In vivo absorption, metabolism, and urinary excretion of alpha,beta-unsaturated aldehydes in experimental animals. Relevance to the development of cardiovascular diseases by the dietary ingestion of thermally stressed polyunsaturate-rich culinary oils.

Thermal stressing of polyunsaturated fatty acid (PUFA)- rich culinary oils according to routine frying or cooking practices generates high levels of cytotoxic aldehydic products (predominantly trans-2-alkenals, trans,trans-alka-2,4-dienals, cis,trans-alka-2, 4-dienals, and n-alkanals), species arising from the fragmentation of conjugated hydroperoxydiene precursors. In this investigation we demonstrate that typical trans-2-alkenal compounds known to be produced from the thermally induced autoxidation of PUFAs are readily absorbed from the gut into the systemic circulation in vivo, metabolized (primarily via the addition of glutathione across their electrophilic carbon-carbon double bonds), and excreted in the urine as C-3 mercapturate conjugates in rats. Since such aldehydic products are damaging to human health, the results obtained from our investigations indicate that the dietary ingestion of thermally, autoxidatively stressed PUFA-rich culinary oils promotes the induction, development, and progression of cardiovascular diseases.

Examination of the metabolic status of rat air pouch inflammatory exudate by high field proton NMR spectroscopy.

High field proton (1H) NMR spectroscopy was employed to investigate the metabolic status of rat air pouch inflammatory exudates obtained subsequent to the induction of inflammation with carrageenan, and the 1H NMR profiles of these fluids were compared and contrasted with those of inflammatory human synovial fluid, rat plasma and human serum. The characteristic biochemical features obtained from 1H NMR analysis of these exudates consisted of (1) substantially elevated levels of lactate (11.40+/-1.46x10-3 mol dm-3 for samples collected at a time point of 24 h post induction) with little or no NMR-detectable glucose, data consistent with a hypoxic environment and consequent anaerobic metabolism in the inflamed air pouch, and (2) high levels of the ketone body 3-d-hydroxybutyrate, providing evidence for an increased utilization of fats for energy by lymphocytes, the predominant leucocytes present in this environment. These phenomena represent a pathological extreme of the abnormal metabolic status of inflammatory human synovial fluids.

High resolution proton NMR investigations of rat blood plasma. Assignment of resonances for the molecularly mobile carbohydrate side-chains of 'acute-phase' glycoproteins.

An intense broad resonance at 2.14 ppm present in high field (400, 500 and 600 MHz) Hahn spin-echo 1H-NMR spectra of rat blood plasma, but absent from those of human blood plasma is attributable to the presence of terminal O-acetylsialate sugars in the molecularly mobile carbohydrate side-chains of 'acute-phase' glycoproteins (predominantly alpha 1-acid glycoprotein). The presence of such alternative acetylsugars in the carbohydrate side-chains of rat plasma glycoproteins are of much physiological and experimental significance in view of the regular use of these animals in model systems of human inflammatory conditions.

Generation of lipid peroxidation products in culinary oils and fats during episodes of thermal stressing: a high field 1H NMR study.

The oxidative deterioration of glycerol-bound polyunsaturated fatty acids (PUFAs) in culinary oils and fats during episodes of heating associated with normal usage (30-90 min at 180 degrees C) has been monitored by high field 1H NMR spectroscopy. Thermal stressing of PUFA-rich culinary oils generated high levels of n-alkanals, trans-2-alkenals, alka-2,4-dienals and 4-hydroxy-trans-2-alkenals via decomposition of their conjugated hydroperoxydiene precursors, whereas only low concentrations of selected aldehydes were produced in oils with a low PUFA content, lard and dripping when subjected to the above heating episodes. Samples of repeatedly used, PUFA-rich culinary oils obtained from restaurants also contained high levels of each class of aldehyde. The dietary, physiological and toxicological ramifications of the results obtained are discussed.

Detection of aldehydes and their conjugated hydroperoxydiene precursors in thermally-stressed culinary oils and fats: investigations using high resolution proton NMR spectroscopy.

High field (400 and 600 MHz) proton NMR spectroscopy has been employed to investigate the thermally-induced autoxidation of glycerol-bound polyunsaturated fatty acids present in intact culinary frying oils and fats. Heating of these materials at 180 degrees C for periods of 30, 60 and 90 min. generated a variety of peroxidation products, notably aldehydes (alkanals, trans-2-alkenals and alka-2,4-dienals) and their conjugated hydroperoxydiene precursors. Since such aldehydes appear to be absorbed into the systemic circulation from the gut in vivo, the toxicological significance of their production during standard frying practices is discussed.

Multicomponent spectroscopic investigations of salivary antioxidant consumption by an oral rinse preparation containing the stable free radical species chlorine dioxide (ClO2.).

