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Biomedical subjects

H Sies

Publications and source records attributed to H Sies.

At least 325 records · Page 18Linked to original sources
PubMed1983

Antioxidant effect of diethyldithiocarbamate on microsomal lipid peroxidation assessed by low-level chemiluminescence and alkane production.

Different thiol-containing compounds, such as diethyldithiocarbamate (DDC), glutathione, penicillamine, and dithioerythritol have been chosen to study their effect on ascorbate/Fe-ADP-induced lipid peroxidation, detected by low-level chemiluminescence and alkane production. In the concentration range used, these thiols exerted a temporary protection against lipid peroxidation by lengthening the induction period; after overcoming this induction period, no substantial inhibition of either chemiluminescence or alkane production was observed. DDC was effective in protecting against lipid peroxidation in the nanomolar range, whereas the group of other thiol-containing molecules operated in the millimolar range.

Adenosine Diphosphate↗
PubMed1983

Oxidation of glutathione by the superoxide radical to the disulfide and the sulfonate yielding singlet oxygen.

The reaction of superoxide with reduced glutathione (GSH) was studied with two O-.2-producing systems: xanthine oxidase using xanthine or acetaldehyde as substrates, and secondly, quinol autoxidation. The capability of GSH to quench superoxide radicals was detected by lowered O-.2-mediated cytochrome c3+ reduction. The formation of the oxidation products, glutathione disulfide (GSSG) and glutathione sulfonate (the latter at levels of about 6-15% compared to GSSG), was dependent on the O-.2 production and was inhibited by superoxide dismutase. The presence of GSH together with an O-.2-producing system led to an extra uptake of oxygen, which was also depressed by superoxide dismutase. The observed O2 uptake was accounted for by the formation of GSSG and GSO-3 from GSH; the data are in accordance with a mechanism involving thiyl radicals. Low-level chemiluminescence measurement indicated the formation of excited oxygen species. The intensity of photoemission was dependent on the GSH concentration and on the O-.2 production rate. Chemiluminescence was inhibited by superoxide dismutase and also by glutathione peroxidase, but not by catalase or OH. quenchers. Spectral analysis and the effects of 1,4-diazabicyclo[2.2.2]octane and sodium azide indicated the contribution of singlet molecular oxygen to the light emission. It is suggested that singlet oxygen results from an intermediate oxygen addition product such as a glutathione peroxysulphenyl radical.

Cytochrome c Group↗
PubMed1983

Identification and quantitation of glutathione in hepatic protein mixed disulfides and its relationship to glutathione disulfide.

The amount of glutathione present in hepatic protein mixed disulfides was determined to be 20-30 nmole/g liver. This was established using two specific enzymatic methods: (a) the coupled assay with DTNB and glutathione (GSSG) reductase and (b) a newly developed test using GSH transferase and 1-chloro-2,4-dinitrobenzene for the estimation of GSH released from proteins after borohydride treatment; further, these results were confirmed by HPLC analysis. Thus, authentic glutathione makes up only 2-6% of the value for total protein mixed disulfides. The latter were determined with the generally employed o-phthalaldehyde assay, which is not necessarily specific for GSH. The amount of glutathione mixed disulfides depends linearly on the content of glutathione disulfide in the liver cell in the range studied. By increasing the GSSG levels from 20 to about 60 nmole/g liver with paraquat, nitrofurantoin or t-butyl hydroperoxide, glutathione protein mixed disulfides are increased by a similar amount.

Animals↗
PubMed1983

Effects of 4-hydroxynonenal on isolated hepatocytes. Studies on chemiluminescence response, alkane production and glutathione status.

