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R Stocker

Publications and source records attributed to R Stocker.

At least 145 records · Page 8Linked to original sources
PubMed1995

Modulation of superoxide generation in in vivo lipopolysaccharide-primed rat alveolar macrophages by arachidonic acid and inhibitors of protein kinase C, phospholipase A2, protein serine-threonine phosphatase(s), protein tyrosine kinase(s) and phosphatase(s).

Ninety minutes after i.v. injection of Escherichia coli lipopolysaccharide (LPS) (1 mg/kg) into rats, phorbol 12-myristate 13-acetate (PMA)-stimulated superoxide anion (O2-) secretion was enhanced in suspensions of in vivo LPS-treated alveolar macrophages (AM phi) when compared with saline (SAL)-treated AM phi. The purpose of this investigation was to dissect the in vitro mechanism of PMA-stimulated O2- generation in both LPS and SAL-treated rat AM phi, with a panel of inhibitors of protein kinase C (PKC), protein serine-threonine phosphatase(s) (PSP), protein tyrosine kinase(s) (PTK) and phosphatase(s) (PTP), phospholipase A2 (PLA2), cyclooxygenase (CO) and 5-lipoxygenase (5-LO). The following agents blocked PMA-stimulated O2- generation in both LPS- and SAL-treated AM phi (expressed as percentage of control): 1) PKC inhibitors: staurosporine: 100 nM, 7.0% (LPS) and 5.6% (SAL); sphingosine: 10 microM, 21% (LPS) and 10.5% (SAL); 2) PTK inhibitor: genistein: 100 microM, 44% (LPS) and 31% (SAL); 3) PTP inhibitors: phenylarsine oxide, 10 microM, 12.1% (LPS) and 18% (SAL); diamide, 1000 microM, 10.1% (LPS) and 10.5% (SAL); and 4) PLA2 inhibitors: manoalide: 1 microM, 29.3% (LPS) and 5.2% (SAL); scalaradial: 1 microM, 7.7% (LPS) and 7.1% (SAL); and WAY 125,984: 10 microM, 17.1% (LPS) and 14.5% (SAL). In addition, it was observed that exogenously added arachidonic acid (AA)-stimulated O2- generation in a time- and dose-dependent manner in both LPS and SAL-treated AM phi. The following inhibitors enhanced or did not affect PMA-stimulated O2- generation in LPS- and SAL-treated AM phi (expressed as percentage of of control): 1) PSP inhibitors: okadaic acid: 0.5 microM, 117% (LPS) and 153% (SAL); calyculin A: 1 microM, 112% (LPS) and 101% (SAL); 2) CO and 5-LO inhibitors: indomethacin: 10 microM, 107% (LPS) and 90% (SAL); WY 50, 295: 1 microM, 99% (LPS) and 103% (SAL); and 3) the PTP inhibitor orthovanadate upon prolonged preincubation. In both in vivo LPS- or SAL-primed AM phi, PMA-stimulated O2- generation appears to be modulated by PKC, PLA2, AA, PTK, PTP and PSP. No modulatory role was evident for either CO or 5-LO metabolites. These findings might bear on the design of therapeutic approaches for the modulation of O2- release by AM phi in the early stages of sepsis and adult respiratory distress syndrome.

Animals↗
PubMed1995

[Results of surgical management of distal femur fractures with joint involvement].

