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There are concerns that weight-loss (WL) diets based on very low carbohydrate (LC) intake have a negative impact on antioxidant status and biomarkers of cardiovascular and metabolic health. Obese men (n 16) participated in a randomised, cross-over design diet trial, with food provided daily, at approximately 8.3 MJ/d (approximately 70 % of energy maintenance requirements). They were provided with two high-protein diets (30 % of energy), each for a 4-week period, involving a LC (4 % carbohydrate) and a moderate carbohydrate (MC, 35 % carbohydrate) content. Body weight was measured daily, and weekly blood samples were collected. On average, subjects lost 6.75 and 4.32 kg of weight on the LC and MC diets, respectively (P < 0.001, SED 0.350). Although the LC and MC diets were associated with a small reduction in plasma concentrations of retinol, vitamin E (α-tocopherol) and β-cryptoxanthin (P < 0.005), these were still above the values indicative of deficiency. Interestingly, plasma vitamin C concentrations increased on consumption of the LC diet (P < 0.05). Plasma markers of insulin resistance (P < 0.001), lipaemia and inflammation (P < 0.05, TNF-α and IL-10) improved similarly on both diets. There was no change in other cardiovascular markers with WL. The present data suggest that a LC WL diet does not impair plasma indices of cardiometabolic health, at least within 4 weeks, in otherwise healthy obese subjects. In general, improvements in metabolic health associated with WL were similar between the LC and MC diets. Antioxidant supplements may be warranted if LC WL diets are consumed for a prolonged period.

Obesity has recently become a focus of research to elucidate diet and lifestyle factors as important risk factors for colon cancer. Altered levels of insulin, leptin, and adiponectin have been identified as potential candidates increasing colon cancer risk within the prevailing obesogenic environment. There has been considerable research to characterize signaling via these hormones in the brain, liver, and adipose tissue; however, very little is known of their emerging role in peripheral signaling, particularly in epithelial tissues. This study profiles insulin, leptin, and adipokine receptors in the rat colon, revealing novel microanatomical location of these receptors and thereby supporting a potential role in regulating colonic tissue. Potential involvement of insulin, leptin, and adiponectin receptors in increased risk of colon cancer was investigated using Sprague-Dawley rats, either resistant or susceptible to diet-induced obesity. Regulation of insulin, leptin, and adiponectin receptors as a consequence of differing levels of adiposity was assessed regionally in the colon in response to treatment with the chemical carcinogen 1,2-dimethylhydrazine (DMH). However, significantly increased fat mass, increased levels of plasma insulin, leptin, and triglycerides, previously associated with an increased risk of colon cancer, were not associated with promotion of precancerous lesions in the experimental rats or deregulation of insulin, leptin, or adiponectin receptors. These findings do not support a direct link between the deregulation of insulin and adipokine levels observed in obese rats and an increased risk of colon carcinogenesis.

Epidemiological studies assessing the health benefits of drinking black tea are equivocal. Such disparity may reflect an inability of semiquantitative assessment to consider how infusion time and addition of milk affect the bioavailability of potentially beneficial antioxidant polyphenols. Six brands of tea demonstrated similar increases in antioxidant capacity and total phenolic and catechin contents with increasing infusion time. These results were unaffected by the addition of milk. Consumption of black tea (400 mL) was associated with significant increases in plasma antioxidant capacity (10%) and concentrations of total phenols (20%), catechins (32%), and the flavonols quercetin (39%) and kaempferol (45%) (all p < 0.01) within 80 min. This was unaffected by adding milk. Infusion time may therefore be a more important determinant in the absorption of polyphenols from black tea. Observational studies assessing the health benefits of tea consumption require recording of brewing methods as well as frequency of consumption.

