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H Esterbauer

Publications and source records attributed to H Esterbauer.

At least 145 records · Page 8Linked to original sources
PubMed1989

A spectrophotometric assay for lipid peroxides in serum lipoproteins using a commercially available reagent.

A method is described for measuring lipid peroxides by means of the color reagent of a commercially available test kit for cholesterol estimation. In principle, this assay makes use of the oxidative capacity of lipid peroxides to convert iodide to iodine, which can be measured photometrically at 365 nm. Calibration curves were obtained using peroxides such as H2O2, t-butyl hydroperoxide, and cumene hydroperoxide. A stoichiometric relationship was observed between the amount of organic peroxides assayed and the concentration of iodine produced. Concentrations of lipid peroxides as small as 1 nmol/ml could be measured. The ability to estimate lipid peroxides of isolated low density lipoprotein was demonstrated.

Humans↗
PubMed1988

Hydroxyl-radical-induced iron-catalysed degradation of 2-deoxyribose. Quantitative determination of malondialdehyde.

The degradation of 2-deoxyribose to thiobarbituric acid-reactive material was investigated with two hydroxyl-radical-generating systems: (i) a defined gamma-radiolysis method and (ii) incubation with FeSO4 in phosphate buffer. In each case the thiobarbituric acid-reactive material can be accounted for by malondialdehyde, as measured by an h.p.l.c. method for free malondialdehyde. In the radiolysis system there is a large post-irradiation increase in free malondialdehyde if iron ions are added to the samples. It is proposed that this is due to iron ions catalysing the formation of hydroxyl radicals from radiolytically generated H2O2 as well as stimulating the breakdown of an intermediate deoxyribose degradation product. A mechanism for the formation of malondialdehyde during deoxyribose degradation is proposed.

Catalase↗
PubMed1988

Metabolism of 4-hydroxynonenal by the rat hepatoma cell line MH1C1.

The metabolism of the toxic lipid peroxidation product 4-hydroxynonenal was investigated in the well-differentiated rat heptoma cell line MH1C1. When exposed to 0.1 mM 4-hydroxynonenal (HNE), MH1C1 cells consumed it in a time-dependent manner. There was a linear relationship between the amount of aldehyde consumed and cell number in the range 0.5 - 4 X 10(6) cells ml-1. This process was unaffected by pyrazole, suggesting that alcohol dehydrogenase is not involved. The whole homogenate of MH1C1 cells consumed added HNE at a rate similar to that in intact cells. Fractionation of the homogenate showed that the highest HNE-metabolizing activity is in the cytosol. The dialysed cytosol had almost no capacity to metabolize HNE, but this was restored by supplementation with NAD, NADH, NADP and NADPH. The metabolism of HNE in MH1C1 cells is thus different from that in hepatocytes, which were shown to utilize cytosolic alcohol dehydrogenase for this process. Both reductive and oxidative pathways could be implicated in the metabolic activity of MH1C1 cells towards HNE as well as binding by glutathione.

Aldehydes↗
PubMed1988

Disturbance of cell proliferation by two model compounds of lipid peroxidation contradicts causative role in proliferative senescence.

Cumene hydroperoxide (Chp), a lipophilic peroxide, and hydroxy-nonenal (HNE), a breakdown product of lipid peroxides, were used as model compounds to assess the effects of lipid peroxidation upon cell proliferation. Amniotic fluid fibroblastlike (AFFL) cells and human diploid skin-derived (HDFL) cells were cultured with the two model compounds and cell proliferation was assayed via bromodeoxyuridine-Hoechst flow cytometry. At low doses Chp elicited an accumulation of cells in the S and G2 phase, while at higher doses the fraction of nonproliferating cells increased as well. Low doses of HNE caused an accumulation of cells in the G1 and G2 phase, whereas an additional increase of cells in S phase and in the nonproliferating fraction was found at an elevated concentration. A delay of onset of proliferation was obtained with both Chp and HNE. Permanent arrests in the S, G2, and G1 compartment are provoked by Chp only when Chp was applied together with serum. HNE, to the contrary, elicited a permanent arrest in the G2 and the G1 compartment even if added to quiescent cell cultures. Additionally, HNE caused a combination of a prolongation of the G1 phase of the cell cycle and an arrest in this compartment, which is reminiscent of cell differentiation. HDFL cells were much more sensitive toward Chp than were AFFL cells, but both cell types showed similar sensitivities toward HNE. We conclude that lipophilic peroxides exert toxic effects upon cell proliferation distinct from the pattern elicited by aldehydic breakdown products of lipid peroxides. The pattern of cell cycle arrest induced by Chp and HNE makes it unlikely that Chp and HNE, or related products of lipid peroxidation, are responsible for the limitation of the proliferative life span of human fibroblasts in culture.

