superoxide generating system (xanthine-xanthine oxidase plus catalase), indicating that superoxide is a vital modulator of the action of NO. These results suggest that NO, but not ONOO(-), can be a regulator of the PG and AA-CoA formation at low substrate concentrations (close to the physiological concentration of AA), and that superoxide may play an important role in the action of NO.

Acyl Coenzyme A↗

PubMed2003

Key parameters for de novo formation of polychlorinated dibenzo-p-dioxins and dibenzofurans.

De novo formation of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDDs and PCDFs) was investigated in an Entrained Flow Reactor (EFR) to simulate combustion conditions. The parameters investigated were carbon content and nature in fly ash; type of gas-phase environment (oxidative versus reducing conditions) influence of combustion gases such as water, carbon monoxide, and carbon dioxide; amount of gas-phase chlorine; reaction temperature (250-600 degrees C); and reaction time (minutes vs hours). The comprehensive data set was further evaluated with principal component analysis (PCA) to statistically determine the role and importance of each parameter for de novo formation of PCDDs and PCDFs. Results revealed that an initial fast de novo formation occurs within the first minutes with a formation rate in the orders of hundreds of pmol per minutes; however, the reactivity of the ash was found to decline with time. An average formation rate as low as 3 pmol/min was measured after 6 h. The slower de novo formation of PCDDs and PCDFs was found to be through different reaction mechanisms and, thus, controlled by different parameters. The amount of Cl2 in the gas phase was observed to be an important parameter for PCDFs formation; meanwhile the levels of O2 were not found to be a PCDF rate controlling parameter. The formation rate of PCDDs was significantly lower than the PCDFs, and two mechanisms appear to be controlling the formation, one depending on the amount of O2 and one on the amount of Cl2 present in the gas phase. Overall the most significant parameter for the rate of formation for both PCDDs and PCDFs was revealed to be the reaction temperature. A maximum rate of formation was observed between 300-400 degrees C for the PCDDs and 400-500 degrees C for the PCDFs.

Benzofurans↗

PubMed2006

Modeling the formation of N-nitrosodimethylamine (NDMA) from the reaction of natural organic matter (NOM) with monochloramine.

This paper presents mechanistic studies on the formation of NDMA, a newly identified chloramination disinfection byproduct, from reactions of monochloramine with natural organic matter. A kinetic model was developed to validate proposed reactions and to predict NDMA formation in chloraminated water during the time frame of 1-5 days. This involved incorporating NDMA formation reactions into an established comprehensive model describing the oxidation of humic-type natural organic matter by monochloramine. A rate-limiting step involving the oxidation of NOM is theorized to control the rate of NDMA formation which is assumed to be proportional to the rate of NOM oxidized by monochloramine. The applicability of the model to describe NDMA formation in the presence of three NOM sources over a wide range in water quality (i.e., pH, DOC, and ammonia concentrations) was evaluated. Results show that with accurate measurement of monochloramine demand for a specific supply, NDMA formation could be modeled over an extended range of experimental conditions by considering a single NOM source-specific value of thetaNDMA, a stoichiometric coefficient relating the amount of NDMA produced to the amount of NOM oxidized, and several kinetic parameters describing NOM oxidation. Furthermore, the oxidation of NOM is the rate-limiting step governing NDMA formation. This suggests that NDMA formation over a 1-5 day time frame may be estimated from information on the chloramine or free chlorine demand of the NOM and the source-specific linear relationship between this demand and NDMA formation. Although the proposed model has not yet been validated for shorter time periods that may better characterize the residence time in some distribution systems, the improved understanding of the important reactions governing NDMA formation and the resulting model should benefit the water treatment industry as a tool in developing strategies that minimize NDMA formation.

Ammonia↗

PubMed1978

A study of regulation of gluconeogenesis and the supply of cytosolic reducing equivalents for lactate formation in rat kidney-cortical-tubule fragments incubated with pyruvate.

