Oxidative catabolism of tetrahydropterins

Mixed labelled folic acid was administerd to rats. Exposure to N2O was used to give an insight into the major route of scission within the monoglutamate pool, results suggest that THF formed during transport from the gut lumen to the plasma is the major route of scission within the gut. Peroxides in corn oil and arising as a result of lipid peroxidation and autoxidation increase catabolism of the monoglutamate pool and decrease incorporation of administered folates into the polyglutamate pool. It is suggested that peroxides may oxidise B12 resulting in inhibition of methionine synthetase, this results in diminished polyglutamation and increased urinary excretion of 5 CH3THF. Fats undergo peroxidation within tissues, the resulting peroxides increase catabolism of the polyglutamate pool. It is suggested that the NBT assay may reflect polyglutamate breakdown. Antioxidants such as vitamin E (and DES) decrease catabolism of the monoglutamate pool. Administration of DES resulted in changes similar to those observed during malignancy, it is suggested that these changes may precede the onset of tumour development. Vitamin E elevates brain DHPR activity. Since lowered DHPR levels and disturbed THB metabolism have been observed in aging and Down's syndrome it is proposed that vitamin E therapy may prove beneficial in situations where oxidative stress is increased. Brain DHPR activity was increased on administration of peroxides suggesting that in situations of oxidative stress (which may result in increased catabolism of THB) the salvage pathway may be stimulated and loss of THB minimised. N2O exposure had no effect on THB metabolism suggesting that the stimulatory role of 5 CH3THF is due to its role as a methyl donor.

Interactions between oligoamines and superoxide anion

1- Oligoamines and EDTA inhibited the reduction of cytochrome-C and nitrobule tetrazolium (NBT) induced by the hypoxanthine/xanthine oxidase superoxide anion generating system in the following order of effectiveness: putrescine > diaminopropane > spermidine > EDTA > spermine > cadaverine. 2- Oligoamines and EDTA did not affect the rate of urate formation from the hypoxanthine/xanthine oxidase system. 3- Oligoamines and EDTA inhibited the reduction of cytochrome-C induced by stimulated PMNL's in the same order of effectiveness as mentioned before. 4- Oligoamines and EDTA inhibited luminol dependent stimulated PMNL's chemiluminescence. 5- Oligoamines and EDTA inhibited the aerobic photoreduction of NBT. 6- Oligoamines-copper sulphate complexes inhibited the reduction of cytochrome-C induced by the hypoxanthine/xanthine oxidase system more effectively than oligoamines or copper sulphate individually. 7- Superoxide anion, hydrogen peroxide and hydroxyl radical induced breakdown of isolated intact guinea pig liver lysosomes. 8- Oligoamines and EDTA protected isolated intact guinea pig liver lysosomes from the lytic effect of superoxide anion generated either by the hypoxanthine/xanthine oxidase system or by stimulated PMNL's. 9- Oligoamines and EDTA have no stabilizing effect on isolated intact guinea pig liver lysosomes. 10- The uptake of oligoamines by lysosomes was in the following order: putrescine > spermidine > spermine. 11- Oligoamines were metabolised into aldehyde compounds either by the hypoxanthine/xanthine oxidase system or stimulated PMNL's. 12- Oligoamines and EDTA have no effect on the activities of free lysosomal enzymes (acid phosphatase and -glucosaminidase). 13- Oligoamines and EDTA inhibited lipid peroxidation in guinea pig liver lysosomes induced either by the hypoxanthine/xanthine oxidase or ascorbic acid-ferrous sulphate. 14- Oligoamines and EDTA have no effect on the release of PGE_2 from stimulated peritoneal guinea pig PMNL's. 15- Oligoamines increased the uptake of (^3H)thymidine and (^3H)leucine by stimulated peritoneal guinea pig macrophages in the following order of effectiveness: spermine > spermidine > putrescine > cadaverine. 16- PGE_2, dibutyryl Cyclic AMP, and theophylline inhibited luminol dependent stimulated peritoneal guinea pig PMNL's chemiluminescence.

Oxidative impairment of plasma and skeletal muscle sarcoplasmic reticulum in rats with adjuvant arthritis - effects of pyridoindole antioxidants