A multicomponent evaluation of the oxidative consumption of salivary biomolecules by a commercially-available oral rinse preparation containing an admixture of the stable free radical species chlorine dioxide (ClO2.) with chlorite anion (ClO2-) has been investigated using high resolution 1H NMR spectroscopy. The results obtained demonstrated that ClO2. and/or ClO2- present in this preparation effected the oxidative decarboxylation of salivary pyruvate (to acetate and CO2). Experiments conducted on chemical model systems confirmed the oxidative decarboxylation of pyruvate by this oral rinse, and also demonstrated that urate, thiocyanate anion, and the amino acids cysteine and methionine (precursors to volatile sulphur compounds responsible for oral malodour), were oxidatively consumed. The biochemical, periodontal and therapeutic significance of the results are discussed.

1H and (13)C NMR spectroscopic analysis of human saliva.

We have explored the ability of high-resolution NMR techniques to (1) index salivary biomolecules and (2) provide valuable data regarding intra- and inter-subject variability in the concentrations of a series of components readily determinable by this technique (organic acids and malodorous amines). Experiments were conducted on 'whole' saliva samples collected from 20 patients, either randomly during their daily activities, or, for investigations involving the quantification of salivary biomolecules, immediately after they woke in the morning throughout a three-day period. These NMR techniques permitted us to detect greater than 60 metabolites, together with agents arising from dietary, oral health care product, and pharmaceutical sources. Highly significant "between-subject" differences in the a.m. waking salivary metabolite concentrations were found for 9 out of 11 components monitored. It is concluded that NMR spectroscopy serves as a powerful technique for the multicomponent analysis of human saliva.

Proinflammatory effects of morphine in the rat adjuvant arthritis model.

Morphine has been shown to alter various aspects of the immune response. We examined its effect on progression of the T-cell-mediated model, rat adjuvant arthritis. Saline, morphine or the opioid antagonist naloxone were administered to male Wistar rats via subcutaneous osmotic pumps implanted three days prior to adjuvant disease induction by an intra-dermal injection of Mycobacterium tuberculosis in oil. The time of disease onset was found to be accelerated (day 11) for the morphine group as compared to the saline control (day 13). In addition morphine produced a significant increase in paw swelling (days 13 and 14), bone demineralization and bone erosions. A significant decrease in body weight as compared to the saline control was also observed. Naloxone had no significant effect on the degree of the inflammation seen, although like morphine it significantly increased bone demineralization and bone erosions as assessed by radiography.

Molecular mechanisms of the bleaching actions associated with commercially-available whitening oral health care products.

An increased public awareness in dental aesthetics has resulted in the wide availability of techniques of tooth bleaching, both in the dental chair and at home. This article reviews the aetiology of tooth discolouration both at the clinical and the molecular level, together with methods of alleviating such discolouration. Much of the therapeutic and aesthetic actions of commercially-available tooth whiteners, gels, oral rinses and other dentifrices are predominantly dependent on their ability to act as oxidants. A novel method of evaluating these aspects of dentifrice activity is also described: high resolution proton nuclear magnetic resonance (NMR) spectroscopy is a virtually non-invasive, multi component bioanalytical technique that can be employed to study oxidation/reduction reactions at the molecular level and is utilised here to investigate the mechanisms of action of a newly developed dentifrice (Ultrawhite Opal, Janina International). Such methodology also offers much potential for studies concerning the numerous chemical reactions occurring within the oral environment.

Vascular turnover during carrageenan synovitis in the rat.

Inhibition of angiogenesis has been proposed as a novel therapeutic approach to the treatment of chronic inflammatory diseases such as rheumatoid arthritis. We have assessed carrageenan synovitis in male Wistar rats as a potential model of the increased vascular turnover observed in human synovitis. Intra-articular injection of 100 microl of 2% carrageenan in the right knee on Day 0 was followed by acute joint swelling through Day 3 and persistent macrophage infiltration at Day 28. Endothelial cell proliferation and endothelial cell death each peaked during the acute phase and remained elevated during persistent synovitis. Vascular densities were high in normal synovia, decreased during the acute phase, transiently increased at Day 14, and then returned to control values by Day 28. Lower doses of carrageenan, which stimulated acute joint swelling and macrophage infiltration but not endothelial cell proliferation at Day 3, were not associated with persistent macrophage infiltration at Day 28. Intra-articular injection of substance P or vascular endothelial growth factor, angiogenic factors that are present in normal synovia or are up-regulated during inflammation, respectively, each stimulated endothelial cell expression of proliferating cell nuclear antigen. Increased vascular turnover during acute and persistent phases of synovitis may be regulated by different factors and may serve different roles. The late phase (Day 28) of carrageenan synovitis displays similarities with rheumatoid synovitis, in which endothelial cell turnover is persistently increased despite little overall change in vascular density.

