Biomedical subjects

E G DeMaster

Publications and source records attributed to E G DeMaster.

At least 19 recordsLinked to original sources

PubMed2001

Cigarette smoke-induced endothelium dysfunction: role of superoxide anion.

OBJECTIVES: Cigarette smoking is strongly associated with coronary artery disease and atherosclerosis. While smoking has been shown to impair endothelium-dependent vasorelaxation, the mechanisms involved are not completely understood. We investigated the role of superoxide anion and vasoconstricting prostanoids in cigarette smoke induced endothelial dysfunction. METHODS: Endothelial function was assessed in rat aortic rings exposed to cigarette smoke-treated Krebs buffer, by measuring agonist stimulated endothelium-dependent vasorelaxation. Treatment with superoxide dismutase (SOD) as well as ifetroban, thromboxane A2/prostaglandin endoperoxide H2 (TxA2/PGH2) receptor blocker and indomethacin (cyclooxygenase inhibitor) was used to investigate the role of superoxide anion and vasoconstricting eicosanoids on cigarette smoke-induced endothelial dysfunction. The effect of cigarette smoke on endothelial nitric oxide synthase (eNOS) catalytic activity was measured by conversion of L-arginine to L-citrulline in rat aortas and rat endothelial cell homogenates supplemented with eNOS cofactors. RESULTS: Relaxations to receptor-dependent agonists, acetylcholine and adenosine diphosphate (ADP), as well as to a receptor-independent agonist, A23187 (Ca2+ ionophore) were significantly impaired by cigarette smoke. Cigarette smoke did not impair relaxations to sodium nitroprusside, indicating preserved guanylate cyclase activity. Further, cigarette smoke did not affect eNOS catalytic activity in homogenates from either endothelial cells or aortas previously exposed to cigarette-smoketreated Krebs buffer. Treatment with SOD or ifetroban and in a lesser degree by indomethacin prevented cigarette-smoke-induced endothelial dysfunction. CONCLUSIONS: Taken together, our results suggest that cigarette smoking causes an increase in vascular superoxide production which results in decreased nitric oxide (NO) bioactivity and concomitantly increases production of cyclooxygenase dependent and independent vasoconstricting eicosanoids.

Animals↗

PubMed2000

N-Terminal dipeptides of D(-)-penicillamine as sequestration agents for acetaldehyde.

Since acetaldehyde (AcH), a toxic oxidation product of ethanol, may play an etiologic role in the initiation of alcoholic liver disease, we had earlier pioneered the development of beta, beta-disubstituted-beta-mercapto-alpha-amino acids as AcH-sequestering agents. We now report the synthesis of a series of N-terminal dipeptides of D(-)-penicillamine, prepared from the synthon 3-formyl-2,2,5,5-tetramethylthiazolidine-4S-carboxylic acid (3), a cyclized N-protected derivative of D(-)-penicillamine. These dipeptides were equally or more effective than penicillamine in trapping AcH in a cell-free system. In experiments using a hepatocyte culture system, two of the dipeptides, D-penicillamylglycine (6a) and D-penicillamyl-beta-alanine (6d), at 1/20 the molar concentration of ethanol, lowered the concentration of ethanol-derived AcH by 79% and 84%, respectively, at 2 h. The presence of cyanamide (an inhibitor of aldehyde dehydrogenase) in the incubation medium resulted in a 45-fold increase in ethanol-derived AcH; nevertheless, dipeptides 6a and 6c (D-penicillamyl-alpha-aminoisobutyric acid) were able to reduce this AcH level by approximately one-third.

Acetaldehyde↗

PubMed2000

Reaction of nitroxyl, an aldehyde dehydrogenase inhibitor, with N-acetyl-L-cysteine.

Nitroxyl (HNO) is the aldehyde dehydrogenase (AIDH) inhibitor produced by catalase action on cyanamide. Incubation of N-acetyl-L-cysteine (NAC), a reagent with a free sulfhydryl group, with Piloty's acid (a nitroxyl generator) suggested that NAC was acting as a competitive "trap" for nitroxyl. Elucidation of the structure of this reaction product should give an insight as to how nitroxyl interacts with AIDH, a sulfhydryl enzyme. We now present evidence that the product formed is N-acetyl-L-cysteinesulfinamide (NACS). We have synthesized NACS and showed that this synthetic product was identical to the product formed in the trapping experiment. Both had identical RT values by reverse phase HPLC and identical RF values by TLC using three different solvent systems. The structural identification of this nitroxyl trapped product as a sulfinamide now allows the chemical confirmation of the active-site cysteine residue of AIDH as Cys-302.

