Christopher J.L. Silwood

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 (CIO2)

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.

Supervised Self Organizing Maps for Classification and Determination of Potentially Discriminatory Variables: Illustrated by Application to Nuclear Magnetic Resonance Metabolomic Profiling

The article describes the extension of the self organizing maps discrimination index (SOMDI) for cases where there are more than two classes and more than one factor that may influence the group of samples by using supervised SOMs to determine which variables and how many are responsible for the different types of separation. The methods are illustrated by an application in the area of metabolic profiling, consisting of a nuclear magnetic resonance (NMR) data set of 96 samples of human saliva, which is characterized by three factors, namely, whether the sample has been treated or not, 16 donors, and 3 sampling days, differing for each donor. The sampling days can be considered a null factor as they should have no significant influence on the metabolic profile. Methods for supervised SOMs involve including a classifier for organizing the map, and we report a method for optimizing this by using an additional weight that determines the relative importance of the classifier relative to the overall experimental data set in order to avoid overfitting. Supervised SOMs can be obtained for each of the three factors, and we develop a multiclass SOM discrimination index (SOMDI) to determine which variables (or regions of the NMR spectra) are considered significant for each of the three potential factors. By dividing the data iteratively into training and test sets 100 times, we define variables as significant for a given factor if they have a positive SOMDI in the training set for the factor and class of interest over all iterations.

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 (1H) NMR spectroscopy was employed to examine the organic acid status of primary root carious lesions. 1H‐NMR analysis of post‐neutralized perchloric acid extracts of active carious lesions revealed that at an operating frequency of 600 MHz, the 1H‐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 1H‐NMR‐visible organic acid/anion expressed as a percentage of total 1H‐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 (Ka), our results demonstrate that formic and pyruvic acids (Ka = 1.77 × 10−4 and 3.20 × 10−3 mol/dm3, respectively) contribute substantially to the decreased pH values associated with active caries lesions (cf. lactate Ka = 1.40 × 10−4 mol/dm3), and hence the pathogenesis of primary root caries. Copyright

Application of high-resolution, two-dimensional 1H and 13C nuclear magnetic resonance techniques to the characterization of lipid oxidation products in autoxidized linoleoyl/linolenoylglycerols

Subjection of polyunsaturated fatty acid (PUFA)-rich culinary oils to standard frying episodes generates a range of lipid oxidation products (LOP), including saturated and α,β-unsaturated aldehydes which arise from the thermally induced fragmentation of conjugated hydroperoxydiene precursors. Since such LOP are damaging to human health, we have employed high-resolution, two-dimensional 1H-1H relayed coherence transfer, 1H-1H total correlation, 1H-13C heteronuclear multiple quantum correlation, and 1H-1H J-resolved nuclear magnetic resonance (NMR) spectroscopic techniques to further elucidate the molecular structures of these components present in (i) a model linoleoylglycerol compound (1,3-dilinolein) allowed to autoxidize at ambient temperature and (ii) PUFA-rich culinary oils subjected to repeated frying episodes. The above techniques readily facilitate the resolution of selected vinylic and aldehydic resonances of LOP which appear as complex overlapping patterns in conventional one-dimensional spectra, particularly when employed in combination with solvent-induced spectral shift modifications. Hence, much useful multi-component information regarding the identity and/or classification of glycerol-bound conjugated hydroperoxydiene and hydroxydiene adducts, and saturated and α,β-unsaturated aldehydes, present in autoxidized PUFA matrices is provided by these NMR methods. Such molecular information is of much value to researchers investigating the deleterious health effects of LOP available in the diet.

The role of N-acetylcysteine in protecting synovial fluid biomolecules against radiolytically-mediated oxidative damage: A high field proton NMR study

High field proton (1H) NMR spectroscopy has been employed to evaluate the abilities of the antioxidant thiol drug N-acetylcysteine and exogenous cysteine to protect metabolites present in intact inflammatory synovial fluid samples against oxidative damage arising from gamma-radiolysis (5.00 kGy) in the presence of atmospheric O2. Although oxidation of urate to allantoin by radiolytically-generated *OH radical was readily circumventable by pre-treatment of synovial fluids with N-acetylcysteine (1.00 or 3.00 x 10(-3) mol x dm(-3)) or cysteine (1.00, 2.00 or 5.00 x 10(-3) mol x dm(-3)), both thiols offered only a limited protective capacity with respect to hyaluronate depolymerisation and the production of formate from carbohydrates in general. Radiolytic products generated from the added thiols (predominantly their corresponding disulphides) were simultaneously detectable in 1H Hahn spin-echo spectra of gamma-irradiated synovial fluids, permitting a quantitative evaluation of the radioprotective capacity of these agents. It is concluded that the multicomponent analytical ability of high field 1H NMR spectroscopy provides much useful molecular information regarding mechanisms associated with the radioprotectant actions of thiols in intact biofluids.