Results of the treatment of 60 intraarticular distal fractures of the femur (AO classification: 33-B and 33-C fractures) are summarized. Frequencies of the different types of fractures were B1, 7; B2, 10; B3, 4; C1, 10; C2, 1; C3, 8. In 36 patients an isolated fracture, was present, in 9 patients, one additional injury, and in 16 patients there was more than one fracture or multiple trauma. We found 16.7% open fractures. Operation was possible within 24 h in 16 patients, in 21 patients between the 2nd and the 5th days, and in the rest after the 5th day. Check-ups took place an average of 46.4 months after the accident. The average age of the patients was 64.1 years (16-93). We encountered 3 cases of infection; we were able to treat 2 of these successfully, while 1 led to chronic osteomyelitis. Further complications were: loosening of implants (6 cases), non-union (1) fractures of plates (2), haematoma requiring operative treatment (1) refracture (1). All fractures resulted in osseous consolidation. Mobilization of patients was achieved an average of 12 days after operation. We evaluated the cases according to the Neer score. In 32 cases the result was excellent, in 18, average and in 4, unsatisfactory. Failure had to be recorded in 1 case. The combination of locking nails and lagscrews is an alternative method of osteosynthesis for distal femural fractures with an intra-articular component, provided strict selection criteria are applied.

Adolescent↗
PubMed1995

[Traumatic aortic rupture: diagnosis using biplanar transesophageal echocardiography].

Acute aortic rupture is a typical consequence of severe blunt chest trauma often associated with rapid deceleration in car accidents. Initial diagnostic findings are often misleading and multiorgan injuries add to the diagnostic complexity; therefore, the natural history of acute rupture is usually fatal during the first 24 h after injury if left untreated. Prompt and simple diagnosis is, hence, of paramount importance for successful treatment of acute aortic rupture. Transesophageal echocardiography, particularly with a biplane or multiplane probe, currently represents the diagnostic tool of choice to meet these criteria; because of its high sensitivity and specificity transesophageal echocardiography will replace aortography as "gold standard" for diagnosis of acute aortic rupture. We report on a 47-year-old woman with severe blunt thoraco-abdominal trauma resulting from a car accident; at hospital admission abdominal injuries were predominant and diagnosis of an acute rupture of the descending thoracic aorta was made only about 18 h after admission using biplane transesophageal echocardiography. Emergency surgical revision confirmed the diagnosis of complete transsection of the descending thoracic aorta immediately after the origin of the left subclavian artery; the site of transsection was surrounded by a large hematoma. Despite successful reconstruction of the descending thoracic aorta by means of graft interposition, a recurrent local bleeding event lead to complete circulatory destabilization and, finally, to the death of the patient.

Aortic Rupture↗
PubMed1995

Exchange of oxidized cholesteryl linoleate between LDL and HDL mediated by cholesteryl ester transfer protein.

This study examines the cholesteryl ester transfer protein (CETP)-mediated exchange of cholesteryl linoleate hydroperoxide (Ch18:2-OOH) and cholesteryl linoleate hydroxide (Ch18:2-OH) between low density lipoprotein (LDL) and high density lipoprotein (HDL). When [3H]Ch18:2-OOH- and [3H]18:2-OH-labeled LDL were incubated at 37 degrees C for 0-24 h with unoxidized HDL and purified CETP, Ch18:2-OOH and Ch18:2-OH accumulated in the HDL. Similarly, when incubations were carried out with [3H]Ch18:2-OOH- and [3H]Ch18:2-OH-labeled HDL, unoxidized LDL, and CETP, Ch18:2-OOH and Ch18:2-OH accumulated in the LDL. Comparable results were obtained for the CETP-mediated transfer of [3H]Ch18:2-OH alone from LDL to HDL. Transfer to HDL of oxidized cholesteryl linoleate from [3H]Ch18:2-OOH- and [3H]Ch18:2-OH-labeled LDL was comparable to that of unoxidized cholesteryl linoleate (Ch18:2). However, the rate of transfer of [3H]Ch18:2-OOH and [3H]Ch18:2-OH from LDL to HDL increased linearly as the molar ratio of acceptor (HDL) to donor (oxidized LDL) particles in the incubation increased from 0.5:1 to 10:1. This increased rate of exchange was accompanied by an increased proportion of the oxidized Ch18:2 being present as the hydroxide rather than hydroperoxide. Further increases in the molar ratio of HDL to oxidized LDL particles neither affected the transfer rate nor the extent of reduction of Ch18:2-OOH to Ch18:2-OH. We therefore conclude that i) CETP mediates bidirectional transfers of Ch18:2-OOH and Ch18:2-OH between HDL and LDL; ii) CETP does not distinguish between Ch18:2-OOH, Ch18:2-OH, and Ch18:2 as it mediates their exchange between HDL and LDL; and iii) association with HDL hastens the reduction of Ch18:2-OOH to Ch18:2-OH.