Glutathione peroxidases (Gpx) are important moderators of oxidative stress that is implicated in the pathogenesis of numerous diseases including colon cancer. Previous studies report limited examinations of cytosolic glutathione peroxidase location of expression in colon tissue. This study reports evidence of both common sites of Gpx1 and Gpx2 expression in rat colon and sites that are exclusive to each isoform. Semi-quantitative PCR performed previously demonstrated RNA expression of Gpx1 and Gpx2 in proximal, transverse and distal colon. Mapping the distribution throughout the entire colon has revealed specific, novel sites of glutathione peroxidase expression in colon lymphatic tissue. In situ hybridisation and immunohistochemistry confirmed micro-anatomical location of Gpx1 within lymphatic tissue and the lamina propria, sub-mucosa, muscularis and serosa, but not the lumenal epithelium. In situ hybridisation and immunohistochemistry were consistent with reports of microanatomical location of Gpx2 in the lumenal epithelium. Novel sites of Gpx2 expression were also observed in lymphatic tissue. Immunolocalisation in the vicinity of aberrant crypt foci was also examined to further investigate the link between glutathione peroxidases and colon cancer. This did not reveal significant abnormalities, nor did measurement of cytosolic glutathione peroxidase activity or gene expression in colon tissue from rats treated with the colontropic chemical, 1,2-dimethylhydrazine. These results support the potential for Gpx1 and Gpx2 redundancy in lymphatic tissue, but not in epithelial cells of the colon crypt or in the lamina propria, sub-mucosa, muscularis or serosa.

Oxidative stress is a characteristic of cancerous colon tissue and inflammatory bowel diseases that increase colon cancer risk. Epidemiological evidence supports a protective effect of plant-derived compounds. Aspirin is also protective against colon cancer. The mechanism of action is unclear although salicylic acid, the main metabolite of aspirin, has been shown to decrease the synthesis of pro-inflammatory and potentially neo-plastic prostaglandins. Salicylic acid is found in significant quantities in a plant-based diet. However, in plants salicylic acid is also reported to modulate the expression of numerous enzymes with antioxidant activity. The aim of this study was to assess whether salicylic acid can modulate pro-cancerous biological pathways in the colon. Oxidative stress, prostaglandins and cytosolic glutathione peroxidase (cyGPX) were analysed in proximal, transverse and distal colon from a rat model of diet-induced oxidative stress. Elevated plasma pyruvate kinase activity (1293+/-206 U/ml) and increased indices of lipid peroxidation in colon (proximal 6.4+/-0.84 nM MDA/mg protein; transverse 6.9+/-0.97 nM MDA/mg protein; distal 5.2+/-0.62 nM MDA/mg protein) from rats fed a Vitamin E deficient diet were significantly decreased on supplementation with salicylic acid (plasma pyruvate 546+/-43 U/ml; salicylic acid proximal 3.6+/-0.39 nM MDA/mg protein; transverse 4.5+/-0.61 nM MDA/mg protein; distal 4.4+/-0.27 nM MDA/mg protein). Reductions in oxidative stress and prostaglandin production on supplementation with salicylic acid were associated with an elevation in glutathione peroxidase activity (Vitamin E deficient proximal 0.056+/-0.013 U/mg protein; transverse 0.073+/-0.008 U/mg protein; distal 0.088+/-0.010 U/mg protein; Vitamin E deficient with salicylic acid proximal 0.17+/-0.01 U/mg protein; transverse 0.23+/-0.016 U/mg protein; distal 0.16+/-0.020 U/mg protein). Gpx1 and Gpx2 gene transcripts were not elevated in association with increased activity of the soluble glutathione peroxidase activity. Glutathione peroxidases are key antioxidant enzymes, catalysing the decomposition of potentially toxic lipid peroxides. Gpx activity and regulation of Gpx gene transcription has been shown previously to be complex with activity not necessarily mirrored by a corresponding elevation in gene transcription. By supplementing the diet of Vitamin E deficient rats with salicylic acid (1 g/kg diet), this study assessed effects of salicylic acid on cytosolic glutathione peroxidase activity in the colon. The ability of salicylic acid to modulate antioxidant enzymes in colon tissue may be an important mechanism in inhibiting colon cancer development.

