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Biomedical subjects

R T Dean

Publications and source records attributed to R T Dean.

At least 19 recordsLinked to original sources
PubMed1995

Biological fate of amino acid, peptide and protein hydroperoxides.

In the course of searching for a suitable marker for studying protein oxidation, we have successfully elucidated the structures of three valine hydroperoxides, i.e. beta-hydroperoxyvaline, (2S,3S)-gamma-hydroperoxyvaline and (2S,3R)-gamma-hydroperoxyvaline, which are novel products of protein oxidation. The corresponding valine hydroxides were obtained by sodium borohydride reduction [Fu, Hick, Sheil and Dean (1995) Free Rad. Biol. Med. 19, 281-292]. We hypothesized that valine hydroxides might be the major biological degradation products of valine hydroperoxides and, as such, could be useful markers for the study of protein oxidation in vivo. The aim of this study was to investigate the fate of valine hydroperoxide in selected biological systems by the use of chemiluminescence detection of hydroperoxides and HPLC analysis of O-phthaldialdehyde derivatives of amino acid residues. The degradation of hydroperoxides present on gamma-radiolysed solutions of valine, Pro-Val-Gly, or BSA occurred in the presence of: (1) transition metals (Fe2+, Fe3+, or Cu2+), (2) the detoxifying enzyme GSH peroxidase, (3) human plasma, and (4) J774 mouse monocyte macrophage cells. The major degradation product of valine hydroperoxide recovered in each case was found to be a valine hydroxide. These results suggest that valine hydroxide (derived from the hydroperoxide) may well be a useful in vivo marker for studying protein damage under oxidative stress.

Amino Acid Sequence↗
PubMed1995

Macrophages require both iron and copper to oxidize low-density lipoprotein in Hanks' balanced salt solution.

The oxidation of low-density lipoprotein (LDL) may be important in the pathogenesis of atherosclerosis. However, the interactions between cells and metals in promoting LDL oxidation are inadequately understood. A sensitive high-performance liquid chromatography analysis of cholesterol, cholesteryl esters, and their oxidation products was used to identify and accurately measure LDL oxidation achieved in thiol-free Hanks' balanced salt solution (HBSS) at pH 7.4. Mouse peritoneal macrophages inhibited LDL oxidation when incubated in HBSS containing either 10 microM iron or 1 microM copper, but were markedly prooxidant in the presence of both metals. The prooxidant effect of macrophages in the presence of both iron and copper did not require the provision of added disulfides or thiols. Both Fe2+ and macrophages were demonstrated to independently reduce Cu2+ to Cu1+ in HBSS, indicating that the direct reduction of copper by cells or iron may underlie the observed promotion of LDL oxidation by macrophages in this system. We conclude that macrophages can either promote or inhibit metal-mediated LDL oxidation and that externally supplied thiols are not essential to the promotion of LDL oxidation by cells. The presence of both iron and copper may be particularly important for macrophages to promote LDL oxidation in vivo.

Animals↗
PubMed1995

Protein hydroperoxides can give rise to reactive free radicals.

Proteins damaged by free-radical-generating systems in the presence of oxygen yield relatively long-lived protein hydroperoxides. These hydroperoxides have been shown by e.p.r. spectroscopy to be readily degraded to reactive free radicals on reaction with iron(II) complexes. Comparison of the observed spectra with those obtained with free amino acid hydroperoxides had allowed identification of some of the protein-derived radical species (including a number of carbon-centred radicals, alkoxyl radicals and a species believed to be the CO2 radical anion) and the elucidation of novel fragmentation and rearrangement processes involving amino acid side chains. In particular, degradation of hydroperoxide functions on the side chain of glutamic acid is shown to result in decarboxylation at the side-chain carboxy group via the formation of the CO2 radical anion; the generation of an identical radical from hydroperoxide groups on proteins suggests that a similar process occurs with these molecules. In a number of cases these fragmentation and rearrangement reactions give rise to further reactive free radicals (R., O2-./HO2., CO2-.) which may act as chain-carrying species in protein oxidations. These studies suggest that protein hydroperoxides are capable of initiating further radical chain reactions both intra- and inter-molecularly, and provide information on some of the fundamental mechanisms of protein alteration and side-chain fragmentation.