A biochemical investigation of auranofin nephrotoxicity by high field proton nuclear magnetic resonance (NMR) spectroscopy.

High-field proton NMR spectroscopic analysis of urine and plasma has been employed to study the biochemical effects and nephrotoxic action of an intramuscular dose of auranofin in rats. Auranofin induced a characteristic profile of proximal tubular damage as evidenced by aminoaciduria, lactic aciduria and increased urinary acetate concentrations. In addition, ethanol was detectable in both urine and plasma obtained from auranofin-treated rats. Auranofin-mediated elevations in the plasma and urine concentrations of 3-D-hydroxybutyrate indicated an increased utilisation of fats for fuel in rats treated with this novel therapeutic agent.

Influence of an intravenous dose of aurothiomalate on the status of low-molecular-mass endogenous metabolites in blood plasma: a proton nuclear magnetic resonance (NMR) study.

The effect of aurothiomalate on the status of a wide range of low-molecular-mass endogenous metabolites in blood plasma obtained from animals treated with an intravenous dose of this second-line agent (150 mg/kg) has been assessed by high field proton Hahn spin-echo NMR spectroscopy. As well as modulating the effective concentrations of NMR-detectable biomolecules, aurothiomalate induces a time-dependent decrease in plasma levels of triacylglycerols with a corresponding elevation in the concentration of the ketone body 3-D-hydroxybutyrate, indicating an increased utilisation of fats for energy in rats treated with this 1:1 gold(I)-thiolate complex. These observations may reflect the toxic side-effects that are associated with aurothiomalate treatment.

Assessment of a human recombinant manganese superoxide dismutase in models of inflammation.

We evaluated a novel human recombinant preparation of manganese superoxide dismutase (MnSOD) for anti-inflammatory and anti-oxidant activity compared with a copper zinc (CuZn) SOD preparation. The results showed that administration of MnSOD (50, 100 and 200 micrograms kg-1) in the Freund's Complete Adjuvant (FCA) mediated paw oedema model suppressed the inflammation at 4 hours by 43, 25 and 43% (P < 0.001, P < 0.01 and P < 0.001 at respective doses). However, 24 hours post-challenge, MnSOD (50 and 100 micrograms kg-1), suppressed the inflammation by 19% (P < 0.001). In contrast, Mn SOD at higher doses (400-800 micrograms kg-1; 2 mgkg-1) exacerbated the inflammatory response at 4 hours. This pro-inflammatory response declined progressively by 24 hours. Furthermore, CuZn SOD produced no significant effects on the inflammatory response. In the carrageenan-induced synovitis model, Mn SOD (25 and 50 micrograms; intra-articular administration) exacerbated the inflammation at 48 hours. In contrast, Mn SOD at 5 micrograms produced a significant suppression (44%, P < 0.05) in knee joint swelling at 24 hours. The CuZn SOD preparation produced marked pro-inflammatory effects in the joints whilst it lacked activity in the FCA-mediated paw oedema model. These findings support a therapeutic potential of MnSOD in inflammatory disorders, however the compound has a complex pharmaco-dynamic profile.

Application of a novel 1:1 gold(I)-chromophoric thiolate complex as a spectrophotometric probe for the thiol-exchange reactions of anti-arthritic gold drugs in biological fluids.

An oligomeric 1:1 gold(I) complex of the chromophoric thiol 5-mercapto (2-nitrobenzoate) has been synthesized and applied as a spectrophotometric probe for the thiol-exchange reactions of structurally-analogous 1:1 gold(I)-thiolate drugs. For low-molecular-mass thiols, results were consistent with the initial formation of a monomeric mixed-ligand bis-thiolato gold(I) complex followed by further ligand substitution by excess thiol to produce 5-mercapto (2-nitrobenzoate) and a monomeric bis-thiolato gold(I) complex. For human serum albumin, however, the spectrophotometric changes were only consistent with the binding of gold(I) to its single cysteine residue (Cys-34) with the retention of the 5-mercapto (2-nitrobenzoate) ligand on gold(I).

A model for desferrioxamine-induced retinopathy using the albino rat.

We describe a rat model of the ocular toxicity induced in patients by the iron chelator desferrioxamine (DFX). The functional integrity of the rat photoreceptor membrane is assessed by measurement of the electroretinogram b wave amplitude. Following single bolus doses of DFX (50, 100 and 150 mg), rats develop dose-related suppression of b wave amplitude within 48 h. Recovery takes place over the subsequent 14 days. This ocular damage is exacerbated by exposure to increased levels of white light and oxygen. Studies in this model also show that darkness and, in particular, red light protect the eye against DFX toxicity. This is of potential use in patient management during DFX therapy.

Attempt to show inhibition by oxygen in the modifying factor assay.