Adult↗
PubMed1994

Human blood cells support the reduction of low-density-lipoprotein-associated cholesteryl ester hydroperoxides by albumin-bound ebselen.

Ebselen, a glutathione peroxidase mimic capable of reducing simple as well as complex hydroperoxides, including those of phospholipids and cholesteryl esters in intact oxidized low-density lipoprotein (LDLox), requires the presence of low-molecular-mass thiols to be active. In plasma, the drug is thought to be transported as an inactive albumin complex. As formation of LDLox is likely to occur extracellularly, we tested under which conditions ebselen can support reduction of LDLox-associated cholesteryl ester hydroperoxides outside cells. We observed that addition of albumin-bound ebselen to whole blood, but not plasma, resulted in reduction of LDLox-associated cholesteryl linoleate hydroperoxides to the corresponding hydroxides. The observed reduction was rapid and its extent increased with increasing concentrations of ebselen. Physical contact of blood cells with LDLox was not required for this reducing activity. These results demonstrate that, in the presence of blood cells, extracellular ebselen is catalytically active. They suggest that ebselen may be considered as a drug for extracellular targets.

Adult↗
PubMed1994

Oxidation of low-density lipoprotein by hypochlorite causes aggregation that is mediated by modification of lysine residues rather than lipid oxidation.

Peroxidation of low-density lipoprotein (LDL) lipid is generally thought to represent the initial step in a series of modification reactions that ultimately transform the protein moiety of the lipoprotein into a form recognized by receptors different from those that bind native LDL. Uptake of LDL via these alternative receptors can lead to the formation of lipid-laden cells, which are typical for the early stages of atherogenesis. We have studied the oxidative modification of LDL by hypochlorite (-OCl), a powerful oxidant produced from H2O2 and chloride via the action of myeloperoxidase which is released from activated neutrophils and monocytes. Exposure of LDL to reagent or enzymically generated -OCl at 4 or 37 degrees C resulted in immediate and preferential oxidation of amino acid residues of apolipoprotein B-100, the single protein associated with LDL. Lysine residues quantitatively represented the major target and, like tryptophan, were oxidized to approximately the same extent with reagent or enzymically generated -OCl. In contrast, LDL lipid oxidation was less favoured than protein oxidation, as judged by the amounts of lipid hydroperoxides, chlorohydrins, cholesterol or fatty acid oxidation products formed. Treatment with -OCl caused aggregation of LDL, as shown by an increased turbidity of the oxidized LDL solution and elution from a size-exclusion h.p.l.c. column of high-molecular-mass LDL complexes. Chemical modification of lysine residues before oxidation with -OCl prevented aggregation, while it enhanced the extent of lipid peroxidation. Treatment of LDL with -OCl also caused the formation of carbonyl groups and release of ammonia; both these modifications were inhibited by lysine-residue modification before oxidation. These results demonstrate that aggregation reactions are dependent on initial lysine oxidation by -OCl, followed by deamination and carbonyl formation, but do not involve lipid (per)oxidation. We propose that the observed -OCl-mediated aggregation of LDL is caused, at least in part, by cross-linking of apoproteins by Schiff-base formation independently of lipid peroxidation.

Adult↗
PubMed1994

Free and albumin-bound bilirubin are efficient co-antioxidants for alpha-tocopherol, inhibiting plasma and low density lipoprotein lipid peroxidation.