Amino Acids↗
PubMed1995

Structural identification of valine hydroperoxides and hydroxides on radical-damaged amino acid, peptide, and protein molecules.

We have previously demonstrated the formation of two reactive moieties on proteins during free radical attack: hydroperoxides, and 3,4-dihydroxyphenylalanine (DOPA). Here we have undertaken the structural elucidation of the hydroperoxides of valine, the amino acid which is most susceptible to peroxidation. Exposure of L-valine to free radicals generated by radiolysis in an oxygen-saturated system produced three valine hydroperoxides. Upon treatment with sodium borohydride these were reduced to their corresponding hydroxides, which can be separated and purified by high performance liquid chromatography (HPLC). Based on spectroscopic data from high resolution chemical ionization (CI) mass spectrometry (MS), electrospray (ES) MS, electron impact (EI) MS, proton (1H) nuclear magnetic resonance (NMR) and carbon-13 (13C) NMR studies, the three valine hydroxides have been identified as beta-hydroxyvaline [(2S)-2-amino-3-hydroxy-3-methyl-butanoic acid], (2S,3S)-gamma-hydroxyvaline [(2S,3S)-2-amino-3-hydroxymethyl-butanoic acid], and (2S,3R)-gamma-hydroxyvaline [(2S,3R)-2-amino-3-hydroxymethyl-butanoic acid]. HPLC analysis of O-phthaldialdehyde (OPA) derivatives of the hydroxyvalines provides a sensitive and accurate method for quantitative measurement. This method enabled hydroxyvalines to be detected in the hydrolysates of a tripeptide (glutamyl-valinyl-phenylalanine) and a protein (bovine serum albumin) that had been gamma-radiolysed and treated with sodium borohydride. Hydroxyvaline may be useful as a marker in studying protein oxidation in some biological systems under oxidative stress.

Amino Acids↗
PubMed1995

Human atherosclerotic plaque contains both oxidized lipids and relatively large amounts of alpha-tocopherol and ascorbate.

We assessed the antioxidant status and contents of unoxidized and oxidized lipids in freshly obtained, homogenized samples of both normal human iliac arteries and carotid and femoral atherosclerotic plaque. Optimal sample preparation involved homogenization of human atherosclerotic plaque for 5 minutes, which resulted in recovery of most of the unoxidized and oxidized lipids without substantial destruction of endogenous vitamins C and E and 87% and 43% recoveries of added standards of alpha-tocotrienol and isoascorbate, respectively. The total protein, lipid, and antioxidant levels obtained from human plaque varied among donors, although the reproducibility of replicates from a single sample was within 3%, except for ubiquinone-10 and ascorbate, which varied by 20% and 25%, respectively. Plaque samples contained significantly more ascorbate and urate than control arteries, with no discernible difference in the vitamin C redox status between plaque and control materials. The concentrations of alpha-tocopherol and ubiquinone-10 were comparable in plaque samples and control arteries. However, approximately 9 mol percent of plaque alpha-tocopherol was present as alpha-tocopherylquinone, whereas this oxidation product of vitamin E was not detectable in control arteries. Coenzyme Q10 in plaque and control arteries was only detected in the oxidized form ubiquinone-10, although coenzyme Q10 oxidation may have occurred during processing. The most abundant of all studied lipids in plaque samples was free cholesterol, followed by cholesteryl oleate and cholesteryl linoleate (Ch18:2). Approximately 30% of plaque Ch18:2 was oxidized, with 17%, 12%, and 1% present as fatty acyl hydroxides, ketones, and hydroperoxides, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1995

Apolipoprotein A-I-mediated efflux of sterols from oxidized LDL-loaded macrophages.

