Biomedical subjects

R Brigelius-Flohé

Publications and source records attributed to R Brigelius-Flohé.

At least 19 recordsLinked to original sources

PubMed2001

Vitamin E kinetics in smokers and nonsmokers.

Does cigarette smoking increase vitamin E utilization in vivo? A trial was carried out in 6 smokers and 5 nonsmokers of comparable ages and serum lipids. Subjects consumed 75 mg each d(3)-RRR and d(6)-all rac-alpha-tocopheryl acetates (natural and synthetic vitamin E, respectively) daily for 7 d with a standardized breakfast. Fasting blood samples were drawn on days -7, -6, -5, -4, -3, -2, -1, 0, 1, 2, 3, 4, 5, 6, 7, 9, 14, 21 (negative days indicate supplementation). In both groups, plasma d(3)-alpha-tocopherol concentrations were approximately double of d(6)-alpha-tocopherol. At day 0, the %d(3) alpha-tocopherols (d(3)-alpha-tocopherol/total-alpha-tocopherol x 100) were similar in both smokers and nonsmokers. Subsequently, there was a trend toward a faster exponential disappearance of the plasma %d(3) alpha-tocopherol in smokers compared with nonsmokers (0.30 +/- 0.04 compared with 0.24 +/- 0.05, p =.0565). The calculated %d(3) half-lives were 55.6 +/- 7.4 h in smokers and 72.1 +/- 17.3 h in nonsmokers (p =.0630). By day 21, the %d(3) in smokers had decreased to 1.4% +/- 0.3% while it was 2.2% +/- 0.7% (p =.0418) in the nonsmokers. These data suggest that smoking increases plasma vitamin E disappearance, but further studies are needed to confirm this finding and to assess its cause.

Adult↗

PubMed2001

An optical method for the detection of oxidative stress using protein-RNA interaction.

The cytosolic 4Fe-4S protein aconitase can be converted under the influence of reactive oxygen species into an iron-regulatory protein (IRP1). Therefore, the IRP1 level is considered as an indirect marker of oxidative stress. An experimental approach is presented here to detect the concentration of this marker protein by surface plasmon resonance. The optical method exploits the natural binding affinity of IRP1 to an iron-responsive element (IRE) which was in vitro transcribed with a linker sequence and subsequently immobilized on a BIACORE sensor chip. The detection was found to be reproducible and sensitive in the range 20-200 nM IRP. Conditions of the binding process, such as pH and thiol concentration, were characterized. Feasibility of the method to detect and quantify IRP1 in physiological media was demonstrated.

Aconitate Hydratase↗

PubMed2001

Stability of gastrointestinal glutathione peroxidase mRNA in selenium deficiency depends on its 3'UTR.

Selenoproteins decrease upon selenium-deprivation according to their hierarchical ranking. Whereas classical glutathione peroxidase (cGPx) responds to decreased selenium supply with a complete loss of protein and a marked reduction of mRNA levels, gastrointestinal glutathione peroxidase (GI-GPx) remains detectable and its mRNA is stable. The impact of the 3'UTR on cGPx and GI-GPx mRNA stability was studied in stably transfected HepG2 cells with combinations of mutually exchanged coding regions and 3'UTRs of human cGPx and GI-GPx. Stability of chimeric mRNAs was measured by competitive RT-PCR. We found that GI-GPx 3'UTR is sufficient to stabilize its own mRNA but not that of cGPx.

3' Untranslated Regions↗

PubMed2001

Functions of GI-GPx: lessons from selenium-dependent expression and intracellular localization.

Gastro intestinal glutathione peroxidase (GI-GPx) is one of the four distinct mammalian selenoperoxidases. It had been reported to be restricted to the gastrointestinal tract but has more recently been identified also in human liver and some tumor cell lines. GI-GPx ranks high in the hierarchy of selenoproteins. The GI-GPx mRNA rather increases than decreases in selenium deficiency. GI-GPx protein responds poorly to selenium deprivation and increases fast upon resupplementation. Putative biological roles of GI-GPx, e.g. protection against food-born hydroperoxides, redox-regulation of proliferation or apoptosis, and modulation of mucosal immunity, are discussed in the light of cellular and subcellular distribution, transcriptional regulation and observations with k.o. mice.

Animals↗

PubMed2001

Cellular and subcellular localization of gastrointestinal glutathione peroxidase in normal and malignant human intestinal tissue.

