Biomedical subjects

J V Hunt

Publications and source records attributed to J V Hunt.

At least 37 records · Page 2Linked to original sources

PubMed1993

Oxidative alterations in the experimental glycation model of diabetes mellitus are due to protein-glucose adduct oxidation. Some fundamental differences in proposed mechanisms of glucose oxidation and oxidant production.

Modification of human serum albumin (HSA) with formaldehyde resulted in a loss of 75% of available lysine residues, but there was no change in histidine content or susceptibility to free-radical-mediated fragmentation. The modified HSA appeared resistant to glycation and glucose-mediated fragmentation. Native HSA inhibited oxidant production by free glucose, as assessed by the hydroxylation of benzoic acid, but modified HSA had little effect. Thus the oxidation of free glucose appeared to be inhibited by glycatable protein, but not by unglycatable protein. Also, a close proximity of glucose to protein (decreased in the case of modified HSA) would seem to be a prerequisite for glucose-mediated protein fragmentation. This latter observation, in particular, led us to examine the role of oxidation of glucose attached to HSA in the production of reactive oxidants and subsequent molecular damage. Glycated HSA, washed free of unbound glucose, became fragmented and generated oxidants capable of hydroxylating benzoic acid and oxidizing cholesteryl linoleate-HSA complexes. Significant levels of benzoate hydroxylation and HSA fragmentation occurred with HSA (10 mg/ml) containing 3.3 mol of glucose bound/mol of HSA. This is equivalent to incubation of 10 mg/ml native HSA with 0.66 mM glucose, conditions which lead to little fragmentation or oxidant formation. The oxidative activity of glycated HSA was dependent on transition-metal concentration. The level of protein-bound glucose appeared to decrease during the oxidant production and protein fragmentation. Thus glucose can oxidize and generate reactive oxidants, whether in solution or attached to protein. We discuss which is the more likely mechanism of glucose oxidation under the near-physiological conditions used to study the effects of protein exposure to glucose in vitro.

Benzoates↗

PubMed1993

Flow cytometric measurement of ceroid accumulation in macrophages.

Flow cytometry has been examined as a method for quantitative measurement of the accumulation in macrophages of ceroid, an autofluorescent polymer composed of oxidised protein and lipid. Murine peritoneal macrophages were cultured in the presence of cholesteryl linoleate- or arachidonate-bovine serum albumin (CL/BSA or CA/BSA) complexes. Ceroid accumulation was greater from CA/BSA than from CL/BSA and was dependent upon both time and cell plating density. Inclusion of vitamin E with the complexes diminished the accumulation of ceroid fluorescence after exposure to either CL/BSA or CA/BSA. Controls included exposure of macrophages to BSA, alone and with vitamin E, both of which led to some fluorescence at a similar wavelength to that used to monitor ceroid accumulation (Ex: 351.1-363.8 nm/Em: 490 nm and upwards). Ceroid accumulation can be monitored semi-quantitatively by staining techniques. However, such methods are relatively crude and give little information about the amount of ceroid within cells. Flow cytometry, on the other hand, can give a quantitative assessment of cellular ceroid accumulation, provided experiments are conducted with appropriate controls. The findings are discussed in the context of human atherosclerosis and of future investigation of cell-mediated lipid oxidation and its potential antagonists.

Animals↗

PubMed1992

Ascorbic acid oxidation: a potential cause of the elevated severity of atherosclerosis in diabetes mellitus?

The exposure of mouse peritoneal macrophages to cholesterol linoleate-containing artificial lipoproteins can lead to intracellular ceroid accumulation. This can be used as a model to study the role of oxidation in macrophage uptake of lipoproteins containing unsaturated fatty acids, considered by many as a primary event in atherosclerotic plaque formation. Our studies show that ascorbic acid can both inhibit and promote the formation of ceroid in such a model system. The transition metal copper (Cu(II)) further elevates ceroid accumulation and EDTA, a metal chelator, inhibits it. When trace levels of transition metals are present, low concentrations of ascorbic acid can elevate ceroid formation. This pro- and antioxidant characteristic of ascorbic acid was confirmed by monitoring the generation of oxidants by various concentrations of ascorbic acid, assessed by benzoic acid hydroxylation or the fragmentation of BSA. We discuss these observations in the context of an apparent increase in ascorbic acid oxidation and elevated severity of atherosclerosis in diabetes mellitus.