An Australian laboratory has demonstrated that antibodies to certain strains of enteric bacteria react with cells from HLA-B27-positive patients with a 'modifying factor' (MF) from these bacteria. These observations have proved difficult to reproduce. We investigated the possibility that oxygen inhibits the test by blocking B27, but we were unable to obtain consistent positive results.

Simulated digestion status of intact and exoskeletally-punctured insects and insect larvae: a spectroscopic investigation.

In this study, we tested the hypothesis that puncturing the chitin exoskeleton of insect and insect larvae food sources aids the ingress of digestive fluids and increases the rate of digestion and energy uptake in insectivorous mammals. For this purpose 10 crickets (Acheta domesticus) and 10 mealworms (Tenebrio molitor larvae) were divided into two groups of 5; one group was punctured using a small blade to mimic the effect of a single bite, the remainder serving as controls. The insects were then individually immersed in 5 ml of a 1 x 10(-2) mol.dm(-3) solution of hydrochloric acid (pH 2.0) for a period of 2 h in order to mimic digestion in the stomach. The matrix was then centrifuged and the supernatant fluid subjected to spectrophotometric and high-resolution proton (1H) NMR analysis. Electronic absorption spectra of these supernatants revealed that puncturing the exoskeleton of mealworms and crickets gave rise to substantial elevations (up to 14-fold) in the concentrations of UV-absorbing biomolecules (p < 0.025 for both species). The 400-MHz 1H NMR profiles of supernatants derived from mealworm and cricket specimens with punctured exoskeletons contained a wide variety of prominent biomolecule resonances, whereas those from unpunctured (control) insects contained signals of a much lower intensity, ascribable only to selected biomolecules. We conclude that puncturing the cuticle of insects and insect larvae prior to swallowing confers significant nutritional advantages over swallowing prey whole.

Amelioration of antigen-induced arthritis in rats by transfer of extracellular superoxide dismutase and catalase genes.

Reactive oxygen species (ROS) have been implicated in the pathogenesis of rheumatoid arthritis (RA), while antioxidant enzymes, such as extracellular superoxide dismutase (EC-SOD) and catalase, block radical-induced events. The present study tested if the ex vivo transfer of EC-SOD and catalase genes alone or in combination in the knee joint of rats with monoarticular antigen-induced arthritis (AIA) was anti-inflammatory, and examined the potential mechanisms involved. Synoviocytes isolated from female Wistar rats were immortalized with a retroviral vector SUV19.5. These cells were permanently transfected with an EC-SOD expression plasmid (pEC-SODZeo) or a catalase expression plasmid (pCatalaseZeo) to create cells overexpressing EC-SOD or catalase, as measured by RT-PCR and Western blots. The cells were engrafted in knee joints of animals at the time of the induction of AIA. Three gene transfer groups, an EC-SOD group, a catalase group and a combined therapy group (EC-SOD and catalase) were included in these experiments. Animals in the control group were engrafted with synoviocytes transfected with the plasmid pZeoSV2 without an insert. Clinical and histological assessments were performed, as well as tissue measurements of SOD, catalase and gelatinase activities. Ex vivo gene transfer of EC-SOD and catalase into rat knee joints produced about a six- to seven-fold increase in EC-SOD activity and a two- to three-fold increase in catalase activity compared with the control animals. Rats treated with cells overexpressing EC-SOD, catalase or a combination of EC-SOD and catalase showed significant suppression of knee joint swelling, decreased infiltration of inflammatory cells within the synovial membrane and reduced gelatinase activity in knee joints, compared with animals receiving cells transfected with the plasmid alone. No statistically significant difference was found between the groups treated with cells overexpressing EC-SOD, catalase or a combination of both. Gene therapy involving the local intra-articular overexpression of two antioxidant enzymes, EC-SOD and catalase, was anti-inflammatory in AIA. One mechanism appears to be the suppression of gelatinase activities by both EC-SOD and catalase.

Influence of disodium aurothiomalate on the activities of xanthine dehydrogenase and xanthine oxidase in endothelial cells.

The effect of aurothiomalate in modulating the conversion of xanthine dehydrogenase to its superoxide producing oxidase form in rat and human liver cytosolic preparations has been investigated. Low concentrations (10(-8)-10(-5) mol.dm-3) of this second-line agent were found to inhibit the conversion of the dehydrogenase to its corresponding oxidase form. High concentrations (10(-4) mol.dm-3), however, accelerated this conversion. It is possible that the influence of aurothiomalate on the relative proportions of xanthine dehydrogenase and xanthine oxidase is a reflection of the gold(I) blockage of critical thiol(ate) or sulphido ligands present in this enzymatic system. These effects may form the basis of aurothiomalate's anti-proliferative action on endothelial cells.