High-resolution 1H NMR investigations of the capacity of dentifrices containing a “smart” bioactive glass to influence the metabolic profile of and deliver calcium ions to human saliva

Dentifrices containing H(2)O-reactive bioactive glasses alleviate hypersensitivity in teeth via the blockage of open dentinal tubules. Here, the ability of two such products to release Ca(2+) ions into human saliva was investigated, together with their influence on the status of this biofluids (1)H NMR-detectable biomolecules. Human salivary supernatants were equilibrated with increasing volumes of those derived from each dentifrice (5.00 min at 37 degrees C). These biofluids were also equilibrated at 37 degrees C with a preselected quantity of the intact products (samples were collected at increasing timepoints). Salivary Ca(2+) concentrations were monitored by a (1)H NMR technique involving ethylenediamine tetra-acetate addition and/or atomic absorption spectrometry. Added Ca(2+)- and dentifrice supernatant volume (DSV)-induced modifications to the salivary (1)H NMR profile were explored by spectral titration. Data acquired demonstrated added DSV-dependent increases in salivary Ca(2+) concentrations and (Ca(2+)-independent) modifications to the intensities of selected salivary (1)H NMR signals, particularly those of the malodorous amines methyl-, dimethyl-, and trimethylamines, which were diminished by up to 80% of their prior values. Time-dependent elevations in salivary Ca(2+) level were observed on equilibration with the intact dentifrices. Added Ca(2+) ions exerted a concentration-dependent influence on a range of resonances (including those of citrate, succinate, pyruvate, and lactate). These data provide valuable information regarding the mechanisms of action of the products tested.

Investigation of the Molecular Nature of Low-molecular-mass Cobalt(II) Ions in Isolated Osteoarthritic Knee-joint Synovial Fluid

High field 1H NMR spectroscopy demonstrated that addition of Co(II) ions to osteoarthritic knee-joint synovial fluid (SF) resulted in its complexation by a range of biomolecules, the relative efficacies of these complexants/chelators being citrate ≫ histidine ∼ threonine≫glycine ∼ glutamate ∼ glutamine ∼ phenylalanine ∼ tyrosine > formate > lactate≫alanine > valine > acetate > pyruvate > creatinine, this order reflecting the ability of these ligands to compete for the available Co(II) in terms of (1) thermodynamic equilibrium constants for the formation of their complexes and (2) their SF concentrations. Since many of these SF Co(II) complexants (e.g. histidinate) serve as powerful •OH scavengers, the results acquired indicate that any of this radical generated from the Co(II) source in such complexes via Fenton or pseudo-Fenton reaction systems will be “site-specifically” scavenged. The significance of these observations with regard to cobalt toxicity and the in vivo corrosion of cobalt-containing metal alloy joint prostheses (e.g. CoCr alloys) is discussed.

Multicomponent evaluations of the oxidising actions and status of a peroxoborate-containing tooth-whitening system in whole human saliva using high resolution proton NMR spectroscopy

High resolution 1H nuclear magnetic resonance (NMR) spectroscopy was employed to conduct a multicomponent investigation of the oxidation of salivary biomolecules by peroxoborate present in a tooth-whitening dentifrice formulation. The results acquired demonstrated that peroxoborate gave rise to the oxidative decarboxylation of the hydrogen peroxide scavenger pyruvate, a reaction generating acetate and CO2 as products. Experiments performed on chemical model systems confirmed the oxidative consumption of pyruvate by dentifrice-derived peroxoborate, and also revealed that the salivary electron donors cysteine and methionine (precursors to volatile sulphur compounds), were oxidised to cystine and methionine sulphoxide respectively. The biochemical and periodontal significance of these results is discussed.

High-resolution 1H NMR investigations of the oxidative consumption of salivary biomolecules by oral rinse peroxides

BACKGROUND A multicomponent evaluation of the oxidative consumption of salivary biomolecules by a tooth-whitening oral rinse preparation has been performed using high-resolution proton ((1)H) nuclear magnetic resonance spectroscopy (NMR). METHODS Unstimulated human saliva samples (n = 12) were treated with aliquots of the oral rinse tested and 600 MHz (1)H NMR spectra acquired on these samples demonstrated that hydrogen peroxide (H(2)O(2)) and/or peroxodisulphate (S(2)O(8) (2-)) present in this product gave rise to the oxidative decarboxylation of the salivary electron-donor pyruvate (to acetate and CO(2)), and also oxidized methionine (a precursor to volatile sulphur compounds responsible for oral malodour), and malodourous trimethylamine to methionine sulphoxide and trimethylamine-N-oxide, respectively (reductions observed in the salivary concentrations of each biomolecular peroxide-scavenging agent were all extremely statistically significant, p < 0.005). RESULTS Experiments conducted on chemical model systems confirmed the consumption of pyruvate by this product, and also revealed that the amino acids cysteine and methionine were oxidatively transformed to cystine and methionine sulphoxide, respectively. CONCLUSIONS High-field (1)H NMR analysis provides much valuable molecular information regarding the fate of tooth-whitening oxidants in human saliva and permits an assessment of the mechanisms of action of oral healthcare products containing these agents. The biochemical and potential therapeutic significance of the results obtained are discussed.