Peroxidation of the lipid moieties of low density lipoproteins (LDL) is regarded as an early event in atherogenesis. Because bilirubin is a physiological reductant with antioxidant activities, we investigated its inhibitory action on the radical-mediated oxidation of LDL and plasma lipids. Exposing fresh human blood plasma to lipophilic peroxyl radicals generated from 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN) resulted in rapid oxidation of ubiquinol-10, followed by that of ascorbate and bilirubin. Plasma lipids were well protected from peroxidation as long as these three antioxidants were present, as assessed by the amounts of cholesterylester hydroperoxides formed during this period. Following consumption of these antioxidants, and in the presence of alpha-tocopherol, the rate of hydroperoxide formation increased sharply with roughly 2 molecules of cholesterylester hydroperoxides being formed for each peroxidation initiating event. Supplementation of AMVN-oxidizing plasma with exogenous bilirubin at the onset of rapid lipid peroxidation, i.e. after depletion of endogenous ubiquinol-10, ascorbate, and bilirubin, led to a halt in both hydroperoxide formation and consumption of alpha-tocopherol. When isolated LDL was incubated with AMVN, approximately 9 molecules of cholesterylester hydroperoxides were formed per peroxidation initiating event and while alpha-tocopherol was consumed. Addition of free or albumin-bound bilirubin to isolated LDL at the onset of oxidation resulted in a strong inhibition of hydroperoxide formation and alpha-tocopherol consumption, the effect being more pronounced with the free pigment. Addition of the corresponding amounts of albumin alone was without effect. In the presence of albumin-bound bilirubin, some 30% of the pigment was initially converted into biliverdin, whereas formation of this oxidation product was not observed with the free pigment. Also, the presence of bilirubin oxidase partially reversed the inhibitory activity of bilirubin on AMVN-induced LDL oxidation in the absence but not presence of albumin. An attenuation of hydroperoxide formation and a temporary increase in LDL's alpha-tocopherol concentration were observed when free- or albumin-bound bilirubin were added to AMVN-oxidizing, alpha-tocopherol-containing LDL. In contrast, hydroperoxide formation was not inhibited significantly when the albumin-bound pigment was added to oxidizing LDL after complete consumption of its alpha-tocopherol. Our results show that bilirubin inhibits oxidation of LDL lipids initiated within the lipoprotein core and indicate that this activity is mediated by interaction of the pigment with LDL's alpha-tocopherol.

Adult↗
PubMed1994

Nitric oxide inhibits indoleamine 2,3-dioxygenase activity in interferon-gamma primed mononuclear phagocytes.

Indoleamine 2,3-dioxygenase (IDO) and nitric oxide synthase are part of the anti-tumor and antimicrobial activities of mononuclear phagocytes induced by interferon-gamma (IFN gamma). As IDO is a heme-containing enzyme and NO, the product of nitric oxide synthase-initiated arginine degradation, is a regulator of heme enzymes, we investigated whether NO is capable of modulating IDO activity in IFN gamma-primed mononuclear phagocytes. Authentic NO gas or the NO-generating compound, diethylamine dinitric oxide adduct, dose-dependently inhibited IDO activity in cell lysates prepared from IFN gamma-primed human peripheral blood mononuclear cells, as assessed by the ascorbate/methylene blue assay for IDO. In contrast, neither nitrite nor nitrate affected IDO activity. Exposure of intact IFN gamma-primed human peripheral blood mononuclear cells or monocyte-derived macrophages to any of the NO-generating compounds, sodium nitroprusside, glyceryl trinitrate, S-nitroso-N-acetylpenicillamine, or diethylamine dinitric oxide adduct, resulted in inhibition of both the consumption of tryptophan from and formation of its metabolite, kynurenine, in the culture medium. The observed inhibition of IDO activity was not due to toxicity of the NO generators and was abrogated by the co-addition of oxyhemoglobin, an antagonist of NO function. Comparable concentrations of nitrite or nitrate did not inhibit IDO activity in intact cells. In contrast to human cells, addition of IFN gamma to murine macrophages, cultured in complete RPMI 1640 medium, readily induced nitric oxide synthase. Others have reported that such treatment does not induce IDO activity in these cells. However, induction of IDO activity was observed in murine macrophages when the synthesis of reactive nitrogen species was inhibited, by using arginine-free medium and/or the nitric oxide synthesis inhibitor, NG-monomethyl-L-arginine. Together, these results demonstrate that both exogenous and endogenous NO inhibit IDO activity and that oxidative arginine and tryptophan metabolism in IFN gamma-primed mononuclear phagocytes are functionally related. Our study thereby provides an insight into how these cells may regulate some of their antimicrobial and anti-tumor activities.