The gastrointestinal glutathione peroxidase (GI-GPx) is believed to prevent absorption of hydroperoxides. GI-GPx is expressed in the intestine together with the other three glutathione peroxidase isoenzymes, raising the question of the physiological role of the different GPx types. We therefore studied the cellular and subcellular distribution of GI-GPx in normal and malignant tissue obtained from patients with colorectal cancer or familial polyposis by immunohistochemistry. In healthy ileum epithelium GI-GPx was preferentially enriched in Paneth cells. In unaffected crypts of colon and rectum, it decreased gradually from the ground to the luminal surface. In crypt ground, GI-GPx was uniformly distributed, whereas in cells at the luminal surface it was concentrated in structures capping the nuclei at the apical pole. In colorectal cancer, GI-GPx expression depended on the stage of malignant transformation. In early stages, GI-GPx was increased and pronouncedly associated with the vesicular structures. In progressed stages of malignancy, structures disintegrated and GI-GPx distribution became more diffuse. These observations support the hypothesis that GI-GPx, apart from being a barrier against hydroperoxide absorption, might be involved in cell growth and differentiation.

Adenomatous Polyposis Coli↗

PubMed2000

The putative glutathione peroxidase gene of Plasmodium falciparum codes for a thioredoxin peroxidase.

A putative glutathione peroxidase gene (Swiss-Prot accession number Z 68200) of Plasmodium falciparum, the causative agent of tropical malaria, was expressed in Escherichia coli and purified to electrophoretic homogeneity. Like phospholipid hydroperoxide glutathione peroxidase of mammals, it proved to be monomeric. It was active with H(2)O(2) and organic hydroperoxides but, unlike phospholipid hydroperoxide glutathione peroxidase, not with phosphatidylcholine hydroperoxide. With glutathione peroxidases it shares the ping-pong mechanism with infinite V(max) and K(m) when analyzed with GSH as substrate. As a homologue with selenocysteine replaced by cysteine, its reactions with hydroperoxides and GSH are 3 orders of magnitude slower than those of the selenoperoxidases. Unexpectedly, the plasmodial enzyme proved to react faster with thioredoxins than with GSH and most efficiently with thioredoxin of P. falciparum (Swiss-Prot accession number 202664). It is therefore reclassified as thioredoxin peroxidase. With plasmodial thioredoxin, the enzyme also displays ping-pong kinetics, yet with a limiting K(m) of 10 microm and a k(1)' of 0.55 s(-)1. The apparent k(1)' for oxidation with cumene, t-butyl, and hydrogen peroxides are 2.0 x 10(4) m(-1) s(-1), 3.3 x 10(3) m(-1) s(-1), and 2.5 x 10(3) m (-1) s(-1), respectively. k(2)' for reduction by autologous thioredoxin is 5.4 x 10(4) m(-1) s(-1) (21.2 m(-1) s(-1) for GSH). The newly discovered enzymatic function of the plasmodial gene product suggests a reconsideration of its presumed role in parasitic antioxidant defense.

Amino Acid Sequence↗

PubMed2000

Translocation of the interleukin-1 receptor-associated kinase-1 (IRAK-1) into the nucleus.

Interleukin-1 (IL-1) signal transduction involves the recruitment of the IL-1 receptor-associated kinase-1 (IRAK-1). Subsequent signaling finally leads to nuclear translocation of NFkappaB. We here show that the association and autophosphorylation of IRAK-1 was already detectable 30 s after IL-1 stimulation of ECV 304 cells. Significant levels of IRAK-1 accumulated in the nucleus 30 min after IL-1 stimulation shown by Western blot analysis and confocal laser scanning microscopy. Nuclear transfer of IRAK-1 upon IL-1 stimulation was confirmed in the murine T cell line EL-4. This characterizes nuclear localization of IRAK-1 as a possibly essential event in the IL-1 signaling cascade.

Animals↗

PubMed2000

Reactive oxygen species and proinflammatory cytokine signaling in endothelial cells: effect of selenium supplementation.

The release of superoxide (O(2)(*-)) and hydrogen peroxide (H(2)O(2)), induced by tumor necrosis factor-alpha (TNF-alpha) or interleukin-1beta (IL-1beta), has been studied in the endothelial cell line ECV 304 in the presence and absence of selenium (Se) supplementation. Both cytokines elicit the production of both species. Selenium supplementation, which increases Se-enzyme activity, decreases the amount of H(2)O(2) but not O(2)(*-) detectable in the extracellular medium. Inhibition of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase by diphenyliodonium (DPI) or phenylarsine oxide (PAO), largely prevents O(2)(*-) production, whereas H(2)O(2) remains above the amount accounted for by disproportion of residual O(2)(*-). Thus, a fraction of H(2)O(2) found in the medium, derives from an intracellular pool, which is under control of selenium-dependent peroxidases. This is further supported by the observation that in Se-supplemented cells, the rate of intracellular glutathione (GSH) depletion induced by cytokine treatment is faster and more extensive. Because Se supplementation decreases cytokine-induced NF-kappaB activity, whereas added H(2)O(2) is inactive and catalase does not affect the activation induced by TNF-alpha, it is concluded that only intracellularly generated H(2)O(2) has a role in transcription factor activation by both TNF-alpha and IL-1beta.

