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.

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.

1H-NMR analysis of microbial-derived organic acids in primary root carious lesions and saliva.

In addition to lowered pH values, the molecular profile and concentrations of microbial-derived organic acids in carious dentin are important demineralization parameters involved in the induction, development and progression of dental caries. High-resolution proton ((1)H) NMR spectroscopy was employed to examine the organic acid status of primary root carious lesions. (1)H-NMR analysis of post-neutralized perchloric acid extracts of active carious lesions revealed that at an operating frequency of 600 MHz, the (1)H-NMR-detectable organic acid composition of carious dentin samples (mean molecular percentage content +/- standard error; the mean molecular percentage content is defined here as the mean of the concentration of each (1)H-NMR-visible organic acid/anion expressed as a percentage of total (1)H-NMR-detectable organic acid/anion level in each sample) was acetate 51 +/- 2%, formate 37 +/- 2%, lactate 5 +/- 1%, propionate 3 +/- 0.8%, pyruvate 2.4 +/- 0.3%, n-butyrate 1.2 +/- 0.2%; succinate 0.1 +/- 0.1%; iso-butyrate, n- and iso-valerate, and n- and iso-caproate (total) <0.2%. Further components detectable included alanine, glycine, choline, phosphorylcholine, trimethylamine oxide, methanol, glycolate and assorted saccharides. In view of their high dissociation constants (K(a)), our results demonstrate that formic and pyruvic acids (K(a) = 1.77 x 10(-4) and 3.20 x 10(-3) mol/dm(3), respectively) contribute substantially to the decreased pH values associated with active caries lesions (cf. lactate K(a) = 1.40 x 10(-4) mol/dm(3)), and hence the pathogenesis of primary root caries.

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.

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.

Evaluating the antioxidant potential of new treatments for inflammatory bowel disease using a rat model of colitis.

BACKGROUND: Reactive oxygen species may mediate tissue injury in inflammatory bowel disease. Aminosalicylates have antioxidant activity and the antioxidants, superoxide dismutase and allopurinol, are of reported benefit in inflammatory bowel disease. AIM: To develop a convenient technique for testing the antioxidant potential of standard and novel therapeutic agents for use in inflammatory bowel disease. METHODS: Amplified chemiluminescence was used to measure reactive oxygen species production by colonic biopsy specimens from rats with acetic acid induced colitis and to assess the in vitro effect of conventional antioxidants, standard therapies and proposed novel therapies for inflammatory bowel disease. RESULTS: The model was validated by demonstrating that the profile of effects on chemiluminescence of acetic acid induced colitis biopsy specimens given by conventional antioxidants (sodium azide, catalase, copper-zinc superoxide dismutase, dimethyl sulphoxide, N-acetylcysteine and ascorbate) and standard therapies (5-aminosalicylate and hydrocortisone) resembled that previously reported using biopsy specimens from ulcerative colitis. Human recombinant manganese superoxide dismutase did not alter chemiluminescence. Two novel compounds, LY231617 (10 mM) and amflutizole (20 mM), reduced chemiluminescence by 98% (n = 5, p = 0.009) and 88% (n = 5, p = 0.03), respectively. CONCLUSIONS: The similarity of the chemiluminescence responses of colonic biopsy specimens from acetic acid induced colitis and ulcerative colitis to a range of conventional antioxidants and standard treatments suggests that this model is a useful method for testing the antioxidant potential of new therapies for inflammatory bowel disease. The antioxidant actions of dimethyl sulphoxide, ascorbate, and the novel compounds, amflutizole and LY231617 in this model suggest that these agents merit further assessment in the treatment of inflammatory bowel disease.

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.

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.

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.

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.

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.

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.

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.

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.

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).

Control of oxidative damage in rheumatoid arthritis by gold(I)-thiolate drugs.

The roles of anti-arthritic gold(I)-thiolate drugs such as disodium aurothiomalate ('Myocrisin') in the modulation or promotion of oxygen radical-mediated oxidative damage in vivo are reviewed. In particular, the precise molecular mechanisms by which these novel second-line agents exert their therapeutic effects are discussed in terms of (i) the direct and indirect control of enzymes involved in the generation or scavenging of reactive oxygen species (ROS) such as superoxide ion, hydrogen peroxide and hydroxyl radical, (ii) the protection of proteins and relevant enzyme systems against attack by ROS and (iii) their direct involvement in the production (at appropriate 'target' sites) or scavenging of ROS in vivo. In addition, the role of the orally-effective gold(I)-phosphine complex auranofin in the control of oxidative damage in rheumatoid arthritis is also discussed.