Acetylcysteine↗

PubMed1999

Diethylcarbamoylating/nitroxylating agents as dual action inhibitors of aldehyde dehydrogenase: a disulfiram-cyanamide merger.

Benzenesulfohydroxamic acid (Piloty's acid) was functionalized on the hydroxyl group with the N,N-diethylcarbamoyl group, and the hydroxylamine nitrogen was substituted with acetyl (1a), pivaloyl (1b), benzoyl (1c), and ethoxycarbonyl (1d) groups. Only compound 1d inhibited yeast aldehyde dehydrogenase (AlDH) in vitro (IC(50) 169 microM). When administered to rats, 1d significantly raised blood acetaldehyde levels following ethanol challenge, thus serving as a diethylcarbamoylating/nitroxylating, dual action inhibitor of AlDH in vivo. A more potent dual action agent was N-(N, N-diethylcarbamoyl)-O-methylbenzenesulfohydroxamic acid (5c), which was postulated to release diethylcarbamoylnitroxyl (9), a highly potent diethylcarbamoylating/nitroxylating agent, following metabolic O-demethylation in vivo. The dual action inhibition of AlDH exhibited by 1d, and especially 9, constitutes a merger of the mechanism of action of the alcohol deterrent agents, disulfiram and cyanamide.

Acetaldehyde↗

PubMed1999

Generation of nitric oxide and possibly nitroxyl by nitrosation of sulfohydroxamic acids and hydroxamic acids.

Diazeniumdiolates (NONOates) and sulfohydroxamic acids are chemical entities that spontaneously generate nitric oxide (NO) and nitroxyl (HNO), respectively, at physiological pH and temperature. By combining the functional aspects of the NONOates with the hydroxamic acids and sulfohydroxamic acids, hybrid NONOate-type compounds that could theoretically generate nitroxyl or nitric oxide can be rationalized. Although the instability of these compounds, viz., the N-nitrosohydroxamic acids and the N-nitrososulfohydroxamic acids, precluded their chemical characterization by actual isolation, their transient existence was deduced by identification of the products of their decomposition. Thus, treatment of benzohydroxamic acid (BHA) with limiting or excess nitrous acid (from NaNO(2) and H(3)PO(4)) gave rise to quantitative generation of N(2)O, possibly via HNO, based on the limiting reactant. Nitrosation of N-t-butyloxycarbonyl hydroxylamine gave similar results. The organic acid produced from BHA was identified as benzoic acid. No nitric oxide was detected from these reactions. In contrast, treatment of Piloty's acid (benzenesulfohydroxamic acid) and methanesulfohydroxamic acid (MSHA) with nitrous acid under the same conditions as above gave 36% of the theoretical quantity of NO from Piloty's acid and 47% of NO from MSHA, although finite quantities of HNO (measured as N(2)O) were also formed. The organic acid produced from Piloty's acid was identified by reverse-phase HPLC as the redox product, benzenesulfinic acid.

Hydrazines↗

PubMed1998

Prodrugs of nitroxyl and nitrosobenzene as cascade latentiated inhibitors of aldehyde dehydrogenase.