The effect of 4-hydroxy-2,3-trans-nonenal, a diffusible product of lipid peroxidation, on isolated hepatocytes was evaluated with two non-invasive techniques measuring low-level chemiluminescence and alkane evolution. Oxygen-induced low-level chemiluminescence and ethane and n-pentane formation by hepatocytes is enhanced over 7-fold in the presence of 4-hydroxynonenal (2 mM). Glutathione-depleted hepatocytes show a higher increase than controls in both low-level chemiluminescence and alkane formation upon supplementation with 4-hydroxynonenal. The effects on both parameters are diminished by vitamin E pretreatment of rats and are absent under anaerobiosis. At variance with chemiluminescence and alkane formation, 4-hydroxynonenal does not elicit a concomitant increase in malonaldehyde or diene-conjugate formation. Addition of 4-hydroxynonenal to a suspension of hepatocytes causes a rapid loss of cellular glutathione in the form of a glutathione conjugate with the alkenal as observed with high-pressure liquid-chromatographic analysis. The reaction between glutathione and 4-hydroxynonenal proceeds also spontaneously in vitro at 1:1 stoichiometry. The cellular effects of 4-hydroxynonenal evaluated by low-level chemiluminescence and alkane formation are independent of the formation of a glutathione conjugate and seem to rely on the remaining not-bound 4-hydroxynonenal. The sensitivity of 4-hydroxynonenal-enhanced chemiluminescence and alkane formation to free-radical quenchers suggests the participation of a free-radical propagation process.

Aldehydes↗
PubMed1983

Ethane release during metabolism of aldehydes and monoamines in perfused rat liver.

Infusion of aldehyde such as acetaldehyde, propionaldehyde or benzaldehyde to perfused rat liver leads to an increase in hepatic ethane production. Half-maximal effect was obtained with about 20 microM acetaldehyde, a concentration range found in plasma during ethanol metabolism. Compounds which metabolically generate aldehydes such as monoamines (benzylamine, phenylethylamine) as substrates for monoamine oxidase or ethanol as substrate for alcohol dehydrogenase [A. Müller and H. Sies (1982) Biochem. J. 206, 153-156] are also able to elicit ethane release. Results obtained with inhibitors of hepatic aldehyde metabolism (pargyline or cyanamide) or of monamine oxidase (pargyline or tranylcypromine) suggest that metabolism of the aldehydes is required for ethane production. Radical scavenging by the addition of the flavonoid, cyanidanol, or by pretreatment with vitamin E (alpha-tocopherol) abolished ethane release, in agreement with lipid peroxidation as a source of alkane production during aldehyde metabolism.

Aldehyde Dehydrogenase↗
PubMed1983

Hepatic low-level chemiluminescence during redox cycling of menadione and the menadione-glutathione conjugate: relation to glutathione and NAD(P)H:quinone reductase (DT-diaphorase) activity.

Formation of excited species such as singlet molecular oxygen during redox cycling (one-electron reduction-oxidation) was detected by low-level chemiluminescence emitted from perfused rat liver and isolated hepatocytes supplemented with the quinone, menadione (vitamin K3). Chemiluminescence was augmented when the two-electron reduction of the quinone catalyzed by NAD(P)H:quinone reductase was inhibited by dicoumarol, thus underlining the protective function of this enzyme also known as DT-diaphorase. Interference with NADPH supply by inhibition of energy-linked transhydrogenase by rhein or of mitochondrial electron transfer by antimycin A led to a depression in the level of photoemission. Unexpectedly, glutathione depletion of the liver led to a lowering of chemiluminescence elicited by menadione, whereas conversely the depletion of glutathione led to increased chemiluminescence levels when a hydroperoxide was added instead of the quinone. As the GSH conjugate of menadione, 2-methyl-3-glutathionyl-1,4-naphthoquinone, studied with microsomes, was shown also to be capable of redox cycling, we conclude that menadione-induced chemiluminescence of the perfused rat liver does not only arise from menadione itself but from the menadione-GSH conjugate as well. Therefore, the conjugation of the quinone with glutathione is not in itself of protective nature and does not abolish semiquinone formation. A biologically useful aspect of conjugate formation resides in the facilitation of biliary elimination from the liver. Nonenzymatic formation of the conjugate from menadione and GSH in vitro was found to be accompanied by the formation of aggressive oxygen species.

Animals↗
PubMed1983

Decreased flux through pyruvate dehydrogenase during calcium ion movements induced by vasopressin, alpha-adrenergic agonists and the ionophore A23187 in perfused rat liver.