OBJECTIVES: To study possible oxidation of proteins and lipids in plasma and sarcoplasmic reticulum (SR) from skeletal muscles and to assess the effects of pyridoindole antioxidants in rats with adjuvant arthritis (AA) and to analyze modulation of Ca-ATPase activity from SR (SERCA). METHODS: SR was isolated by ultracentrifugation, protein carbonyls in plasma and SR were determined by ELISA. Lipid peroxidation was analyzed by TBARS determination and by mass spectrometry. ATPase activity of SERCA was measured by NADH-coupled enzyme assay. Tryptophan fluorescence was used to analyze conformational alterations. RESULTS: Increase of protein carbonyls and lipid peroxidation was observed in plasma of rats with adjuvant arthritis. Pyridoindole antioxidant stobadine and its methylated derivative SMe1 decreased protein carbonyl formation in plasma, effect of stobadine was significant. Lipid peroxidation of plasma was without any effect of pyridoindole derivatives. Neither protein oxidation nor lipid peroxidation was identified in SR from AA rats. SERCA activity from AA rats increased significantly, stobadine and SMe1 diminished enzyme activity. Ratio of tryptophan fluorescence intensity in SR of AA rats increased and was not influenced by antioxidants. CONCLUSION: Plasma proteins and lipids were oxidatively injured in rats with AA; antioxidants exerted protection only with respect to proteins. In SR, SERCA activity was altered, apparently induced by its conformational changes, as supported by study of tryptophan fluorescence. Stobadine and SMe1 induced a decrease of SERCA activity, elevated in AA rats, but they did not affect conformational changes associated with tryptophan fluorescence.

Measurement of HNE-protein adducts in human plasma and serum by ELISA-Comparison of two primary antibodies

There is increasing evidence that non-enzymatic post-translational protein modifications might play key roles in various diseases. These protein modifications can be caused by free radicals generated during oxidative stress or by their products generated during lipid peroxidation. 4-Hydroxynonenal (HNE), a major biomarker of oxidative stress and lipid peroxidation, has been recognized as important molecule in pathology as well as in physiology of living organisms. Therefore, its detection and quantification can be considered as valuable tool for evaluating various pathophysiological conditions.The HNE-protein adduct ELISA is a method to detect HNE bound to proteins, which is considered as the most likely form of HNE occurrence in living systems. Since the earlier described ELISA has been validated for cell lysates and the antibody used for detection of HNE-protein adducts is non-commercial, the aim of this work was to adapt the ELISA to a commercial antibody and to apply it in the analysis of human plasma samples.After modification and validation of the protocol for both antibodies, samples of two groups were analyzed: apparently healthy obese (n=62) and non-obese controls (n=15). Although the detected absolute values of HNE-protein adducts were different, depending on the antibody used, both ELISA methods showed significantly higher values of HNE-protein adducts in the obese group. © 2013 The Authors.

Studies of phospholipid oxidation by electrospray mass spectrometry: from analysis in cells to biological effects

The oxidation of lipids is important in many pathological conditions and lipid peroxidation products such as 4-hydroxynonenal (HNE) and other aldehydes are commonly measured as biomarkers of oxidative stress. However, it is often useful to complement this with analysis of the original oxidized phospholipid. Electrospray mass spectrometry (ESMS) provides an informative method for detecting oxidative alterations to phospholipids, and has been used to investigate oxidative damage to cells, and low-density lipoprotein, as well as for the analysis of oxidized phosphatidylcholines present in atherosclerotic plaque material. There is increasing evidence that intact oxidized phospholipids have biological effects; in particular, oxidation products of 1-palmitoyl-2-arachidonoyl-sn-glycerophosphocholine (PAPC) have been found to cause inflammatory responses, which could be potentially important in the progression of atherosclerosis. The effects of chlorohydrin derivatives of lipids have been much less studied, but it is clear that free fatty acid chlorohydrins and phosphatidylcholine chlorohydrins are toxic to cells at concentrations above 10 micromolar, a range comparable to that of HNE and oxidized PAPC. There is some evidence that chlorohydrins have biological effects that may be relevant to atherosclerosis, but further work is needed to elucidate their pro-inflammatory properties, and to understand the mechanisms and balance of biological effects that could result from oxidation of complex mixtures of lipids in a pathophysiological situation.

Detection of phospholipid oxidation in oxidatively stressed cells by reversed-phase HPLC coupled with positive-ionization electrospray MS

Measurement of lipid peroxidation is a commonly used method of detecting oxidative damage to biological tissues, but the most frequently used methods, including MS, measure breakdown products and are therefore indirect. We have coupled reversed-phase HPLC with positive-ionization electrospray MS (LC-MS) to provide a method for separating and detecting intact oxidized phospholipids in oxidatively stressed mammalian cells without extensive sample preparation. The elution profile of phospholipid hydroperoxides and chlorohydrins was first characterized using individual phospholipids or a defined phospholipid mixture as a model system. The facility of detection of the oxidized species in complex mixtures was greatly improved compared with direct-injection MS analysis, as they eluted earlier than the native lipids, owing to the decrease in hydrophobicity. In U937 and HL60 cells treated in vitro with t-butylhydroperoxide plus Fe2+, lipid oxidation could not be observed by direct injection, but LC-MS allowed the detection of monohydroperoxides of palmitoyl-linoleoyl and stearoyl-linoleoyl phosphatidylcholines. The levels of hydroperoxides observed in U937 cells were found to depend on the duration and severity of the oxidative stress. In cells treated with HOCl, chlorohydrins of palmitoyloleoyl phosphatidylcholine were observed by LC-MS. The method was able to detect very small amounts of oxidized lipids compared with the levels of native lipids present. The membrane-lipid profiles of these cells were found to be quite resistant to damage until high concentrations of oxidants were used. This is the first report of direct detection by LC-MS of intact oxidized phospholipids induced in cultured cells subjected to oxidative stress.