Background: Experimentally imposed dietary restriction is known to extend the lifespan of rodents, perhaps by slowing the accumulation of oxidative damage that is thought to be one of the causes of aging., Aim of the Study: We examined the effects of restricted total food intake, and protein and calorie restriction, on DNA oxidation and related biomarkers in rats., Methods: From 1 to 17 months, rats in group 1 received normal diet ad libitum. Group 2 received 70% of the quantity consumed by the first group. Group 3 had the same quantity as group 2, but with a reduction in protein (from 18% to 10% of the diet by weight), and group 4 were further restricted with a 30% decrease in calories. Lymphocytes were isolated from blood samples taken every two months. DNA breaks, oxidised pyrimidines, resistance to H2O2-induced damage, and strand break repair were measured with the comet assay. Organs were isolated from rats killed at 17 months, with 1 month-old rats for comparison; DNA oxidation and antioxidant enzyme activities were measured., Results: DNA breaks in lymphocytes increased from 1 to 3 months but thereafter declined with age, except in ad libitum fed rats. Oxidised pyrimidines did not change significantly. Resistance to H2O2-induced damage was least at 3 months, and increased with age. Repair of DNA strand breaks was efficient at all ages. Diet had little effect on these endpoints. Diet had no influence on 8-oxo-7.8-dihydroguanine levels in DNA from liver, testis and brain of 17 month old rats. Combining data from all four groups, the levels in brain and liver were significantly higher at 17 months compared with 1 month. Antioxidant enzyme activities tended to increase between 1 and 17 months; effects of diet were not so consistent., Conclusions: While DNA damage shows a modest increase with age in some organs, antioxidant status and DNA strand break repair do not decline with age. Restricted diets (including protein and calorie restriction) have no effect on any of these markers of genetic stability.

Background: Fruit and vegetable consumption protects against cancer. This is attributed in part to antioxidants such as vitamin E combating oxidative DNA damage. Anthocyanins are found in significant concentrations in the human diet. However, it remains to be established whether they are bioactive in vivo., Aim: To investigate the consequence both of vitamin E deficiency on oxidative damage to DNA and lipids and the cytoprotective effect of nutritionally relevant levels of cyanidin-3-glycoside both in vivo in rats and in vitro in human colonocytes., Methods: Male Rowett Hooded Lister rats were fed a diet containing less than 0.5 mg/kg vitamin E or a vitamin E supplemented control diet containing 100 mg d alpha-tocopherol acetate/kg. Half of the controls and vitamin E-deficient rats received cyanidin-3-glycoside (100 mg/kg). After 12 weeks endogenous DNA stability in rat lymphocytes (strand breaks and oxidised bases) and response to oxidative stress ex vivo (H2O2; 200 microM) was measured by single cell gel electrophoresis (SCGE). Tissue levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-Oxo-dG) were measured by HPLC with EC detection. D alpha-tocopherol and lipid peroxidation products (thiobarbituric acid reactive substances; TBARS) were measured by HPLC. Rat plasma pyruvate kinase and the production of reactive oxygen by phagocytes were detected spectrophotometrically and by flow cytometry respectively. Immortalised human colon epithelial cells (HCEC) were preincubated in vitro with the anthocyanins cyanidin and cyanidin-3-glycoside and the flavonol quercetin (all 50 microM) before exposure to H2O2 (200 microM). DNA damage was measured by SCGE as above., Results: Plasma and liver d alpha-tocopherol declined progressively over 12 weeks in rats made vitamin E deficient. Lipid peroxidation was increased significantly in plasma, liver and red cells. Reactive oxygen levels in phagocytes and plasma pyruvate kinase were increased. Vitamin E deficiency did not affect DNA stability in rat lymphocytes, liver or colon. Cyanidin-3-glycoside did not alter lipid peroxidation or DNA damage in rats. However, it was chemoprotective against DNA damage in human colonocytes.DNA strand breakage was decreased 38.8 +/- 2.2% after pretreatment with anthocyanin., Conclusion: While it is accepted that vitamin E alters lipid oxidation in vivo, its role in maintaining DNA stability remains unclear. Moreover, whereas cyanidin-3-glycoside protects against oxidative DNA damage in vitro, at nutritionally relevant concentrations it is ineffective against oxidative stress in vivo.