Vasopressin or alpha-adrenergic agents such as phenylephrine or adrenaline, but not glucagon, elicited an initial decrease in flux through pyruvate dehydrogenase assayed by 14CO2 production from [1-14C]pyruvate in perfused rat liver. This rapid decrease in 14CO2 production was maximal within 1-2 min of exposure, concomitant with a rise in effluent pyruvate concentration: a subsequent return towards initial values in both parameters was completed well before 5 min. This time course was superposed with Ca2+ efflux from perfused liver, maximal (at 116 nmol/min per g wet wt. of liver) at 1-2 min of exposure. The percentage of the active (dephospho) form of pyruvate dehydrogenase was not decreased at 2 min of exposure. The effect on flux through pyruvate dehydrogenase by phenylephrine was abolished by prazosine, phentolamine or phenoxybenzamine. Ionophore A23187 also caused a depression in 14CO2 production from [1-14C]pyruvate and a rise in effluent pyruvate concentration, but this effect was stable for longer times, and it was delayed when Ca2+ was omitted from the perfusion medium. Responses of phenylephrine and A23187 were not additive. The results demonstrate that under the experimental conditions employed in intact perfused liver, the mitochondrial multienzyme system of pyruvate dehydrogenase is sensitive to vasopressin, alpha-adrenergic agents and A23187. The similar time course in Ca2+ efflux may be indicative of the involvement of Ca2+ in mediating this effect.

Adrenergic alpha-Agonists↗
PubMed1983

Regulation of flux through glutaminase and glutamine synthetase in isolated perfused rat liver.

1. Glutaminase and glutamine synthetase are simultaneously active in the intact liver, resulting in an energy consuming cycling of glutamine at a rate up to 0.2 mumol per g per min. 2. An increase in portal glutamine concentration was followed by an increased flux through glutaminase, but flux through glutamine synthetase remained unchanged. Glutaminase flux was also increased by ammonium ions or glucagon; these effects were additive. 3. Glutamine synthetase flux was increased by ammonium ions, but this activation was partly overcome by increasing portal glutamine concentrations. Glutamine synthetase flux was slightly increased by glucagon at portal glutamine concentrations of about 0.2-0.3 mM, but was strongly inhibited above 0.6 mMs. 4. During experimental metabolic acidosis there was an increased net release of glutamine by the liver, being due to opposing changes of flux through glutaminase and glutamine synthetase. Conversely, an increased glutamine uptake by the liver during metabolic alkalosis was observed due to an inhibition of glutamine synthetase and an activation of glutaminase. However, the two enzyme activities respond differently depending on whether glucagon or ammonium ions are present.

Ammonium Chloride↗
PubMed1983

Inhibition of ethanol- and aldehyde-induced release of ethane from isolated perfused rat liver by pargyline and disulfiram.

Acute addition of ethanol or acetaldehyde to the isolated, perfused rat liver leads to an increase in ethane and n-pentane release. These volatile hydrocarbons are known to originate from the peroxidation of polyunsaturated fatty acids. The effects are half-maximal at 0.5 mM ethanol or 20 microM acetaldehyde in the entering perfusate. Propionaldehyde and benzaldehyde are also able to elicit ethane release. Pargyline and disulfiram, inhibitors of aldehyde oxidation, inhibited the extra ethane release in all cases. The inhibitory effect of pargyline is suppressed during addition of metyrapone. The study indicates that the oxidation of acetaldehyde and not of ethanol itself is the step responsible for increased ethane formation by the perfused rat liver during ethanol infusion.

Acetaldehyde↗
PubMed1983

Oxidant- and lipid-induced pulmonary vasoconstriction mediated by arachidonic acid metabolites.

In experiments using isolated rabbit lungs perfused with Krebs-Henseleit buffer in a nonrecirculating manner, we found that administration of an organic peroxide, tert-butyl hydroperoxide (t-bu-OOH), or Intralipid, an esterified mixture of unsaturated fatty acids, caused a marked vasopressor response which was associated with increased levels of thromboxane in the effluent perfusate. The vasopressor response to t-bu-OOH was greater in the lungs of vitamin E-deficient animals, and this could be correlated with an attenuated ability to increase prostacyclin production in these lungs. Conversely, administration of Intralipid to normal lungs caused marked increases in prostacyclin and a smaller pressor response. The magnitude of the pressor response was strongly correlated with the ratio of thromboxane B2 to the prostacyclin metabolite. No release of these mediators was associated with pulmonary vasoconstriction caused by administration of angiotensin II. The pressor response could be completely blocked by administration of indomethacin. We propose that this activation of the cyclooxygenase pathway of arachidonic acid metabolism may be important in the pathophysiology of oxidant lung damage.

6-Ketoprostaglandin F1 alpha↗
PubMed1983

Singlet oxygen formation detected by low-level chemiluminescence during enzymatic reduction of prostaglandin G2 to H2.