Although oxidized low-density lipoprotein (Ox-LDL) can accumulate in macrophages in vitro, generating cholesterol-loaded cells, little attention has been paid to the capacity of such macrophages loaded with OxLDL to export cholesterol and oxidized sterol moieties. In vitro lipid-loaded cells were generated by incubating primary cultures of mouse peritoneal macrophages with acetylated LDL (AcLDL) or OxLDL for 24 hours. The cellular content of native cholesterol, individual cholesteryl esters, and 7-ketocholesterol was determined by high-performance liquid chromatography. These cells were then incubated with medium containing apolipoprotein (apo) A-I and albumin or albumin alone for up to 24 hours; cholesterol and oxidized sterol efflux were measured both in terms of intracellular depletion and extracellular accumulation. Macrophages loaded with AcLDL accumulated cholesterol and large quantities of cholesteryl esters, whereas OxLDL-loaded cells accumulated cholesterol, a number of oxidized compounds (predominantly 7-ketocholesterol), and a relatively small quantity of cholesteryl esters. AcLDL-derived cells released approximately 50% of their total cholesterol (unesterified and esterified) to apo A-I-containing medium over 24 hours in the form of unesterified cholesterol, whereas OxLDL-derived cells released approximately 30% of their total cholesterol and 7% of their total content of 7-ketocholesterol over the same period. There was minimal efflux of any sterol in the absence of apo A-I. The proportions of cholesterol and 7-ketocholesterol released by either AcLDL- or OxLDL-loaded cells were not reduced by inhibiting cellular acyl-CoA:cholesterol acyl transferase using Sandoz 58-035, despite substantial alterations in the proportions of both free cholesterol and (in OxLDL-loaded cells) free 7-ketocholesterol in these cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

EDTA differentially and incompletely inhibits components of prolonged cell-mediated oxidation of low-density lipoprotein.

The extent to which cells can oxidize LDL may be underestimated because of the use of standard and arbitrary 24 hour in vitro incubations of cells with LDL. Such incubations have resulted in inconsistent results regarding the ability of cell-mediated LDL oxidation to generate relatively advanced oxidation products such as 7-ketocholesterol (7-KC). We studied prolonged oxidation of low density lipoprotein (LDL) by mouse peritoneal macrophages using HPLC measurement of cholesterol, cholesteryl esters and their oxidation products 7-KC and cholesteryl linoleate hydroperoxide (CL-OOH). Cell-mediated oxidation in Ham's F10 consistently followed the successive stages previously described during 24 hour-10 microM copper-mediated LDL oxidation, always generating 7-KC if allowed to proceed for sufficient time. The degree of inhibition of LDL oxidation achieved by metal chelators EDTA and DTPA at more advanced stages of cell-mediated LDL oxidation was not predictable from the published effects of such chelators upon early stages of metal-mediated and cell-mediated LDL oxidation. EDTA and DTPA only incompletely prevented the consumption of cholesteryl esters and the loss of performed CL-OOH when added after cell-mediated LDL oxidation was established, while effectively concurrently inhibiting the generation of 7-KC. These data indicate that progressive cell-mediated peroxidation of LDL cholesteryl esters and decomposition of CL-OOH may be less dependent upon a continuing supply of redox active metals than is the generation of 7-KC. In addition, they confirm the plausibility of prolonged cell-mediated oxidation of LDL as a source of oxysterols found in human atherosclerotic plaque, and imply that active redox cycling of metals is particularly important for their generation in vivo.

Animals↗
PubMed1995

Augmentation of vascular endothelial barrier function by heparin and low molecular weight heparin.

Glycosaminoglycans (GAGs) are an important component of endothelial barrier function. Early passage human umbilical vein endothelial cells were grown to confluence on transparent micropore filters and barrier function assessed as transendothelial electrical resistance (TEER) and permeability to albumin and sucrose. Unfractionated heparin and the LMW heparin Clexane decreased endothelial permeability to both sucrose and albumin and increased TEER. Chondroitin 6-sulphate also augmented barrier function, but other GAGs had no effect. Interleukin-1 increased permeability to albumin and sucrose and decreased TEER. Although heparin attenuated the effect of IL-1 on TEER and sucrose permeability, it could not restore the barrier to albumin transfer. Denuded endothelial matrix presented a negligible barrier, which was not enhanced by heparin. When sulphation of endogenous GAGs was inhibited by chlorate, barrier function was compromised and was not restored by exogenous heparin. Thus heparin enhances the barrier function of resting endothelium, but cannot completely overcome the increased permeability resulting from exposure to IL-1 or substitute for endogenous GAGs.