The development of colon cancer is characterised by alterations in multiple genetic and epigenetic pathways in colon tissue leading ultimately to deregulation of colon epithelial cells. Early detection is an important factor in decreasing colon cancer deaths. Proteomic techniques were used to identify potential early markers in colon tissue exhibiting pre-cancerous activity that may characterise pathological changes in a chemically induced colon cancer rat model. Protein profiles were assessed in soluble and insoluble fractions prepared from distal colon of rats treated with the colonotropic carcinogen, dimethylhydrazine. Alterations in protein profiles were associated with the presence of aberrant crypt foci, hyperplasia and dysplasia, microanatomical changes, and metabolic changes in rat colon. These changes may have a potential role in the identification of pre-pathological features preceding colon tumorigenesis.

Inflammatory disorders of the bowel and colon cancer are associated with elevated indices of oxidative stress. Analogous elevations in markers of oxidative stress and loss of cell-membrane integrity are also observed in the colons of rats deficient in vitamin E (D-alpha-tocopherol), the major lipid-soluble antioxidant in biological systems. The causal relationship between colon pathologies associated with oxidative stress and dietary deficiency in antioxidant vitamins such as vitamin E is still uncertain. Investigation of potential mechanisms by which lack of dietary vitamin E may lead to clinically relevant pathological changes in colon tissue was conducted using gene expression profiling strategies on vitamin E-sufficient and -deficient rats. Morphological changes and increased indices of lipid peroxidation were linked to vitamin E deficiency. These changes in colon tissue are potentially important in disease pathogenesis of the colon linked with oxidative stress or other direct consequences of inadequate levels of vitamin E.

The flavonol myricetin, reacts with oxygen-centred galvinoxyl radicals 28 times faster than d-alpha-tocopherol (vitamin E), the main lipid-soluble antioxidant in biological membranes. Moreover, each myricetin molecule reduces twice as many such radicals as vitamin E. However, myricetin fails to protect vitamin E-deficient microsomes from lipid peroxidation as assessed by the formation of thiobarbituric acid reactive substances (TBARS). Novel and potentially therapeutic antioxidants have been prepared that combine the radical-scavenging ability of a myricetin-like head group with a lipophilic chain similar to that of vitamin E. C(6)-C(12) alkyl chains are attached to the A-ring of either a 3,3',4',5'-tetrahydroxyflavone or a 3,2',4',5'-tetrahydroxyflavone head group to give lipophilic flavonoids (C log P = 4 to 10) that markedly inhibit iron-ADP catalysed oxidation of microsomal preparations. Orientation of the head group as well as total lipophilicity are important determinants of antioxidant efficacy. MM2 models indicate that our best straight chain 7-alkylflavonoids embed to the same depth in the membrane as vitamin E. The flavonoid head groups are prepared by aldol condensation followed by Algar-Flynn-Oyamada (AFO) oxidation or by Baker-Venkataraman rearrangement. The alkyl tails are introduced by Suzuki or Negishi palladium-catalysed cross-coupling or by cross-metathesis catalysed by first generation Grubbs catalyst, which tolerate phenolic hydroxyl and ketone groups.