Amino Acid Oxidoreductases↗
PubMed1994

Reduction of HDL- and LDL-associated cholesterylester and phospholipid hydroperoxides by phospholipid hydroperoxide glutathione peroxidase and Ebselen (PZ 51).

The reaction of phospholipid hydroperoxide glutathione peroxidase (PHGPx) and Ebselen with phospholipid and cholesterylester hydroperoxides associated with HDLox and LDLox was investigated using specific HPLC assays for the hydroperoxides of phosphatidylcholine (PCOOH) and cholesteryllinolate (Ch18:2-OOH) and for cholesteryllinolate hydroxides (Ch18:2-OH). HDLox and LDLox were formed from the corresponding isolated native lipoproteins by controlled and limited oxidation initiated by aqueous peroxyl radicals. Incubation of HDLox or LDLox in the presence of PHGPx/GSH or Ebselen/GSH resulted in rapid degradation of both classes of lipid hydroperoxides, with equimolar amounts of Ch18:2-OH formed from Ch18:2-OOH. No pronounced differences were observed between PCOOH and Ch18:2-OOH in terms of substrate specificity, whereas HDLox-associated PCOOH and Ch18:2-OOH appeared to be slightly better substrates for PHGPx/GSH as compared to those in LDLox. Also, Ch18:2-OOH associated with HDLox but not LDLox were reduced by Ebselen or GSH alone. These in vitro findings indicate that the enzymatic PHGPx/GSH and the nonenzymatic Ebselen/GSH systems can efficiently reduce hydroperoxides of phospholipids and cholesterylesters associated with intact lipoproteins.

Azoles↗
PubMed1994

Radical-mediated oxidation of isolated human very-low-density lipoprotein.

Oxidative modification of human low-density lipoprotein (LDL) has received much attention because of its suggested involvement in the early events of atherogenesis. In contrast, little data exist concerning the oxidation of human very-low-density lipoprotein (VLDL), although such modification promotes foam cell formation by these lipoproteins. We therefore investigated the radical-mediated oxidation of VLDL by using controlled oxidizing conditions and sensitive and specific methods to assess lipoprotein lipid oxidation and antioxidation. We observed that the ratio of alpha-tocopherol to coenzyme Q10 in VLDL was close to that of LDL, suggesting that these lipoproteins may transport some coenzyme Q10 to extrahepatic tissues, as they do tocopherol. Most of the coenzyme Q10 associated with VLDL was present in its reduced, antioxidant active form, ubiquinol-10. The small amounts of ubiquinol-10 in VLDL provided the lipoprotein lipids with a highly efficient antioxidant protection. Also, the kinetics of radical-mediated lipid peroxidation in VLDL resembled that in LDL and therefore also probably proceeded via the recently described tocopherol-mediated peroxidation mechanism. Oxidation competition experiments using aqueous radicals and physiological concentrations and molar ratios of LDL and VLDL indicated that in contrast to the situation with high-density lipoproteins, lipid peroxidation was initiated and detected simultaneously in the former two lipoprotein particles. However, once initiated, peroxidation propagated at an approximately twofold higher rate in VLDL than LDL. Our studies suggest that radical-mediated lipid (per)oxidation proceeds via similar mechanisms in isolated LDL and VLDL. We conclude that efficient LDL antioxidants are also likely to be effective protective agents for VLDL.