Biphenyl Compounds↗

PubMed2000

Overexpression of PHGPx inhibits hydroperoxide-induced oxidation, NFkappaB activation and apoptosis and affects oxLDL-mediated proliferation of rabbit aortic smooth muscle cells.

Rabbit abdominal aortic smooth muscle cells (SMC) were stably transfected with the cDNA of porcine phospholipid hydroperoxide glutathione peroxidase (PHGPx) by means of a retroviral gene transfer technique, to create a model for studying cellular processes relevant to atherogenesis. The transfected cells (SMC/PHGPx) had approximately 4-fold higher PHGPx activity when cultured in the presence of selenite whereas the parental cells did not show any significant increase in PHGPx or total GPx activity upon selenium supplementation. In situ functionality of PHGPx was validated by inhibition of linoleic acid hydroperoxide-induced toxicity, dihydrorhodamine oxidation, NFkappaB activation and apoptosis. SMC grown in 1% FCS responded to oxidized LDL (oxLDL) with a marked proliferation, as measured by [3H]thymidine incorporation, irrespective of selenium supplementation. In SMC/PHGPx grown with or without selenite under control conditions or exposed to native LDL, thymidine incorporation was generally depressed. Also, oxLDL-induced proliferation was lower in SMC/PHGPx compared to untransfected SMC up to 24 h of incubation. After 40 h, however, selenite supplementation restored maximum proliferation response to oxLDL in SMC/PHGPx. The results suggest a proliferative effect of endogenous hydroperoxides in SMC. They further reveal that hydroperoxy lipids of oxLDL contribute to the induction of proliferation, but also suggest involvement of hydroxy lipids in the response to oxLDL.

Animals↗

PubMed2000

Gastrointestinal glutathione peroxidase prevents transport of lipid hydroperoxides in CaCo-2 cells.

BACKGROUND & AIMS: Gastrointestinal glutathione peroxidase (GI-GPx), 1 of the 4 types of selenium-dependent glutathione peroxidases, is expressed exclusively in the gastrointestinal system and has therefore been suggested to function as a barrier against the absorption of dietary hydroperoxides. METHODS: The selenium-dependent expression of GI-GPx and cytosolic GPx (cGPx) was analyzed by Western blotting. Transport of 13-hydroperoxy octadecadienoic acid (13-HPODE) was investigated in a CaCo-2 cell monolayer modulated in GI-GPx and cGPx by selenium restriction or repletion. Localization of GI-GPx in rat intestine was visualized by immunohistochemistry. RESULTS: Low but significant GI-GPx levels were detected in selenium-deficient CaCo-2 cells and in the gastrointestinal tract of selenium-deficient rats, whereas cGPx was completely absent. Selenium supplementation of CaCo-2 cells resulted in a 5-fold increase of GI-GPx protein, whereas total GPx activity increased by a factor of 13, with most of the GPx activity under selenium-adequate conditions being cGPx. Irrespective of the selenium status, 13-HPODE did not reach the basolateral side of an intact CaCo-2 cell monolayer. Depending on the selenium status, hydroperoxides damaged the monolayer as evidenced by loss of transepithelial resistance and paracellular diffusion of lucifer yellow. Only under these conditions was unmetabolized 13-HPODE detectable at the basolateral side. CONCLUSIONS: Low GI-GPx levels, as present in selenium deficiency, suffice to prevent transport of 13-HPODE. GI-GPx may thus function as a barrier against hydroperoxide absorption. cGPx contributes to balance major oxidative challenge.

Animals↗

PubMed2000

Selenium, the element of the moon, in life on earth.

The present status of selenium biochemistry is reviewed with particular emphasis on biomedical problems related to the selenium status of humans and experimental animals. Historical milestones of selenium biochemistry starting from the identification of the first selenoenzymes up to the elucidation of prokaryotic and eukaryotic selenoprotein biosynthesis are compiled. Topical hypotheses on the biological role of selenium in general and of individual selenoproteins in respect to antioxidant defense, redox regulation of metabolic processes, thyroid function, spermatogenesis, oncogenesis, and atherogenesis are critically evaluated.