Addition of arachidonic acid to a suspension of ram vesicular gland microsomes or purified prostaglandin synthase, causes a rapid burst of light emission in the range 600-750 nm, as detected by single-photon counting. Maximal light emission intensity is obtained within 15-30 s after the addition of arachidonic acid and is followed by a rapid decay to the background level. The intensity of chemiluminescence is dependent on the amount of ram vesicular gland microsomes or isolated prostaglandin synthase and arachidonic acid concentration (Km about 6 microM). Spectral analysis of arachidonic acid-induced photoemission of isolated prostaglandin synthase in the range 600-750 nm showed two distinctive peaks at about 634 and 703 nm. The similar relative intensities of these peaks, along with the lower intensity at about 668 nm is indicative of singlet oxygen dimol emission. Chemiluminescence with arachidonate is enhanced by 1,4-diazabicyclo[2,2,2]octane and inhibited by azide, indomethacin, acetylsalicylic acid and beta-carotene. Cooxygenation substrates such as phenol, hydroquinone and reduced glutathione, inhibited the arachidonic acid-induced chemiluminescence. Dioxygen is a requirement for the observation of singlet oxygen dimol emission with arachidonic acid as a substrate for ram vesicular gland microsomes or purified prostaglandin synthase. However, when prostaglandin G2 is substituted for arachidonic acid, light emission is not dependent on oxygen. Thus, singlet oxygen can be formed in the dismutation reaction, 2 PGG2 leads to 2 PGH2 + 1O2, catalysed by prostaglandin hydroperoxidase.

Animals↗
PubMed1983

Decrease of flux through pyruvate dehydrogenase and branched-chain 2-oxo-acid dehydrogenase by nitrofurantoin in perfused rat liver.

Addition of nitrofurantoin to isolated perfused rat liver leads to an inhibition of 14CO2 production from [1-14C]pyruvate, 2-oxo-[1-14C]isocaproate and 2-oxo-[1-14C]isovalerate, indicating a decreased flux through the corresponding mitochondrial 2-oxo-acid dehydrogenases. This is in agreement with a decreased tissue level of the active (dephospho)form of pyruvate dehydrogenase in presence of nitrofurantoin. 2) Evidence is presented for an inhibition of the monocarboxylate translocator in the mitochondrial membrane during addition of nitrofurantoin by comparing the effects of alpha-cyanocinnamate as a transport inhibitor with those of nitrofurantoin. 3) It is concluded that nitrofurantoin-induced hepatocyte toxicity may include mitochondrial effects due to decreased oxo-acid dehydrogenase flux.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1983

Decreased flux through pyruvate dehydrogenase by thiol oxidation during t-butyl hydroperoxide metabolism in perfused rat liver.

Addition of t-butyl hydroperoxide to isolated perfused rat liver leads to a decreased flux through pyruvate dehydrogenase, shown by a decreased 14CO2 release from [1-14C]pyruvate. The effect is observed at rates of infusion of t-butyl hydroperoxide exceeding 0.7 mumol per min per g liver in normal livers and at significantly lower rates in glutathione-depleted livers. The effect is absent in livers from Se-deficient rats in which the hepatic Se-dependent glutathione peroxidase activity is very low, indicating that reduction of t-butyl hydroperoxide by glutathione peroxidase is a necessary prerequisite for the inhibition. With isolated mitochondria, decreased 14CO2 release from [1-14C]pyruvate during t-butyl hydroperoxide metabolism correlates with decreased GSH and increased GSSG contents, respectively. The addition of various disulfide compounds, including GSSG, inhibits activity of the enzyme in mitochondrial extracts. In both mitochondria and perfused liver, t-butyl hydroperoxide-mediated decrease of pyruvate dehydrogenase flux is relieved by thiol reductants. The active (dephospho)form of pyruvate dehydrogenase as measured in freeze-stopped liver samples is actually increased from 46% to 72% during t-butyl hydroperoxide metabolism. The tissue levels of ATP and ADP and perfusate beta-hydroxybutyrate/acetoacetate ratio are not markedly perturbed by addition of the hydroperoxide (10 min). It is concluded that the decreased flux through pyruvate dehydrogenase during t-butyl hydroperoxide metabolism results from oxidation of critical thiol group(s) of the enzyme complex consequential to a decrease in mitochondrial GSH/GSSG.

Animals↗