Adult↗
PubMed1994

Inhibition by interferon-gamma of human mononuclear cell-mediated low density lipoprotein oxidation. Participation of tryptophan metabolism along the kynurenine pathway.

In this study we examined the potential inhibition by interferon-gamma (IFN gamma) of the early stages of low density lipoprotein (LDL) oxidation mediated by human peripheral blood mononuclear cells (PBMC) and monocyte-derived macrophages (MDM) in Ham's F-10 medium supplemented with physiological amounts of L-tryptophan (Trp). We assessed LDL oxidation by measuring the consumption of LDL's major antioxidant (i.e., alpha-tocopherol) and targets for oxidation (cholesteryllinoleate and cholesterylarachidonate), together with the accumulation of cholesterylester hydroperoxides and the increase in relative electrophoretic mobility of the lipoprotein particle. Exposure of PBMC or MDM to IFN gamma induced the degradation of extracellular Trp with concomitant accumulation of kynurenine, anthranilic and 3-hydroxyanthranilic acid (3HAA) in the culture medium. Formation of 3HAA, but neither Trp degradation nor formation of kynurenine and anthranilic acid, was inhibited by low amounts of diphenylene iodonium (DPI) in a concentration-dependent manner. In contrast to oxidative Trp metabolism, exposure of human PBMC or MDM to IFN gamma failed to induce degradation of arginine, and nitrite was not detected in the cell supernatant, indicating that nitric oxide synthase was not induced under these conditions. Incubation of LDL in Trp-supplemented F-10 medium resulted in a time-dependent oxidation of the lipoprotein that was accelerated in the presence of PBMC or MDM but inhibited strongly in the presence of both cells and IFN gamma, i.e., when Trp degradation and formation of 3HAA were induced. In contrast, when IFN gamma was added to PBMC or MDM in F-10 medium that was virtually devoid of Trp, inhibition of cell-accelerated LDL oxidation was not observed. Exogenous 3HAA added to PBMC or purified monocytes in the absence of IFN gamma also strongly and in a concentration-dependent manner inhibited LDL oxidation. Selective inhibition of IFN gamma-induced formation of 3HAA by DPI caused reversion of the inhibitory action of this cytokine on both PBMC- and MDM-mediated LDL oxidation. These results show that IFN gamma treatment of human PBMC or MDM in vitro attenuates the extent of LDL oxidation caused by these cells, and indicate that Trp degradation with formation of 3HAA is a major contributing factor to this inhibitory activity.

3-Hydroxyanthranilic Acid↗
PubMed1994

Transformation of normal thyroids into colloid goiters in rats and mice by diphenylthiohydantoin.

Some years ago, we reported that colloid goiters could be produced experimentally in mice and rats by injection of TSH over a few days in the presence of ample iodine supply. This clearly showed that colloid accumulation and intense TSH stimulation are not mutually exclusive. In the present study, large colloid goiters, sharing many morphological and biochemical characteristics with human colloid goiters, were induced in rats and mice by treatment with 5,5-diphenyl-2-thiohydantoin (DPTH). This drug increases fecal loss of thyroid hormone and inhibits conversion of T4 to T3. Thus, DPTH raises TSH and induces macrofollicular colloid-rich goiters. In contrast to this, goiters induced by combined treatment with methimazole (MMI) or sodium perchlorate and DPTH are microfollicular, although serum TSH is increased to the same level as in rats treated with DPTH alone. The degree of iodine organification obviously determines if the follicle will sprout and form daughter follicles or if it will expand its hull. Thyroglobulin content of DPTH goiters is lower than that of normal glands but considerably higher than after MMI treatment, whereas total iodine content of DPTH goiters is only slightly lower than in normal glands, but also much higher than in MMI goiters. In DPTH goiters, a high proportion of total iodine is in the particulate fraction which probably contains the periodic acid Schiff-positive bodies floating in the colloid of DPTH treated glands. Acute DPTH administration does not inhibit iodide organification, but after treatment with DPTH for 1 day, chromatography suggests some inhibition of iodine organification and hormone synthesis by DPTH, but much less than by MMI. DPTH treatment causes considerable tissue damage and repair, such as follicular cell necrosis and invasion of the colloid by macrophages and granulation tissue. Therefore, DPTH goiters might well be a useful model not only for colloid goiter formation but also for inflammatory processes in the thyroid gland.