The prototypic aromatic C-nitroso compound, nitrosobenzene (NB), was shown previously to mimic the effect of nitroxyl (HN=O), the putative active metabolite of cyanamide, in inhibiting aldehyde dehydrogenase (AlDH). To minimize the toxicity of NB in vivo, pro-prodrug forms of NB, which were designed to be bioactivated either by an esterase intrinsic to AlDH or the mixed function oxidase enzymes of liver microsomes, were prepared. Accordingly, the prodrug N-benzenesulfonyl-N-phenylhydroxylamine (3) was further latentiated by conversion to its O-acetyl (1a), O-methoxycarbonyl (1b), O-ethoxycarbonyl (1c), and O-methyl (2) derivatives. Similarly, pro-prodrug forms of nitroxyl were prepared by derivatization of the hydroxylamino moiety of methanesulfohydroxamic acid with N, O-bis-acetyl (7a), N,O-bis-methoxycarbonyl (7b), N, O-bis-ethoxycarbonyl (7c), and N-methoxycarbonyl-O-methyl (7d) groups. It was expected that the bioactivation of these prodrugs would initiate a cascade of nonenzymatic reactions leading to the ultimate liberation of NB or nitroxyl, thereby inhibiting AlDH. Indeed, the ester pro-prodrugs of both series were highly active in inhibiting yeast AlDH in vitro with IC50 values ranging from 21 to 64 microM. However, only 7d significantly raised ethanol-derived blood acetaldehyde levels when administered to rats, a reflection of the inhibition of hepatic mitochondrial AlDH-2.

Acetaldehyde↗

PubMed1998

Mechanisms of inhibition of aldehyde dehydrogenase by nitroxyl, the active metabolite of the alcohol deterrent agent cyanamide.

Nitroxyl, produced in the bioactivation of the alcohol deterrent agent cyanamide, is a potent inhibitor of aldehyde dehydrogenase (AIDH); however, the mechanism of inhibition of AlDH by nitroxyl has not been described previously. Nitroxyl is also generated from Angeli's salt (Na2N2O3) at physiological pH, and, indeed, Angeli's salt inhibited yeast AlDH in a time- and concentration-dependent manner, with IC50 values under anaerobic conditions with and without NAD+ of 1.3 and 1.8 microM, respectively. Benzaldehyde, a substrate for AlDH, competitively blocked the inhibition of this enzyme by nitroxyl in the presence of NAD+, but not in its absence, in accord with the ordered mechanism of this reaction. The sulfhydryl reagents dithiothreitol (5 mM) and reduced glutathione (10 mM) completely blocked the inhibition of AlDH by Angeli's salt. These thiols were also able to partially restore activity to the nitroxyl-inhibited enzyme, the extent of reactivation being dependent on the pH at which the inactivation occurred. This pH dependency indicates the formation of two inhibited forms of the enzyme, with an irreversible form predominant at pH 7.5 and below, and a reversible form predominant at pH 8.5 and above. The reversible form of the inhibited enzyme is postulated to be an intra-subunit disulfide, while the irreversible form is postulated to be a sulfinamide. Both forms of the inhibited enzyme are derived via a common N-hydroxysulfenamide intermediate produced by the addition of nitroxyl to active site cysteine thiol(s).

Alcoholism↗

PubMed1998

Reaction between S-nitrosothiols and thiols: generation of nitroxyl (HNO) and subsequent chemistry.

S-Nitrosothiols have been implicated to play key roles in a variety of physiological processes. The potential physiological importance of S-nitrosothiols prompted us to examine their reaction with thiols. We find that S-nitrosothiols can react with thiols to generate nitroxyl (HNO) and the corresponding disulfide. Further reaction of HNO with the remaining S-nitrosothiol and thiol results in the generation of other species including NO, sulfinamide, and hydroxylamine. Mechanisms are proposed that rationalize the observed products.

Aerobiosis↗

PubMed1998

Utility of a nitric oxide electrode for monitoring the administration of nitric oxide in biologic systems.

Amperometric techniques for the detection of nitric oxide (NO) are commercially available, but their sensitivity and specificity are not well described. We evaluated the sensitivity and specificity of a Clark-style, platinum NO electrode. The electrode has a lower limit of detection for NO of <25 pmol/ml in vitro and is linear over the range from 25 pmol/ml to 4 nmol/ml. The electrode is specific for NO so long as the protective membrane that covers the electrode is intact. Any defect in this membrane results in the detection of other redox agents such as hydrogen peroxide. Because of its ease of handling, specificity, and sensitivity, the NO electrode is a useful tool for quantification of administered NO in vitro and in various biologic systems.

Animals↗

PubMed1997

Novel prodrugs of cyanamide that inhibit aldehyde dehydrogenase in vivo.