Animals↗

PubMed1992

Ferrous ion oxidation in the presence of xylenol orange for detection of lipid hydroperoxide in low density lipoprotein.

A simple and sensitive method for the direct measurement of lipid peroxides in lipoprotein and liposomes is described. The method is based on the principle of the rapid peroxide-mediated oxidation of Fe2+ to Fe3+ under acidic conditions. The latter, in the presence of xylenol orange, forms a Fe(3+)-xylenol orange complex which can be measured spectrophotometrically at 560 nm. Calibration with standard peroxides, such as hydrogen peroxide, linoleic hydroperoxide, t-butyl hydroperoxide, and cumene hydroperoxide gives a mean apparent extinction coefficient of 4.52 x 10(4) M-1 cm-1 consistent with a chain length of approximately 3 for ferrous ion oxidation by hydroperoxides. Endoperoxides are less reactive or unreactive in the assay. The assay has been validated in the study of lipid peroxidation of low density lipoprotein and phosphatidyl choline liposomes. By pretreatment with enzymes known to metabolize peroxides, we have shown that the assay measures lipid hydroperoxides specifically. Other methods for measuring peroxidation, such as the assessment of conjugated diene, thiobarbituric acid reactive substances and an iodometric assay have been compared with the ferrous oxidation-xylenol orange assay.

Ferrous Compounds↗

PubMed1992

Formation of hydrogen peroxide by lens proteins: protein-derived hydrogen peroxide as a potential mechanism of oxidative insult to the lens.

The exposure of dialyzed preparations of lens crystallins to copper (II) ions causes a decrease in protein surface thiol and the production of hydrogen peroxide (H2O2). H2O2 production by gamma and beta crystallin subfractions (which contain the greatest level of thiol) is the predominant source of this H2O2. Protein surface thiols are probable sources of H2O2 formation since N-ethyl maleimide treatment of lens proteins and zinc ions inhibit H2O2 production. These data are consistent with a hypothesis that transition metal-catalyzed oxidation of protein contributes to cataractogenic lens protein oxidations.

Animals↗

PubMed1991

Spirohydantoin inhibitors of aldose reductase inhibit iron- and copper-catalysed ascorbate oxidation in vitro.

Transition metal-catalysed oxidations have been implicated in the complications of diabetes. We report here that some experimental inhibitors of the enzyme aldose reductase (implicated in diabetes mellitus via its ability to catalyse glucose reduction to sorbitol) are also potent inhibitors of transition metal-catalysed ascorbate oxidation. The inhibition appears to be dependent upon the presence of a spirohydantoin group. It is conceivable that the copper- and iron-binding capacity of these compounds may contribute to some of their observed biological effects and may provide a starting point for a new generation of experimental drugs for the treatment of diabetes mellitus.

Aldehyde Reductase↗

PubMed1991

Protein glycation and oxidative stress in diabetes mellitus and ageing.

Hyperglycemia is increasingly regarded as the cause of the diabetic complications, in particular via the ability of glucose to glycate proteins and generate Maillard browning products which cross-link proteins and render them brown and fluorescent in vitro. Similar changes occur in vivo to long-lived proteins in diabetes mellitus as well as in ageing. The evidence supporting this route of glucose toxicity is discussed in the context of the ability of glucose to oxidize in vitro (catalyzed by trace amounts of transition metal) generating hydrogen peroxide, highly reactive oxidants, and protein-reactive ketoaldehyde compounds. It is suggested that protein browning in vivo may not result from the reactions of glucose with protein but from the transition metal-catalyzed reactions of other small autoxidisable substrates, such as ascorbate, with protein. Overall, studies of glycation and protein browning suggest a critical role for oxidative processes perhaps involving decompartmentalized transition metals and a variety of low molecular weight reducing agents in diabetes mellitus and ageing.