Animals↗
PubMed1994

Lipoprotein oxidation: mechanistic aspects, methodological approaches and clinical relevance.

The oxidation of LDL is now commonly implicated as an initiator of atherosclerosis and a standard in-vitro LDL 'oxidizability' test is required. This review will discuss current problems and advances that have been made in our understanding of the molecular mechanisms of radical-mediated LDL oxidation and antioxidation, how they relate to the in-vitro assessment of the 'oxidizability' of LDL and how they may be relevant to in-vivo LDL oxidation. Tocopherol-mediated peroxidation is used as a novel model of LDL lipid oxidation to discuss why terms such as 'lag time' are features of the in-vitro oxidation conditions, rather than being inherent to LDL oxidation per se. In addition, we will also cover why it is premature, at present, to use one particular LDL oxidizability test as a standard.

Antioxidants↗
PubMed1994

[Patient management in polytrauma with injuries of the cervical spine].

Complex unstable cervical spine injuries in polytraumatized patients are stabilized ventro-dorsally in a two-stage procedure. The ventral stabilization is a day-one surgery with the goal to get primary stability for intensive care, early spinal decompression and protection against secondary damage of the spinal cord. The additional dorsal stabilization allows early functional treatment or in case of spinal cord lesions early neurorehabilitation. The combination of severe brain injury and unstable cervical spine injury is especially demanding concerning diagnostic and therapeutic procedures.

Adolescent↗
PubMed1993

Bilirubin attenuates radical-mediated damage to serum albumin.

Oxidative damage to biological macromolecules has been implicated in a number of diseases. Much interest has focused on how non-proteinaceous, low-molecular weight antioxidants prevent oxidative damage to lipids, while comparatively little is known about protein antioxidation. Here we show that bilirubin (BR), the end-product of heme catabolism, when bound to bovine serum albumin (BSA), is oxidised by hydroxyl (.OH), hydroperoxyl (HO2.), and superoxide anion (O2-.) radicals to so far mostly uncharacterised products. The initial oxidation rates of BSA-bound BR decreased in the order OH > HO2. > O2-.. BR protected its carrier protein from oxidative damage inflicted by .OH radicals. This protective action included a reduction in the .OH-mediated cleavage of BSA, conversion of Trp into kynurenine and formation of 'bityrosine-specific' fluorescence. BR also strongly inhibited .OH-mediated formation of protein carbonyls, whereas ascorbate and Trolox (a water-soluble analogue of vitamin E) were much less effective. These results support an antioxidant-protective function of BR and point towards significant differences in the efficacies of various antioxidants in the prevention of oxidative damage to lipids and proteins.

Antioxidants↗
PubMed1993

Greater selective uptake by Hep G2 cells of high-density lipoprotein cholesteryl ester hydroperoxides than of unoxidized cholesteryl esters.