Molecular and biochemical characterization of postharvest senescence in broccoli

Postharvest senescence in broccoli (Brassica oleracea L. var Italica) florets results in phenotypic changes similar to those seen in developmental leaf senescence. To compare these two processes in more detail, we investigated molecular and biochemical changes in broccoli florets stored at two different temperatures after harvest. We found that storage at cooler temperatures delayed the symptoms of senescence at both the biochemical and gene expression levels. Changes in key biochemical components (lipids, protein, and chlorophyll) and in gene expression patterns occurred in the harvested tissue well before any visible signs of senescence were detected. Using previously identified senescence-enhanced genes and also newly isolated, differentially expressed genes, we found that the majority of these showed a similar enhancement of expression in postharvest broccoli as in developmental leaf senescence. At the biochemical level, a rapid loss of membrane fatty acids was detected after harvest, when stored at room temperature. However, there was no corresponding increase in levels of lipid peroxidation products. This, together with an increased expression of protective antioxidant genes, indicated that, in the initial stages of postharvest senescence, an orderly dismantling of the cellular constituents occurs, using the available lipid as an energy source. Postharvest changes in broccoli florets, therefore, show many similarities to the processes of developmental leaf senescence.

Dapsone induces oxidative stress and impairs antioxidant defenses in rat liver

Dapsone (DDS) is currently used in the treatment of leprosy, malaria and in infections with Pneumocystis jirovecii and Toxoplasma gondii in AIDS patients. Adverse effects of DDS involve methemoglobinemia and hemolysis and, to a lower extent, liver damage, though the mechanism is poorly characterized. We evaluated the effect of DDS administration to male and female rats (30 mg/kg body wt, twice a day, for 4 days) on liver oxidative stress through assessment of biliary output and liver content of reduced (GSH) and oxidized (GSSG) glutathione, lipid peroxidation, and expression/activities of the main antioxidant enzymes glutathione peroxidase, superoxide dismutase, catalase and glutathione S-transferase. The influence of DDS treatment on express ion/activity of the main DDS phase-II- metabolizing system, UDP-glucuronosyltransferase (UGT), was additionally evaluated. The involvement of dapsone hydroxylamine (DDS-NHOH) generation in these processes was estimated by comparing the data in male and female rats since N-hydroxylation of DDS mainly occurs in males. Our studies revealed an increase in the GSSG/GSH biliary output ratio, a sensitive indicator of oxidative stress, and in lipid peroxiclation, in male but not in female rats treated with DDS. The activity of all antioxidant enzymes was significantly impaired by DDS treatment also in male rats, whereas UGT activity was not affected in any sex. Taken together, the evidence indicates that DDS induces oxidative stress in rat liver and that N-hydroxylation of DDS was the likely mediator. Impairment in the activity of enzymatic antioxidant systems, also associated with DDS-NHOH formation, constituted a key aggravating factor.

Clinical oxidative stress during leprosy multidrug therapy:impact of dapsone oxidation

This study aims to assess the oxidative stress in leprosy patients under multidrug therapy (MDT; dapsone, clofazimine and rifampicin), evaluating the nitric oxide (NO) concentration, catalase (CAT) and superoxide dismutase (SOD) activities, glutathione (GSH) levels, total antioxidant capacity, lipid peroxidation, and methemoglobin formation. For this, we analyzed 23 leprosy patients and 20 healthy individuals from the Amazon region, Brazil, aged between 20 and 45 years. Blood sampling enabled the evaluation of leprosy patients prior to starting multidrug therapy (called MDT 0) and until the third month of multidrug therapy (MDT 3). With regard to dapsone (DDS) plasma levels, we showed that there was no statistical difference in drug plasma levels between multibacillary (0.518±0.029 μg/mL) and paucibacillary (0.662±0.123 μg/mL) patients. The methemoglobin levels and numbers of Heinz bodies were significantly enhanced after the third MDTsupervised dose, but this treatment did not significantly change the lipid peroxidation and NO levels in these leprosy patients. In addition, CAT activity was significantly reduced in MDT-treated leprosy patients, while GSH content was increased in these patients. However, SOD and Trolox equivalent antioxidant capacity levels were similar in patients with and without treatment. These data suggest that MDT can reduce the activity of some antioxidant enzyme and influence ROS accumulation, which may induce hematological changes, such as methemoglobinemia in patients with leprosy. We also explored some redox mechanisms associated with DDS and its main oxidative metabolite DDS-NHOH and we explored the possible binding of DDS to the active site of CYP2C19 with the aid of molecular modeling software. © 2014 Schalcher et al.