Albumins↗
PubMed1995

Detecting deep venous thrombosis with technetium-99m-labeled synthetic peptide P280.

UNLABELLED: Scintigraphy, using small, thrombus-avid, synthetic peptides labeled with gamma-emitting nuclides is an innovative approach to the noninvasive detection of acute deep venous thrombosis (DVT). The goal of this study was to evaluate clinically 99mTc-P280 for imaging DVT. The peptide P280 is a 26 amino acid dimer that binds with high affinity to the GPIIb/IIIa receptor expressed on activated platelets and can be labeled with 99mTc. METHODS: Scintigraphy with 99mTc-P280 (10-22 mCi) was performed in nine patients with clinical suspicion and diagnostic evidence of DVT. Planar and tomographic images of the legs, abdomen/pelvis, chest and head were obtained immediately, 1, 2, 4 and 24 hr after injection. RESULTS: No adverse effects were noted after 99mTc-P280 administration in any patient. Positive visualization of thrombi occurred in eight of nine cases with confirmed DVT within 1 hr of tracer injection. The majority of the patients had recent onset of DVT symptoms (less than 3 wk), while the only negative case was diagnosed 42 days earlier and was likely related to an accident 7 mo earlier. Thrombi-to-background ratios were essentially constant over the study. Technetium-99m-P280 accumulation was also discernible in two patients with pulmonary embolism, while in a third patient the radiotracer concentrated in a cerebellar hemangioblastoma. CONCLUSION: These human studies indicate that 99mTc-P280 is a potentially safe and sensitive procedure for diagnosing DVT and pulmonary embolism. It also may have substantial utility in monitoring active venous thrombosis.

Adult↗
PubMed1994

Human macrophage-mediated oxidation of low-density lipoprotein is delayed and independent of superoxide production.

There is growing evidence that oxidatively modified low-density lipoprotein (LDL) accumulates in the atherosclerotic intima of arteries. Cells present in the intima (including the monocyte/macrophage) are capable of oxidizing LDL in vitro, but the mechanisms by which this occurs are unknown. Several reports have claimed a crucial role for superoxide as a cell-derived radical species capable of enhancing the rate of LDL oxidation. We have used a sensitive h.p.l.c. system with chemiluminescence detection to measure LDL cholesteryl ester hydroperoxides at early stages of LDL oxidation. During the initial stages of LDL oxidation, there is at least a 2 h delay before human monocyte-derived macrophages enhance this process. Stimulation of these cells to produce large fluxes of superoxide does not increase the rate of LDL oxidation or decrease the delay of its onset. Prior exposure of LDL to a high flux of superoxide does not increase its susceptibility to oxidation by human monocyte-derived macrophages. We also show that the thiobarbituric acid-reactive substances (TBARS) assay does not always correlate with more direct methods of assessing LDL oxidation and confirm recent reports that superoxide dismutase only partially inhibits cell-mediated LDL oxidation. We conclude that superoxide does not play a major role in human monocyte-derived macrophage-mediated LDL oxidation under the conditions that we describe.

Cholesterol Esters↗
PubMed1994

Apolipoprotein B of oxidized LDL accumulates in the lysosomes of macrophages.

We have studied the intracellular fate of the apolipoprotein B of copper-oxidized LDL in cultured J774 macrophages, using subcellular fractionation and immunofluorescence techniques. The oxidized apolipoprotein B, using cell fractionation, was located primarily in secondary lysosomes (identified using the lysosomal marker-enzyme aryl sulfatase). Light microscopy using antibodies to the mannose-6-phosphate receptor, the lysosomal membrane protein lgp 120, and oxidized LDL (biotinylated) confirmed that apo B of oxidized LDL did accumulate in secondary lysosomes rather than in endosomes. We conclude from these results that the oxidized apolipoprotein B of LDL reaches the secondary lysosomes, but is not efficiently degraded, leading to intracellular accumulation within this compartment. If this occurs in vivo it may influence the physiology of the macrophage and their subsequent roles in forming foam cells and the development of the fatty streaks of early atherosclerosis.