Aldehydes↗
PubMed1988

Formation of a pyridinium derivative by reaction of 4-hydroxypentenal with glycine.

4-Hydroxypentenal reacts with glycine in a slightly alkaline (pH 8.8) aqueous solution to 1-(1-carboxymethyl)-3-(2-hydroxypropyl)-pyridinium betaine. The yield of the pyridinium betaine isolated by preparative HPLC was 20 mol% and its structure was ascertained by 13C-NMR, IR, UV and mass spectroscopy. Several other products could be detected by HPLC in the 4-hydroxpentenal-glycine reaction mixture, their instability, however, impeded their preparative isolation. 4-Hydroxyalkenals are toxic products generated during lipid peroxidation and the results described explain in part the mechanism how these aldehydes react with nucleophilic amino groups in tissue.

Aldehydes↗
PubMed1988

Reversible inhibition of DNA and protein synthesis by cumene hydroperoxide and 4-hydroxy-nonenal.

To test the possible role of lipid peroxidation in the process of in vitro ageing, human diploid skin fibroblasts were cultured with the lipophilic hydroperoxide cumene hydroperoxide (Chp) or the breakdown product of lipid peroxidation 4-hydroxy-2,3-trans-nonenal (HNE). Both compounds inhibited cellular DNA and protein synthesis in a dose-dependent way. Cells exposed to Chp or to HNE during growth inhibition recovered DNA and protein synthesis within 24 h upon removal of Chp or HNE from the culture medium. Continuously proliferating cells showed only a partial recovery of DNA and protein synthesis. Pre-culturing cells with the lipophilic free radical scavenger vitamin E did not abolish the effect of Chp upon DNA synthesis. Cellular levels of reduced glutathione (GSH) rose slightly during 1 week of culture with HNE, but remained unaltered with Chp. Neither ATP levels nor cellular energy charges were affected during culture with Chp or HNE. So, DNA synthesis is not impaired due to a shortage of nucleotides nor does GSH protect DNA synthesis against the effects of Chp or HNE. These results suggest that oxygen free-radical induced lipid peroxidation is not the cause of the irreversible loss of proliferation occurring during in vitro ageing.

Aging↗
PubMed1987

Structure-activity relationships of 4-hydroxyalkenals in the conjugation catalysed by mammalian glutathione transferases.

The substrate specificities of 15 cytosolic glutathione transferases from rat, mouse and man have been explored by use of a homologous series of 4-hydroxyalkenals, extending from 4-hydroxypentenal to 4-hydroxypentadecenal. Rat glutathione transferase 8-8 is exceptionally active with the whole range of 4-hydroxyalkenals, from C5 to C15. Rat transferase 1-1, although more than 10-fold less efficient than transferase 8-8, is the second most active transferase with the longest chain length substrates. Other enzyme forms showing high activities with these substrates are rat transferase 4-4 and human transferase mu. The specificity constants, kcat./Km, for the various enzymes have been determined with the 4-hydroxyalkenals. From these constants the incremental Gibbs free energy of binding to the enzyme has been calculated for the homologous substrates. The enzymes responded differently to changes in the length of the hydrocarbon side chain and could be divided into three groups. All glutathione transferases displayed increased binding energy in response to increased hydrophobicity of the substrate. For some of the enzymes, steric limitations of the active site appear to counteract the increase in binding strength afforded by increased chain length of the substrate. Comparison of the activities with 4-hydroxyalkenals and other activated alkenes provides information about the active-site properties of certain glutathione transferases. The results show that the ensemble of glutathione transferases in a given species may serve an important physiological role in the conjugation of the whole range of 4-hydroxyalkenals. In view of its high catalytic efficiency with all the homologues, rat glutathione transferase 8-8 appears to have evolved specifically to serve in the detoxication of these reactive compounds of oxidative metabolism.