Anthocyanins are secondary plant metabolites responsible for the blue, purple, and red color of many plant tissues. The phenolic structure of anthocyanins conveys marked antioxidant activity in model systems via donation of electrons or hydrogen atoms from hydroxyl moieties to free radicals. Dietary intakes of anthocyanins may exceed 200 mg/day, however, little is known about their antioxidant potency in vivo. Consequently, the aim of this study was to establish whether anthocyanins could act as putative antioxidant micronutrients. Rats were maintained on vitamin E-deficient diets for 12 weeks in order to enhance susceptibility to oxidative damage and then repleted with rations containing a highly purified anthocyanin-rich extract at a concentration of 1 g/kg diet. The extract consisted of the 3-glucopyranoside forms of delphinidin, cyanidin, petunidin, peonidin, and malvidin. Consumption of the anthocyanin-repleted diet significantly improved (p <.01) plasma antioxidant capacity and decreased (p <.001) the vitamin E deficiency-enhanced hydroperoxides and 8-Oxo-deoxyguanosine concentrations in liver. These compounds are indices of lipid peroxidation and DNA damage, respectively. Dietary consumption of anthocyanin-rich foods may contribute to overall antioxidant status, particularly in areas of habitually low vitamin E intake.

Quercetin is a plant polyphenol which is present in the diet as an aglycone and as sugar conjugates. Despite potent vasodilatory and antioxidant effects in vitro, destruction by intestinal organisms has been assumed to limit its nutritional relevance in the rat. However, we have refined extraction techniques using beta-glucuronidase followed by acid hydrolysis. Following this with HPLC methodology with post-column derivatisation, we have detected significant concentrations of quercetin and its metabolite, isorhamnetin, in tissues of rats maintained on quercetin-rich diets. Percentage recoveries are greater than 95% and intra-batch variation does not exceed 7% suggesting that the method may be useful in further studies of the biological role of this flavonoid.

Phytoestrogens (PEs) are diphenolic compounds from plants which can bind to estrogen receptors and have estrogen and antiestrogen effects in man and animals. Like other plant phenolics, PEs may have antioxidant properties through hydrogen/electron donation via hydroxyl groups. They might therefore act as free radical scavengers and inhibit development of coronary heart disease and cancers. The hydrogen-donating ability of a range of phytoestrogens was assessed using electron spin resonance spectroscopy, the ferric-reducing ability of plasma assay, and the Trolox equivalent antioxidant capacity. In addition, the ability of compounds to inhibit lipid peroxidation was examined in vitamin E-deficient liver microsomes. Genistein had the highest activity of the isoflavones; however, the isoflavones were relatively poor hydrogen donors compared with the other estrogenic compounds examined. Coumestrol and equol were more effective antioxidants than genistein but had relatively limited activity in comparison with Trolox. The only estrogenic compound with significant antioxidant activity was kaempferol which is better known as a dietary antioxidant than a phytoestrogen. As the concentrations of PEs used in this study exceed the estimated serum concentrations, their relatively poor antioxidant ability in vitro may indicate little significance as antioxidants in vivo., (Copyright 1998 Academic Press.)

1. During all aerobic metabolism, free radicals generated by the partial reduction of oxygen are potentially injurious to cells. Highly efficient antioxidant defence systems exist to inhibit oxidative damage to cellular lipids and proteins. Specific enzymes have a crucial role in these antioxidant defences, and their activity may be induced by regulatory mechanisms that respond to oxygen metabolite concentration. 2. To assess whether smoking induces an additional adaptive response, we compared antioxidant defence systems in erythrocytes from smokers and non-smokers and assessed whether a high intake of vitamin E (280 mg/day), a major lipophilic free-radical-scavenging antioxidant, affects the activity of antioxidant enzymes. 3. A total of 100 men, 50 smokers and 50 non-smokers, were allocated to four treatment groups in a 2 x 2 factorial design (smokers versus non-smokers and placebo versus vitamin E). For 10 weeks each subject took one capsule per day of either 280 mg dl-alpha-tocopherol acetate or a visually identical placebo (hydrogenated coconut oil with negligible vitamin E content). 4. Despite increased erythrocyte cytosolic antioxidant enzyme activities in smokers compared with non-smokers, erythrocytes from smokers were more susceptible to hydrogen peroxide-induced lipid peroxidation in vitro. 5. Vitamin E supplementation further increased erythrocyte catalase (EC 1.11.1.6) activity in both smokers and non-smokers (P < 0.001) and erythrocyte glutathione peroxidase (EC 1.11.1.9) and glutathione reductase (EC 1.6.4.2) activities in non-smokers (P < 0.001). After supplementation with vitamin E there was a concomitant fall in erythrocyte superoxide dismutase (EC 1.15.1.1) activity (P < 0.001) and total glutathione concentration (P < 0.01). Furthermore, in both smokers and non-smokers there was a significant decrease in the susceptibility of erythrocytes to peroxidation (P < 0.001). 6. Various endogenous and exogenous factors exert control over cellular protection against reactive oxygen species, and our data suggest that one such factor is the supply of vitamin E.