Acylation↗
PubMed1994

Enhanced LDL oxidation by murine macrophage foam cells and their failure to secrete nitric oxide.

Foam cells were produced in vitro by incubation of mouse peritoneal macrophages with acetylated or copper-oxidized LDL. Nitric oxide synthesis was stimulated by exposure of the cells to IFN gamma and LPS. Nitric oxide production, detected by measurement of nitrite in the culture medium, was unchanged in Ac-LDL loaded cells as compared with non-loaded cells. However, Ox-LDL foam cells produced 68-99% less nitrite than non-loaded cells. Failure to detect nitric oxide synthase (NOS) products from macrophages previously loaded with Ox-LDL appeared to result from lack of NOS activity, as little active enzyme could be recovered from Ox-LDL loaded cells. However, addition of Ox-LDL to an active cell-free NOS preparation had no direct effect on enzymic activity. When native LDL was subsequently incubated with these various IFN gamma/LPS stimulated cells, cells pre-loaded with Ox-LDL promoted, on average, a 2-fold greater increase in oxidative modification of the LDL added than either non-loaded or Ac-LDL loaded cells. That is, there was an inverse correlation between NOS activity and the ability of the cells to promote LDL oxidation. Unstimulated Ox-LDL loaded foam cells also oxidized LDL better than unstimulated non-loaded or Ac-LDL loaded foam cells, and the extent of oxidative modification was generally greater than seen with the equivalent IFN gamma/LPS stimulated cells. This suggests that Ox-LDL loading also affects some additional factor(s) responsible for cell-mediated LDL oxidation.

Acetylation↗
PubMed1994

The action of nine chelators on iron-dependent radical damage.

Nine iron chelators were tested in five systems for their effects on radical-generation and conversion at chelator: iron molar ratios from 0.1 to 10. Stimulatory actions might distinguish toxic from safer chelators. Radical-generating reactions which represent different aspects of iron (ferrous and ferric) availability were studied: a) the reaction with hydrogen peroxide to hydroxylate benzoate; b) the oxidation of ascorbate; c) the reaction with hydrogen peroxide to fragment proteins; d) the reaction with hydrogen peroxide to permit amplified chemiluminescence; and e) the induction of peroxidation of mitochondrial membrane lipids. The compounds used were HBED, CP130, Desferal, EDTA, pyridine-hydrazone (CGP 43'902B), Ferrozine, CP94 (CGP 46'700), L1 (CGP 37,391) and rhodotorulic acid (CGP 45 274). Only the hexadentate compounds HBED, CP130 and Desferal were uniformly inhibitory ("protective"). The protective compounds were also apparently more stable during radical fluxes than the other chelators.

Animals↗
PubMed1993

A method for defining the stages of low-density lipoprotein oxidation by the separation of cholesterol- and cholesteryl ester-oxidation products using HPLC.

A new high-performance liquid chromatographic system for the identification of some of the lipid oxidation products of low-density lipoprotein (LDL) oxidized by copper is described. Using a reversed-phase C-18 column and an isocratic solvent system of acetonitrile/isopropanol/water (44/54/2, v/v/v), a number of oxidized lipid moieties were resolved and detected simply by their 234-nm absorbance. The nature of several of these compounds was determined by chromatographic criteria, chemiluminescence, and mass spectrometry. The production of compounds within 4 h oxidation corresponded to the production of lipid hydroperoxides, the quantitatively most important of which is cholesteryl linoleate hydroperoxide, and to the rapid decrease in the cholesteryl ester content of LDL detected at 210 nm. More prolonged copper oxidation (up to 48 h) of LDL resulted in decreased quantities of lipid hydroperoxide moieties and increased amounts of a number of other, nonhydroperoxide, compounds. 7-Ketocholesterol and cholesterol linoleate hydroxide are two of the major products of prolonged oxidation. The detection of oxidation products correlates with the modification of LDL protein, permits a four-stage definition of metal-mediated LDL oxidation, and enables the calculation of a quantitative index of oxidation (lipoprotein oxidation index). This method will be generally applicable to cell- and copper-mediated oxidation, and will enable standardization of, and direct comparison between, different preparations of oxidized LDL.