Aldehydes↗
PubMed1987

Quantitative measurement of 5-, 12-, and 15-hydroxyeicosatetraenoic acid together with 12-hydroxyheptadecatrienoic acid by stable isotope dilution gas chromatography-negative ion chemical ionization-mass spectrometry.

A stable isotope dilution gas chromatography-negative ion chemical ionization-mass spectrometry assay for simultaneous quantitative measurement of 5-hydroxyeicosatetraenoic acid (HETE), 12-HETE, 15-HETE, and 12-hydroxyheptadecatrienoic acid in one single GC/MS run was established. 18O2-Labeled analogs as internal standards, prepared according to conventional procedures, were found to be useful for this application. A sample processing and derivatization sequence providing highly purified compounds with a recovery of 42.7% was elaborated. The detection limit was in the femtomole range.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1987

Influence of cumene hydroperoxide and 4-hydroxynonenal on the glutathione metabolism during in vitro ageing of human skin fibroblasts.

Cumene hydroperoxide (Chp) and 4-hydroxynonenal (HNE) were used to investigate the effect of peroxidative challenge upon the glutathione (GSH) metabolism of human skin fibroblasts. Cellular GSH contents decreased during short-term incubations with Chp and oxidised glutathione (GSSG) was formed concomitantly. During longer incubations the GSH level was restored and the substrate flux through the pentose phosphate shunt increased. So in the presence of hydroperoxides the GSH level is maintained by reduction of GSSG. HNE caused a strong decrease in cellular GSH contents. Prolonged incubation with HNE lead to a rise in GSH contents above the basal level. The flux through the pentose phosphate shunt did not change during exposure to HNE. Hence, during incubation with HNE the cell maintains its GSH content by de novo synthesis of GSH. This conclusion is further substantiated by the findings with a cell strain deficient in GSH synthetase. These cells survived if incubated with Chp but not if exposed to HNE. GSH contents of normal cells from phase II (young) cultures and from phase III (aged) cultures responded similarly to Chp during short-term incubations and during a week of culture with the test compound. The flux through the pentose phosphate shunt rose much more in phase III than in phase II cells when incubated with the same concentration series of Chp. We conclude that during in vitro ageing the amount of NADPH needed to maintain cellular GSH levels in the presence of hydroperoxides increases, while the capacity to respond to such a challenge is not affected.

Adult↗
PubMed1987

Effects of the lipid peroxidation product 4-hydroxynonenal on the aggregation of human platelets.

The stimulation by ADP or arachidonic acid of the aggregation of human platelets in plasma was inhibited by 4-hydroxynonenal (HNE). This reduction of aggregation was time related, and was increased by prolonged preincubation of the platelets with the aldehyde. HNE was more potent than its homologue 4-hydroxypentenal (HPE). HNE was less active in decreasing the aggregation induced by calcium ionophore A23187 or collagen in comparison with ADP. HNE was inactive against aggregation of platelet-rich plasma (PRP) stimulated by thrombin whereas it potently inhibited the aggregation of washed platelets in response to both thrombin and collagen. Platelets were found to degrade HNE, and mechanisms additional to covalent binding to glutathione are indicated by the results obtained. The aldehydes, including HNE, generated by platelets originated principally from arachidonic acid metabolism.

Adenosine Diphosphate↗
PubMed1987

Detection of sulphite in plants.