Aging↗

PubMed1991

Free radical damage to proteins: the influence of the relative localization of radical generation, antioxidants, and target proteins.

Free radicals were generated at known rates in the aqueous phase (by means of 2,2'-azobis (2-amidinopropane) dihydrochloride [AAPH]) and in a membranous (lipid) phase (by means of 2,2'-azobis (2,4-dimethylvaleronitrile [AMVN]). A soluble protein (bovine serum albumin: BSA), and membranes of lysed mitochondria containing radioactively labeled monoamine oxidase (MAO), were exposed to the resultant radical fluxes. Antioxidants were added to the system, either in the aqueous phase (Trolox) or in a liposomal membrane phase (alpha-tocopherol). Protein damage was assessed as tryptophan oxidation and conformational changes in tryptophan fluorescence of the soluble protein, BSA, and as fragmentation of both BSA and monoamine oxidase. Radicals generated in the aqueous phase, by AAPH, were effective in damaging BSA and MAO. Radicals generated within the liposome membrane phase (by AMVN) were less effective against BSA than those deriving from AAPH. Liposomal AMVN radicals could damage MAO, present in a separate membranous phase, though again, less effectively than could AAPH-derived radicals. BSA could be protected by Trolox, the aqueous soluble antioxidant, but hardly by tocopherol itself. Damage to MAO was limited by Trolox, and also by the hydrophobic antioxidant, tocopherol. Damaging reactions due to radicals generated in a membrane phase were significantly accelerated when the membrane was peroxidizable (soybean phosphatidylcholine) rather than nonperoxidizable (saturated dimyristoyl phosphatidylcholine). Thus lipid radicals also played some role in protein damage in these systems. BSA was attacked similarly in the presence or absence of liposomes by AAPH. Correspondingly, BSA could inhibit the peroxidation of liposomes induced by AAPH and less efficiently that induced by AMVN.(ABSTRACT TRUNCATED AT 250 WORDS)

Amidines↗

PubMed1991

The role of histidine residues in the nonenzymic covalent attachment of glucose and ascorbic acid to protein.

Copper ions have been suggested to play a role in the non-covalent glycosylation (glycation) of proteins via transition metal-catalysed oxidations. We have further investigated "autoxidative glycosylation" by comparison of the behaviour of dog and bovine serum albumin with respect to the oxidative reactions of glucose and ascorbate. The proteins possess similar numbers of total amino residues available for glucose attachment but dog serum albumin contains fewer histidine groups and also lacks a high affinity copper-binding site. We find that the higher copper-binding capacity of bovine serum albumin is reflected in a lower rate of ascorbate oxidation as well as less protein oxidative damage than is the case for dog serum albumin. We also observe that modification of bovine serum albumin histidine groups by diethylpyrocarbonate enhances ascorbate-mediated protein fluorophore formation.

Animals↗

PubMed1991

Oxidative glycation and free radical production: a causal mechanism of diabetic complications.

Glucose may oxidise under physiological conditions and lead to the production of protein reactive ketoaldehydes, hydrogen peroxide and highly reactive oxidants. Glucose is thus able to modify proteins by the attachment of its oxidation derived aldehydes, leading to the development of novel protein fluorophores, as well as fragment protein via free radical mechanisms. The fragmentation of protein by glucose is inhibitable by metal chelators such as diethylenetriamine pentaacetic acid (DETAPAC) and free radical scavengers such as benzoic acid, and sorbitol. The enzymic antioxidant, catalase, also inhibits protein fragmentation. Protein glycation and protein oxidation are inextricably linked. Indeed, using boronate affinity chromatography to separate glycated from non-glycated material, we demonstrate that proteins which are glycated exhibit an enhanced tryptophan oxidation. Our observation that both glycation and oxidation occur simultaneously further supports the hypothesis that tissue damage associated with diabetes and ageing has an oxidative origin.

Aging↗

PubMed1990

Is glucose the sole source of tissue browning in diabetes mellitus?