Apolipoproteins B↗
PubMed1993

Protein-bound 3,4-dihydroxyphenylalanine is a major reductant formed during hydroxyl radical damage to proteins.

Proteins and aromatic amino acids previously exposed to hydroxyl radicals reduced cytochrome c, free iron, and copper ions. A major product of hydroxyl radical addition to tyrosine is 3,4-dihydroxyphenylalanine (DOPA), which has these reducing properties. The reduction of nitro blue tetrazolium by radical-damaged protein was consistent with the generation of quinones in the protein. By acid hydrolysis followed by high-performance C18 reversed-phase liquid chromatography we have shown that hydroxyl radical-damaged proteins contain significant amounts of protein-bound DOPA (PB-DOPA). The authenticity of the DOPA measured was confirmed by gas chromatography-mass spectrometry. PB-DOPA was also generated enzymatically using mushroom tyrosinase, which catalyzes the hydroxylation of tyrosine residues. By comparing the levels of DOPA in radical-damaged or enzyme-treated protein with that of cytochrome c reduction, we show that PB-DOPA is a major source of the observed reducing activity. PB-DOPA may have a role in the replenishment of reduced transition metal ions involved in free radical generating systems in vivo.

Catechols↗
PubMed1993

Free radical and enzymatic mechanisms for the generation of protein bound reducing moieties.

We have previously shown that exposure of many proteins, and free aromatic amino acids (particularly tyrosine) to free radical fluxes generates a stable activity capable of reducing protein bound and free transition metal ions. Here we define the capacity of several radical generating systems (gamma irradiation of water, UV irradiation, metal-dependent sugar autoxidation and Haber-Weiss systems) to produce protein-bound reducing moieties (PBRedM), and also reducing derivatives of tyrosine. Under the defined conditions of the gamma radiolysis system, reductive activity was generated under both oxic and anoxic irradiations and specific gassing regimes as well as the inclusion of specific radical scavengers established that hydroxyl radicals were responsible. When BSA was irradiated anoxically in the presence of formate a reductive activity related to the exposure of protein thiol groups was generated; all other reductive activities we detected were not thiol-related. Incubations of tyrosinase with BSA or insulin also generated reductive activity. All the conditions we have studied can convert tyrosine into DOPA and we suspect that protein-bound DOPA is the main reductive activity generated on proteins.

Amidines↗
PubMed1993

External imaging of atherosclerosis in rabbits using an 123I-labeled synthetic peptide fragment.

The oligopeptide fragment of apolipoprotein B, SP-4, has demonstrated pronounced uptake in the healing edges of balloon-injured rabbit aortic endothelium. To assess 123I-labeled SP-4 for identification of atherosclerotic plaques by gamma camera imaging, 14 Watanabe heritable hyperlipidemic (WHHL) and 5 normal rabbits were imaged 5 minutes and 12 and 24 hours after intravenous injection of 123I-SP-4. In addition, two WHHL and two normal rabbits were injected with 125I-SP-4 for autoradiography. Twelve of the 14 WHHL, but none of the normal, rabbits had visually apparent focal radioiodine accumulation in the region of the aorta. Focus-to-lung and focus-to-heart count ratios were 2.4 +/- 1.3 and 1.0 +/- 0.4, respectively. Five of the visually positive WHHL rabbits were reimaged 4 and 8 weeks later with 123I-NaI and 123I-SP-2 (an apo E peptide), respectively, as negative controls. Perceptible, but faint, aortic localization of 123I-NaI and of 123I-SP-2 was seen in only one animal each. The distributions of atherosclerotic lesions on photographs of the opened WHHL aortas and of film blackening on 125I-SP-4 autoradiograms were identical. In contrast, the two normal rabbit aortas did not exhibit plaques on photographs or film blackening on autoradiograms. Thus, in an animal model closely simulating human atherosclerotic disease, SP-4 localizes specifically in aortic atherosclerotic lesions.

Amino Acid Sequence↗