Animals↗

PubMed2000

A rapid method for the extraction and determination of vitamin E metabolites in human urine.

A method for the direct extraction and routine analysis of the vitamin E metabolites gamma- and alpha-carboxyethyl hydroxychroman (gamma- and alpha-CEHC) from human urine has been developed. A relatively small sample volume (5 ml) can be used and, after enzymatic hydrolysis of the conjugated forms and acidification, the metabolites are extracted with diethyl ether. Recovery of alpha- and gamma-CEHC was compared to that of trolox, used as an internal standard, added to 24-h urine collections from vitamin E-unsupplemented volunteers. Various solvent conditions were initially tested; acidification and ether extraction gave the highest recovery. It was found that after addition and extraction from urine, trolox, alpha- and gamma-CEHC are recovered to a similar extent, hence trolox is viable as an internal standard. The samples were analyzed by both GC and HPLC with electrochemical detection (ECD). HPLC-ECD was found to give higher selectivity and higher sensitivity compared to GC or HPLC with UV detection at 290 nm. The HPLC-ECD detection limit was 10 fmol, linearity (r(2) > 0.98) was achieved in the range of 40 to 200 fmol, which was found to be optimal for 24-h urines from unsupplemented subjects. Inter-sample variability was typically 2-5%. This greater sensitivity and selectivity means that vitamin E metabolites can be analyzed even in unsupplemented subjects. It is also possible to measure unconjugated forms of the metabolites. Typically these were found to represent approximately 10% of the total alpha- and gamma-CEHC. This method can be used routinely for the determination of vitamin E metabolites in urine. The new extraction and detection methods described are relatively quick, less laborious, and more cost-effective than previously available methods.

Calibration↗

PubMed2000

Urinary alpha-tocopherol metabolites in alpha-tocopherol transfer protein-deficient patients.

Patients with alpha-tocopherol transfer protein (alpha-TTP) defects experience neurological symptoms characteristic of vitamin E deficiency and depend on continuous high alpha-tocopherol supplements. We investigated the excretion of 2,5,7, 8-tetramethyl-2(2'-carboxyethyl)-6-hydroxychroman (alpha-CEHC), a urinary metabolite of alpha-tocopherol, as a putative marker for the alpha-tocopherol status of alpha-TTP-deficient patients and control subjects. In three patients vitamin E supplementation was stopped for short periods of time, during which plasma alpha-tocopherol concentrations and urinary alpha-CEHC excretion were measured. In the patients, plasma alpha-tocopherol decreased below normal (<5 micromol/l) but alpha-CEHC excretion remained above the range of unsupplemented control subjects (0.118-0.306 mg/day, n = 6). In healthy subjects, however, alpha-CEHC excretion was increased only after surpassing a plasma alpha-tocopherol threshold of 30-40 micromol/l. Such a threshold did not exist in patients. The general mechanism of alpha-tocopherol degradation did not appear to differ between patients and control subjects. The presumed mechanism of omega- and subsequent beta-oxidation was supported by the detection of alpha- CPHC, an alpha -CEHC homolog with a side chain longer by 3 carbon atoms, both in supplemented patients and in control subjects.

Adolescent↗

PubMed1999

Quantification of the alpha- and gamma-tocopherol metabolites 2,5,7, 8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman and 2,7, 8-trimethyl-2-(2'-carboxyethyl)-6-hydroxychroman in human serum.

alpha- and gamma-tocopherol are the major vitamin E compounds found in human blood and tissues. The metabolites are 2,5,7, 8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman (alpha-CEHC) and 2,7,8-trimethyl-2-(2'-carboxyethyl)-6-hydroxychroman (gamma-CEHC, LLU-alpha), respectively. alpha-CEHC is excreted mainly as glucuronide or sulfate conjugates in the urine. Here we describe a sensitive and reliable method to analyze alpha- and gamma-CEHC in human serum. The concentration of alpha-CEHC in human serum is in the range of 5-10 pmol/ml but increases significantly up to 200 pmol/ml upon supplementation with RRR-alpha-tocopherol. About one-third of the alpha-CEHC circulating in the blood is present as a glucuronide conjugate. Baseline levels of gamma-CEHC are about 50 to 85 pmol/ml.

Adult↗

PubMed1999

ATP-sensitive association of mortalin with the IL-1 receptor type I.