S-Methylisothiourea (4), when administered to rats followed by a subsequent dose of ethanol, gave rise to a 119-fold increase in ethanol-derived blood acetaldehyde (AcH) levels-a consequence of the inhibition of hepatic aldehyde dehydrogenase (A1DH)-when compared to control animals not receiving 4. The corresponding O-methylisourea was totally inactive under the same conditions, suggesting that differential metabolism may be a factor in this dramatic difference between the pharmacological effects of O-methylisourea and 4 in vivo. The S-n-butyl- and S-isobutylisothioureas (8 and 9, respectively) at doses equimolar to that of 4 were nearly twice as effective in raising ethanol-derived blood AcH, while S-allylisothiourea (10) was slightly less active. However, blood ethanol levels of all experimental groups were indistinguishable from controls. Pretreatment of the animals with 1-benzylimidazole, a known inhibitor of the hepatic mixed function oxidases, followed sequentially by either 8, 9, or 10 plus ethanol, reduced blood AcH levels by 66-88%, suggesting that the latter compounds were being oxidatively metabolized to a common product that led to the inhibition of AcH metabolism. In support of this, when 8 was incubated in vitro with rat liver microsomes coupled to catalase and yeast A1DH, the requirement for microsomal activation for the inhibition of A1DH activity was clearly indicated. We suggest that S-oxidation is involved and that the S-oxides produced in vivo inhibited A1DH directly, or spontaneously released cyanamide, an inhibitor of A1DH. Indeed, incubation of 8 with rat liver microsomes and NADPH gave rise to cyanamide as metabolite, identified as its dansylated derivative. Cyanamide formation was minimal in the absence of coenzyme. Extending the side chain was detrimental, since S-benzylisothiourea (11) and S-n-hexadecylisothiourea (12) were toxic, the latter producing extensive necrosis of the liver and kidneys when administered to rats.

Acetaldehyde↗

PubMed1997

Inhibition of S-nitrosation of reduced glutathione in aerobic solutions of nitric oxide by phosphate and other inorganic anions.

Reduced glutathione is nitrosated in aerobic solutions of nitric oxide under physiological conditions; however, the extent of S-nitrosation was found to be dependent on the inorganic anions present. Of nine anions tested, the bifunctional anions, arsenate, phosphate, and pyrophosphate (40 mM), inhibited the S-nitrosation reaction from 20 to 40%, whereas SO4(2-), H3BO3, SCN-, NO3-, Cl-, and acetate inhibited this reaction < or = 15%. A mechanism of inhibition is presented that involves the catalytic hydrolysis of N2O3 by the bifunctional anions; however, using [18O]phosphate as inhibitor, only 10% of the theoretically produced N2O3 was found to be hydrolyzed to nitrite via this mechanism as calculated from the loss of 18O from phosphate. We conclude that this mechanism accounts for only a minor part of the increased inhibition of S-nitrosation by these bifunctional anions.

Aerobiosis↗

PubMed1997

Use of measurements of ethanol absorption from stomach and intestine to assess human ethanol metabolism.

Controversy exists concerning the site (stomach vs. liver) and magnitude of first-pass metabolism of ethanol. We quantitated gastric and total ethanol absorption rates in five male subjects and utilized these measurements to evaluate first-pass metabolism. Gastric emptying of ethanol (0.15 g/kg) was determined via a gamma camera and gastric absorption from the ratio of gastric ethanol to [14C]polyethylene glycol. Gastric absorption accounted for 30% and 10% of ethanol administered with food and water, respectively. With food, estimated gastric mucosal ethanol concentrations fell from 19 to 5 mM over 2 h. Calculations using these concentrations and kinetic data for gastric alcohol dehydrogenase showed <2% of the dose underwent gastric metabolism. Application of observed ethanol absorption rates to a model of human hepatic ethanol metabolism indicated that only 30% and 4% of the dose underwent first-pass metabolism when administered with food and water, respectively. We conclude that virtually all first-pass ethanol metabolism occurs in the liver and first-pass metabolism accounts for only a small fraction of total clearance.

Absorption↗

PubMed1996

Nitrosyl cyanide, a putative metabolic oxidation product of the alcohol-deterrent agent cyanamide.