1. Tubule fragments were isolated after treatment of rat kidney cortex with collagenase. The formation of glucose and lactate on incubation with 5mM-pyruvate was then measured under various conditions. 2. When tubule fragments were isolated from fed rats in the absence of Ca2+ and then incubated with various Ca2+ concentrations, an incubation period of 15--30 min was necessary to establish a metabolic steady state. Under these conditions glucose formation was increased by Ca2+, adrenaline or 3':5'-cyclic AMP to a greater extent than was lactate formation. Data show that appreciable lactate formation could not have resulted from glycolytic metabolism of glucose formed by gluconeogenesis during incubation. 3. When tubule fragments were isolated from fed rats in the presence of 1.27 mM-Ca2+ and adjustments made to the Ca2+ concentration at the commencement of incubation, metabolic steady state was rapidly established. Under these conditions lactate formation was almost insensitive to Ca2+ concentration (0.16--4.5 mM), whereas glucose formation varied with Ca2+ concentration in a sigmoidal manner. 3':5'-Cyclic AMP decreased this sigmoidicity. 4. Ca2+ depletion of the tissue before incubation appeared to change permanently the relationship between extracellular Ca2+ concentration and the measured rates of metabolic processes. 5. Under conditions of metabolic steady state, glucose formation by tubule fragments from fed rats was less sensitive than lactate formation to inhibition by 3-mercaptopicolinate or 2-n-butylmalonate. Lactate formation by tubule fragments prepared from 48 h-starved rats was more sensitive to these inhibitors. 6. Estimates were made of the rate of futile cycling of C3 species through pyruvate kinase. This was greater in the starved than in the fed state, was decreased by 3':5'-cyclic AMP in both the fed and the starved state, but was unaffected by Ca2+. 7. These results suggested that formation of lactate and glucose is less tightly linked in kidney cortex than in liver. A considerable amount of the supply of reducing equivalents for lactate formation did not appear to be associated with an energy-dependent translocation from mitochondria to cytosol involving a pyruvate leads to oxaloacetate leads to phosphoenolpyruvate leads to pyruvate cycle.

Aminooxyacetic Acid↗

PubMed1991

Hydroxylation of salicylate by microsomal fractions and cytochrome P-450. Lack of production of 2,3-dihydroxybenzoate unless hydroxyl radical formation is permitted.

Attack by hydroxyl radicals (.OH) upon salicylate (2-hydroxybenzoate) leads to formation of both 2,3-dihydroxybenzoate (2,3-DHB) and 2,5-dihydroxybenzoate (gentisate, 2,5-DHB). It has been suggested that formation of 2,3-DHB from salicylate is a means of monitoring .OH formation. Production of 2,3-DHB and 2,5-DHB by liver microsomal fractions and isoforms of cytochrome P-450 was investigated. Liver microsomes prepared from variously treated rats and rabbits catalysed the formation of 2,5-DHB but not 2,3-DHB. Formation of 2,5-DHB was inhibited by CO, metyrapone and SKF-525A, but not by the .OH scavengers mannitol and formate or by the iron chelator desferrioxamine. Purified P-450s IIE1, IIB4 or IA2 from rabbit liver microsomes, reconstituted together with NADPH-cytochrome P-450 reductase, led to formation of equal amounts of 2,3-DHB and 2,5-DHB in reactions that were almost completely inhibited by mannitol or formate. Addition of Fe3+/EDTA either to microsomes or to membranes containing reconstituted P-450 caused formation of approximately equal amounts of 2,3-DHB and 2,5-DHB, consistent with an .OH-dependent attack on salicylate. The data indicate that the microsomal P-450 system catalyses hydroxylation of salicylate to 2,5-DHB, but not formation of 2,3-DHB. Hence measurement of 2,3-DHB might provide a means of monitoring .OH formation. Care must be taken in studies of substrate hydroxylation by microsomes or reconstituted P-450 systems to avoid artefacts resulting from .OH generation.

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