Reactions between glucose and protein are held to be responsible for the protein 'browning' reactions which occur in diabetes mellitus. In vitro, however, the formation of such novel protein fluorophores is dependent upon the metal-catalysed oxidation of the monosaccharide (glucose 'autoxidation'). Since other small oxidisable molecules are capable of 'browning' proteins via similar metal-catalysed oxidative mechanisms we suggest that protein modification in diabetes may not be restricted to reactions with monosaccharides but may also include other small autoxidisable molecules.

Arachidonic Acid↗

PubMed1990

Autoxidative glycosylation and possible involvement of peroxides and free radicals in LDL modification by glucose.

It has been postulated that the etiology of the complications of diabetes involves oxidative stress, perhaps as a result of hyperglycemia. Consistent with this hypothesis, it has been shown that glucose, under physiological conditions, produces oxidants that possess reactivity similar to the hydroxyl free radical. These oxidants hydroxylate benzoic acid, fragment protein, and induce peroxidation in phosphatidylcholine liposomes and low-density lipoprotein (LDL) when LDL is incubated with hyperglycemic levels of glucose in vitro. These reactions are accelerated by transition metals and inhibited by a metal-chelating agent. The atherosclerotic potential of LDL in diabetes mellitus is often discussed in terms of protein glycosylation, which may affect cellular interactions. Our studies demonstrate, however, that peroxidative reactions also accompany LDL glycosylation in vitro. Peroxidative modification of LDL has also been implicated in LDL atherogenicity. Our studies indicate that glycosylation and peroxidation occur concomitantly in LDL modified by glucose in vitro and may both contribute to the behavioral changes of this lipoprotein.

Free Radicals↗

PubMed1989

Free radical-mediated degradation of proteins: the protective and deleterious effects of membranes.

Lipid membranes have been shown to scavenge free radicals generated by various means. However, under oxidative conditions, unsaturated lipids within membranes can produce damaging free radicals. We have determined the relative importance of these two conflicting properties of lipid membranes with the use of liposomal membrane studies. (1) Liposome membranes can protect extra-liposomal albumin from free radicals derived from sources other than peroxidizing lipid. When albumin or copper (an essential component of the free radical generating systems used) were encapsulated, protein damage was further reduced. (2) Using sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE) we demonstrate that the exposure of albumin to peroxidizing liposome membranes results in both cross-linking and degradation. Our results indicate that protein damage is substantially less than in the case of other biologically relevant free radical generating systems. We discuss our findings with respect to membrane function and the in vivo exposure of cells to free radicals.

Benzene Derivatives↗

PubMed1989

"Autoxidative glycosylation": free radicals and glycation theory.

Studies have shown that glycation in vitro is complicated by the ability of glucose to oxidise, in the presence of trace amounts of transition metal, generating protein-reactive ketoaldehydes, hydrogen peroxide and diverse free radicals. Protein exposed to glucose undergoes fragmentational and conformational alterations, and these, as well as thiol oxidation, appear to be caused by hydroxyl radicals. Glycofluorophore formation is dependent upon ketoaldehyde formation. It is suggested that glucose autoxidation contributes to oxidative stress in pathophysiology associated with diabetes and ageing via this newly described process of "autoxidative glycosylation".

Chelating Agents↗

PubMed1988

Hydroxyl radical production and autoxidative glycosylation. Glucose autoxidation as the cause of protein damage in the experimental glycation model of diabetes mellitus and ageing.

Protein exposed to glucose is cleaved, undergoes conformational change and develops fluorescent adducts ('glycofluorophores'). These changes are presumed to result from the covalent attachment of glucose to amino groups. We have demonstrated, however, that the fragmentation and conformational changes observed are dependent upon hydroxyl radicals produced by glucose autoxidation, or some closely related process, and that antioxidants dissociate structural damage caused by the exposure of glucose to protein from the incorporation of monosaccharide into protein. We have also provided further evidence that glycofluorophore formation is dependent upon metal-catalysed oxidative processes associated with ketoaldehyde formation. If experimental glycation is an adequate model of tissue damage occurring in diabetes mellitus, then these studies indicate a therapeutic role for antioxidants.