When incubated with catalase/glucose-glucose oxidase, 13C-labeled cyanamide gave rise not only to 13C-labeled cyanide, but also to 13C-labeled CO2. Moreover, a time-dependent formation of nitrite was observed when cyanamide was oxidized in this system. These results suggested that the initial product of cyanamide oxidation, viz. N-hydroxycyanamide, was being further oxidized by catalase/H2O2 to nitrosyl cyanide (O = N-C = N). Theoretically, nitrosyl cyanide can hydrolyze to the four end-products detected in the oxidative metabolism of cyanamide in vitro, viz. nitroxyl, cyanide, nitrite, and CO2. Accordingly, both unlabeled and 13C-labeled nitrosyl cyanide were synthesized by the low temperature (-40 to -50 degrees) nitrosylation of K-(18-crown-6)cyanide with nitrosyl tetrafluoroborate. The product, a faint blue liquid at this temperature, was transferred as a gas to phosphate-buffered solution, pH 7.4, where it was solvolyzed. Analysis of the headspace by gas chromatography showed the presence of N2O, the dimerization/dehydration product of nitroxyl, while cyanide was detected in the aqueous solution, as measured colorimetrically. [13C]CO2 was analyzed by GC/MS. An oxidative biotransformation pathway for cyanamide that accounts for all the products detected and involving both N-hydroxycyanamide and nitrosyl cyanide as tandem intermediates is proposed.

Alcohol Deterrents↗

PubMed1995

Reaction of nitric oxide with the free sulfhydryl group of human serum albumin yields a sulfenic acid and nitrous oxide.

Nitric oxide (NO) generated by diethylamine nonoate (DEA/NO), an NO donor, readily oxidized the free sulfhydryl group of human serum albumin (HSA) as well as the sulfhydryl groups of reduced glutathione (GSH) and dithiothreitol (DTT) at pH 7.4 and 37 degrees C. Under anaerobic conditions, the major products of the oxidation of HSA thiol by NO were the sulfenic acid (RSOH) of HSA and nitrous oxide (N2O). The stoichiometry for this reaction, viz., 1 mol of HSA sulfhydryl oxidized to 1 mol of N2O produced, is consistent with a net two-electron oxidation of the protein thiol to a sulfenic acid. The sulfenic acid product of HSA was shown to react with dimedone and GSH, two known reactions of sulfenic acids. In contrast, anaerobic oxidation of GSH and DTT by NO gave a stoichiometry close to the expected ratio of 2:1 (sulfhydryl oxidized to N2O produced) for the oxidation of these thiols to their disulfides and N2O. Under aerobic conditions, significant fractions of the sulfhydryl groups of HSA, GSH, and DTT were oxidized to their respective thionitrites, presumably by N2O3. Thionitrite formation was not observed in the absence of oxygen. The production of HSA-sulfenic acid by NO, as well as by other oxidizing agents such as H2O2 and peroxynitrite, followed by its reaction with circulating GSH or L-Cys may account for the mixed disulfides of HSA observed in plasma.

Humans↗

PubMed1995

Prodrugs of nitroxyl as potential aldehyde dehydrogenase inhibitors vis-a-vis vascular smooth muscle relaxants.

The synthesis and the chemical/biological properties of N-hydroxysaccharin (1) (2-hydroxy-1,2-benzisothiazol-3(2H)-one 1,1-dioxide), a nitroxyl prodrug, are described. When treated with 0.1 M aqueous NaOH, 1 liberated nitroxyl (HN=O), a known inhibitor of aldehyde dehydrogenase (AlDH), in a time-dependent manner. Nitroxyl was measured gas chromatographically as its dimerization/dehydration product N2O. Under these conditions, Piloty's acid (benzenesulfohydroxamic acid) also gave rise to HNO. However, whereas Piloty's acid liberated finite quantities of nitroxyl when incubated in physiological phosphate buffer, pH 7.4, formation of nitroxyl from 1 was minimal. This was reflected in the differential inhibition of yeast AlDH (IC50 = 48 and > 1000 microM) and the differential relaxation of preconstricted rabbit aortic rings in vitro (EC50 = 1.03 and 14.0 microM) by Piloty's acid and 1, respectively. The O-acetyl derivative of 1, viz., N-acetoxysaccharin (13a), was much less active in both assays. It is concluded that N-hydroxysaccharin (1) is relatively stable at physiological pH and liberates nitroxyl appreciably only at elevated pH's. As a consequence, neither 1 nor its O-methyl (8a) and O-benzyl (8b) derivatives were effective AlDH inhibitors in vivo when administered to rats at 1.0 mmol/kg.