Artificially SO(2)-fumigated plant material was investigated for its sulphite content. Experiments with moderate concentrations of SO(2) (0.15-0.45 mg m(-3) lasting for 3 weeks, showed no detectable amounts of sulphite present in peas. Considering the detection limit of the method, this indicates that the steady state level in plant tissues is below 4 x 10(-5)M sulphite. Only in pea plants exposed to very high SO(2)-doses of 2.6-26mg m(-3) for 12-48 h, was sulphite recorded. We conclude from our results that sulphite dioxide, taken up by pea plants, is very effectively removed by enzymic and non-enzymic processes, and this 'detoxification' prevents accumulation of sulphite in the plant tissue, and so only very small amounts of sulphite are to be expected in SO(2)-damaged plant material. The possible role of very low sulphite concentrations in plant tissues is discussed.

Journal Article↗
PubMed1987

Investigation of lipid peroxidation in human low density lipoprotein.

Human plasma low density lipoprotein (LDL) exposed to oxygen saturated buffer becomes depleted of alpha-tocopherol within 3 to 6 hours. Thereafter, lipid peroxidation commences as evidenced by the loss of 18:2 (67 nmol/mg LDL) and 20:4 (12 nmol/mg LDL) and the concomitant formation of 4-hydroxynonenal (0.28 nmol/mg LDL) and fluorescent compounds. The major fluorophor in apo B of oxidized LDL has an excitation maximum at 355 nm and an emission maximum at 430 nm. A fluorophor with the same spectral properties is produced in apo B, if LDL is incubated with 4-hydroxynonenal, whereas malonaldehyde gives a fluorophor with excitation and emission maxima at 400/470 nm. Three-dimensional fluorescence spectroscopy proved to be an useful tool in analysing the complex fluorescence of apo B.

Aldehydes↗
PubMed1987

Effect of methoxy-p-benzoquinones and methoxy-p-hydroquinones on DNA synthesis in Ehrlich ascites tumor cells.

The effects of various methoxy-p-quinones and methoxy-p-hydroquinones on DNA synthesis and thymidine transport in Ehrlich Ascites Tumor Cells was investigated. The inhibitory concentrations (IC50) were in the range of 5 to 65 microM. Adriamycin, used as positive control, had an IC50 of 6 microM. 2,3-Dimethoxy-benzoquinone and -hydroquinone and 2,3,5,6-tetramethoxy-benzoquinone clearly inhibited DNA synthesis at concentrations where thymidine transport was not affected. 2-Methoxy-hydroquinone, 2-methoxy-benzoquinone, 2,6-dimethoxy-benzoquinone, 2,5-dimethoxy-benzoquinone had IC50-values close to the dose inhibiting also thymidine transport. 2,5-Dimethoxy-benzoquinone, 2,3,5-trimethoxy-hydroquinone, 2,3,5-trimethoxy-benzoquinone and 2,3,5,6-tetramethoxy-hydroquinone strongly inhibited thymidine transport without a significant effect on DNA synthesis. Addition of ascorbate enhanced the inhibitory activity of 2,6-dimethoxy-benzoquinone.

Animals↗
PubMed1987

Chemotactic activity of aldehydes. Structural requirements. Role in inflammatory process.

The lipid peroxidation product 4-hydroxy-2,3-transnonenal (HNE) and its homologous compounds were found to induce a high significant stimulation of rat neutrophil oriented migration and morphological polarization at concentrations within a pico-micromolar range. These effects are weak with respect to the potent chemoattractant formyl-peptides. The structural requirements for the chemotactic activity of aldehydes were studied and the trans C-C double bond was found to be the essential requirement. The problem of the mechanism by which alpha, beta-unsaturated aldehydes interact with neutrophils is unsolved. With regard to this point, some hypotheses are discussed. The possibility that chemotactic unsaturated aldehydes are present in vivo in the inflammatory site at a concentration at which they are chemotactic in vitro was studied. Rat pleural exudates collected 1,2,3,4 hours after a pleurisy induction were analyzed and HNE was detected both in the supernatant of exudate and in cells. Its concentration in the liquid part of the exudate increased with time, in parallel with an increase in the number of cells in the exudate. It is reasonable that this aldehyde might be formed as a consequence of lipid peroxidation reactions concomitant to the phlogistic process. The hypothesis is proposed that HNE could participate in the neutrophil recruitment in the inflammatory area.

Aldehydes↗