Aging↗

PubMed1988

Hydroperoxide-mediated fragmentation of proteins.

1. Chemiluminescence and benzoic acid hydroxylation were used to detect oxygen-centred free-radical production by 2.5 mM-H2O2 and 100 microM-Cu2+. Free radicals could not be detected by these methods when H2O2 was replaced with 10 mM-t-butyl hydroperoxide (TBH) or 10 mM-cumene hydroperoxide (CH). The inclusion of the thiol compound dithioerythritol (DTET; 100 microM) increased radical production by H2O2 and Cu2+ as judged by both assays. Mannitol scavenged radicals in the chemiluminescence system in a dose-dependent manner. 2. H2O2, TBH and CH, each with Cu2+, gave rise to substantial fragmentation of the protein bovine serum albumin (BSA). This fragmentation could be increased by the inclusion of DTET. Omission of Cu2+ or the addition of the chelator DETAPAC (diethylenetriaminepenta-acetic acid; 1 mM) lead to virtual abolition of fragmentation. Autoxidized lipid in the presence of Cu2+ caused protein fragmentation by reactions of lipid hydroperoxides. 3. Polyacrylamide-gel electrophoresis in the presence of SDS confirmed that production of fragments had occurred. 4. Susceptibility of BSA to enzymic hydrolysis by two different proteinases acting at pH 5 and pH 7.2 was increased after a limited exposure to hydroperoxides in the presence of Cu2+. 5. These results may have biological significance, particularly for proteins in lipid environments (e.g. membrane proteins and lipoproteins).

Benzoates↗

PubMed1988

Very low birth weight infants at 8 and 11 years of age: role of neonatal illness and family status.

The intellectual and educational status of 108 children with very low birth weight (less than or equal to 1,500 g), born from 1965 to 1978, was evaluated and tested on standard tests (eight children with severe handicaps were excluded) at 8 years of age. Fifty-seven were further evaluated at 11 years of age. Six categorical outcomes were defined a priori, based on the Wechsler Intelligence Scale for Children-Revised IQ and discrepancies between Verbal and Performance scores and Bender Gestalt Test score. Proportions at 8 years of age were: 4.6% very low IQ (below 70), 13.9% low IQ (70 to 84), and, for those with IQ greater than 84, 12.0% language disability, 12.0% performance disability, 21.4% visual-motor disability, and 36.1% normal. Learning disabilities, determined by discrepancies between IQ and Wide Range Achievement Test scores, included 16.7% of all children. Outcome proportions at 11 years of age were essentially comparable to those at 8 years of age; outcome constancy was present in 52.6%. Ratings of neonatal illness and parent education level strongly influenced the likelihood of outcome at 8 years of age. When ratings were dichotomized (ie, low v high neonatal illness and low v high parent education), the level of neonatal illness primarily influenced the likelihood of normal outcome, whereas the level of parent education influenced the degree of severity of the disability.

Achievement↗

PubMed1987

Developmental outcome following posthemorrhagic hydrocephalus in preterm infants. Comparison of twins discordant for hydrocephalus.

The mental development and neurologic outcome of four sets of preterm twins, in whom one infant within each pair developed posthemorrhagic hydrocephalus during the neonatal period, were assessed. Each hydrocephalic infant presented with signs of increased intracranial pressure and required placement of a ventriculoperitoneal shunt. Three of the four hydrocephalic infants required revision or replacement of the shunt during the first year. The four nonhydrocephalic infants had normal neurologic outcomes. All four hydrocephalic infants had some neurologic abnormalities on follow-up examination. Neurologic abnormalities were transient in one infant and persistent in three others, each of whom showed evidence of mild right hemiparesis. Within each twin pair, developmental rates and mental test scores were strikingly similar throughout infancy and early childhood. Two of the twin pairs have undergone psychological assessments at 8 and 11 years of age. Relatively lower IQ scores were obtained for the hydrocephalic child in each twin pair at these ages.

Cerebral Hemorrhage↗