We have observed recently that high-density lipoproteins (HDL) are the predominant carriers of cholesteryl ester hydroperoxides (CEOOH), the major class of lipid hydroperoxides detectable at nanomolar concentrations in the plasma of healthy fasting humans. The present study investigates the effect of such very low levels of CEOOH in apolipoprotein E-free HDL3 on lipoprotein particle metabolism and 'selective uptake' of its CE by human Hep G2 cells. Minimal oxidation with aqueous peroxyl radicals had a negligible effect on the binding, internalization and degradation of 125I-labelled HDL3. In contrast, with an increasing degree of radical-mediated oxidation of labelled HDL3, [3H]cholesteryl linoleate ([3H]Ch18:2) was taken up at an increasingly greater rate than were 125I-apoproteins. When [3H]cholesteryl linoleate hydroperoxide ([3H]Ch18:2-OOH was incorporated into unoxidized HDL3 by exchange from donor liposomes, it was taken up at a more than 8-fold higher rate than was incorporated [3H]Ch18:2. The same degree of preferential uptake of oxidized CE was observed when HDL3 was used that was doubly labelled with [3H]Ch18:2-OOH and cholesteryl [14C]oleate ([14C]Ch18:1). In both situations, uptake of [3H]Ch18:2-OOH exceeded that of 125I-apolipoprotein A-I some 40-fold. This increased selective uptake of [3H]Ch18:2-OOH from very mildly oxidized HDL3 was accompanied by a parallel increase in the intracellular levels of labelled free cholesterol. In contrast, lipid hydroperoxides were not detectable within Hep G2 cells, suggesting efficient detoxification of CEOOH by these cells. Neither the increased selective uptake of Ch18:2-OOHs nor the levels of intracellular free cholesterol were influenced by the presence of 50 microM chloroquine, suggesting extralysosomal hydrolysis of oxidized CEs. These results show that the selective uptake of HDL CEOOH by Hep G2 cells is more efficient than that of unoxidized CE, and support a protective role for rapid selective uptake in the removal of circulating HDL CEOOH.

Biological Transport↗
PubMed1993

The oxidation of blood plasma and low density lipoprotein components by chemically generated singlet oxygen.

Human blood plasma and freshly isolated LDL were exposed to singlet oxygen (1O2) by thermal decomposition of synthetic endoperoxides. Exposure of blood plasma to 20 mM water-soluble 1O2 generator resulted in the depletion of ascorbate (100%), urate (75%), ubiquinol-10 (65%), protein thiols (50%), and bilirubin (25%), whereas under these conditions the levels of alpha-tocopherol, beta-carotene, and lycopene remained unchanged. The following rates of depletion were obtained by kinetic analysis (moles depleted per 100 mol of 1O2 consumed): protein thiols (5), urate (5), ascorbate (4), bilirubin (1), and ubiquinol-10 (0.008). In contrast, the rates of depletion using the lipid-soluble 1O2 generator were faster for bilirubin (13-fold), protein thiols (9-fold), ubiquinol-10 (8-fold), and ascorbate (5-fold), and slower for urate (2-fold). The formation of lipid hydroperoxides, including mostly cholesteryl linoleate hydroperoxide, was observed in 1O2-treated plasma (0.007-0.009 mol/100 mol 1O2) and LDL solutions (0.086 mol/100 mol 1O2). Based on competition kinetics, we estimate that 98% of 1O2 generated in the aqueous phase of plasma is quenched by components in this phase, mostly by plasma protein (63%; 6% by protein thiols), urate (9%; 5% by chemical quenching), and bilirubin (5%; 1% by chemical quenching). Ascorbate and ubiquinol-10 do not contribute to 1O2 quenching in plasma, and their oxidation is probably mediated secondary species. The remaining 1O2 generated in plasma (2%) diffuses into lipoprotein leading to the formation of lipid hydroperoxides with an efficiency of about 100-fold greater than that compared to aqueous generated 1O2. The principal 1O2 quenchers in LDL include apoB (42%), lycopene and beta-carotene (40%), and alpha-tocopherol (17%). The importance of carotenoids in the quenching of 1O2 in lipoprotein suggest that the beneficial effects of these compounds in health may in part be due to the elimination of this species in biology and medicine.

Adult↗