Aldehyde Dehydrogenase↗

PubMed1995

Carbethoxylating agents as inhibitors of aldehyde dehydrogenase.

N,O-Dicarbethoxy-4-chlorobenzenesulfohydroxamate (1c) and O-carbethoxy-N-hydroxysaccharin (6), both potential carbethoxylating agents, inhibited yeast aldehyde dehydrogenase (AlDH) with IC50's of 24 and 56 microM, respectively. The esterase activity of the enzyme was commensurably inhibited. AlDH activity was only partially restored on incubation with mercaptoethanol (20 mM) for 1 h. On incubation with rat plasma, 1c liberated nitroxyl, a potent inhibitor of AlDH. Under the same conditions, nitroxyl generation from 6 was minimal, a result compatible with a previous observation that nitroxyl generation from N-hydroxysaccharin (7), the product of the hydrolysis of the carbethoxy group of 6, was minimal at physiological pH. Since chemical carbethoxylating agents represented by the O-carbethoxylated N-hydroxyphthalimide, 1-hydroxybenzotriazole, and N-hydroxysuccinimide (8, 9, and 10, respectively) likewise inhibited yeast AlDH, albeit with IC50's 1 order of magnitude higher, we postulate that 1c and 6 act as irreversible inhibitors of AlDH by carbethoxylating the active site of the enzyme.

Aldehyde Dehydrogenase↗

PubMed1994

Selective, prolonged alteration of complement-mediated immune clearance after acute exposure of mice to ethanol.

A selective and prolonged alteration in complement-mediated immune clearance was found in mice given a single intraperitoneal injection of ethanol. Rate constants for the separate components of complement- and IgG Fc gamma-mediated clearance were determined using a branched series, first-order reaction sequence model and measurements of the disappearance of radiolabeled IgG-opsonized murine erythrocytes from the circulation of BALB/c mice. The rate constant governing immune clearance mediated by IgG Fc gamma receptors (k3) decreased to 16% of control at 1 hr after ethanol injection but returned to normal in 72 hr. A > 50% decrease in complement-mediated clearance occurred, with a nadir of complement-mediated sequestration (k1) and complement-dependent phagocytosis (k4) at 1 hr (P < 0.001). In this case, however, k1 and k4 rate constant values did not return to control levels until 6 weeks after the injection of ethanol. The rate constant governing C3b deactivation and release of deactivated, sensitized cells back to the circulation before they undergo phagocytosis (k2) was initially normal, but decreased in Week 6 and remained low to the end of the observation period at 22 weeks (P < 0.0001). These changes resulted in a major reduction in overall complement-mediated immune clearance up to 4 weeks after the ethanol injection. The change to normal rates for sequestration and phagocytosis coupled with decreased deactivation and release at 6 weeks postinjection resulted in a small increase in overall complement-mediated clearance that persisted through Week 22.

Alcohol Dehydrogenase↗

PubMed1994

Magnitude, origin, and implications of the discrepancy between blood ethanol concentrations of tail vein and arterial blood of the rat.

The rat is widely used as an animal model for experiments involving ethanol, and alcohol concentrations in blood obtained from the tail routinely are used to monitor ethanol exposure and metabolism. The present study demonstrates that during periods of rising and declining ethanol levels, the alcohol concentrations in tail vein blood lags far behind that of arterial, jugular, or femoral vein blood. As a result, tail vein ethanol concentrations markedly underestimate the concentration in arterial blood and rapidly perfused tissue during periods of increasing body ethanol, whereas the reverse is true as body ethanol declines. This discrepancy, which appeared to result from the low blood perfusion:tissue water ratio in the tail, disappeared when the tail was heated to 37 degrees C. Compared with arterial blood, alcohol measurements performed on tail vein blood yielded a much higher apparent Km for ethanol clearance and a somewhat lower estimate of ethanol reaching the peripheral circulation. We conclude that, for a variety of studies, analyses of arterialized blood from the heated tail should yield a more accurate and reproducible measure of ethanol exposure and/or metabolism than does the conventional collection from the unheated tail.

Alcohol Drinking↗