Interleukin-1 (IL-1) is a major proinflammatory cytokine mediating local and systemic responses of the immune system. Two types of IL-1 receptors are known, but only the IL-1 receptor type I initiates biological responses. Here we show that two proteins with nucleic acid binding potential and mortalin, a member of the HSP70-family, are associated with the IL-1 receptor type I irrespective of IL-1 binding. The association of mortalin with the IL-1 receptor type I is specifically reversed by ATP concentrations in the physiological range. Other nucleotides are not or much less effective. The in vitro dissociation of mortalin effects neither the receptor association nor the activity of IRAK, which initiates the IL-1-dependent phosphorylation cascade. The roles of the receptor-associated proteins are therefore discussed in the context of receptor internalisation.

Adenosine Triphosphate↗

PubMed1999

13-HPODE and 13-HODE modulate cytokine-induced expression of endothelial cell adhesion molecules differently.

Expression of cellular adhesion molecules (CAMs) at endothelial surfaces represents a physiological response to vascular damage and mediates the initiation of inflammation and possibly of atherogenesis. The cytokines TNF alpha and IL-1 are potent inducers of CAMs in endothelial cells. Reactive oxygen species comprising lipid oxidation products have been implicated in the signaling pathways of both TNF alpha and IL-1 and accordingly could modulate atherogenic events. We, therefore, investigated the potential role of the lipoxygenase product, 13-hydroperoxyoctadecadienoic acid (13-HPODE), which has also been identified in oxidized low density lipoproteins on CAM expression in HUVEC. 13-HPODE induced the expression of ICAM-1 in a concentration dependent manner up to 75 microM. Higher concentrations were toxic. Similar effects were observed with H2O2 and phosphatidylcholine hydroperoxide. VCAM-1 and E-selectin were not induced by 13-HPODE. 13-HPODE administered simultaneously with IL-1 or TNF alpha induced ICAM-1 additively, suggesting that hydroperoxides and cytokines act on the same signaling pathways. In contrast, pretreatment of cells with 50 microM 13-HPODE for 1 hour rather inhibited subsequent cytokine-induced ICAM-1 and E-selectin expression. Surprisingly, the reduction product of 13-HPODE, 13-hydroxyoctadecadienoic acid (13-HODE) proved to be an even better inducer of ICAM-1 than 13-HPODE. Pretreatment with 13-HODE did not show any inhibitory effect on ICAM-1 expression. Our data show that lipoxygenase products differentially affect CAM expression. 13-HPODE is stimulatory by itself and can positively or negatively affect cytokine signaling depending on time of exposure. 13-HODE induces CAM expression by itself but does not inhibit cytokine signaling. Thus, the interplay of lipoxygenase products with proinflammatory cytokines can not simply be explained by an oxidant-mediated facilitation of cytokine signaling.

Cell Adhesion Molecules↗

PubMed1999

Interleukin-1 signaling is dependent on free thiols.

Stimulation of the Interleukin-1 receptor type I (IL-1-RI) with IL-1 activates an associated serine/threonine kinase, IRAK, which phosphorylates downstream targets, resulting in NFkappaB activation. The signaling cascade is accompanied by oxidative processes and contains putative targets for redox regulation. Preincubation of the murine T cell line EL-4 and the human umbilical cord vein endothelial cell line ECV 304 with thiol modifying compounds like diamide, menadione or phenylarsine oxide inhibited the IL-1-induced phosphorylation of an endogenous substrate with a molecular mass of 60 kD. In the endothelial cell line, a second target of about 85 kD was phosphorylated after IL-1 stimulation, which was also inhibited by thiol modification. These data suggest that IL-1 signal transduction depends on free thiols which might be targets for redox regulation not only in lymphocytes, but also in endothelial cells.

Animals↗

PubMed1999

Gastrointestinal glutathione peroxidase.

The gastrointestinal glutathione peroxidase (GI-GPx) is the fourth member of the GPx family. In rodents, it is exclusively expressed in the gastrointestinal tract, in humans also in liver. It has, therefore, been discussed to function as a primary barrier against the absorption of ingested hydroperoxides. A vital function of GI-GPx can be deduced from the unusual stability of its mRNA under selenium-limiting conditions, the presence of low amounts of GI-GPx protein in selenium deficiency where cGPx is absent, and the fast reappearance of the GI-GPx protein upon refeeding of cultured cells with selenium compared to the slower reappearance of cGPx protein. Furthermore, the Secis efficiency of GI-GPx is low when compared to cGPx and PHGPx. It is, however, almost independent of the selenium status of the cells tested. All these characteristics rank GI-GPx high in the hierarchy of selenoproteins and point to a role of GI-GPx which might be more crucial than that of cGPx, at least in the gastrointestinal system.

Animals↗