Homogenates of cardiac left ventricle from malignant hyperthermia-susceptible (MHS) pigs produced a circa 72% more pentane than those from malignant hyperthermia-resistant (MHR) animals, indicating enhanced peroxidation of n-6 fatty acids. This is consistent with the observed circa 70% decrease in total phospholipid polyunsaturated fatty acids (PUFA) in MHS compared with MHR tissue, a decrease mainly due to the quantitatively greater loss of n-6 PUFA. Although the percentage loss of n-3 PUFA was greater than that of n-6 PUFA (90% vs 60%), absolute amounts were insufficient to register as ethane production. Three-fold greater phospholipid content of MHS compared with MHR ventricles indicates reduced neutral lipid content probably due to increased catecholamine stimulation. These findings were associated with a small but significant decrease in superoxide dismutase activity in MHS tissues.

Ex vivo studies have been carried out on roe and red deer erythrocytes using electron spin resonance spectroscopy. Free radical formation in cells challenged with the brassica-derived haemolysin, dimethyldisulphide, was measured using spin trapping techniques. Significantly greater amounts of radical were trapped in the roe deer cells which may relate to differences in the antioxidant profile of the two genotypes. Results suggest that roe deer have a greater risk of developing oilseed rape poisoning than red deer.

We tested the antioxidant hypothesis of coronary heart disease (CHD) by comparing blood antioxidants, indices of lipid peroxidation and classic (CHD) risk factors of 25 subjects with stable angina pectoris with 200 matched controls. Angina subjects had significantly increased plasma concentrations of total cholesterol, low density lipoproteins and triglycerides although body mass index, plasma cotinine concentration and blood pressure were similar to those of the control group. Plasma concentrations of vitamin A, vitamin C and cholesterol- adjusted vitamin E did not differ between the groups although subjects with angina had significantly decreased plasma uric acid concentrations and elevated indices of lipid peroxidation. Although the results are compatible with the antioxidant hypothesis, it is unclear whether the increased oxidative stress in angina sufferers is a cause or consequence of the disease.

Finnish Landrace sheep with a genetic lesion which results in restricted cysteine transport across the red cell membrane have total glutathione concentrations in their red blood cells that are approximately 40% of those in normal sheep of the same breed. However, dimethyldisulphide-challenged red blood cells from both phenotypes produce an ESR-spin adduct at similar rates. The resistance of the low glutathione phenotype red cells to oxidant challenge is reflected by increases in the activities of antioxidant enzymes. Sheep with a genotypic disorder in cysteine transport may be a suitable model for studying the genetic expression of antioxidant enzymes in response to oxidant loads.

Homogenates of semitendinosus muscle from malignant hyperthermia (MH)-susceptible pigs produced threefold more pentane than those from MH-resistant pigs, indicating enhanced free radical-mediated peroxidation of n-6 fatty acids. This did not reflect a deficiency in tissue antioxidants or antioxidant-enzymes but glutathione concentrations and glutathione peroxidase activities were increased in the tissue from MH-susceptible swine, consistent with an adaptive response to a sustained oxidant stress. A lower proportion of linoleic acid (18:2 n-6) in phospholipids and neutral lipids in muscle from MHS pigs indicated increased peroxidation or metabolism (desaturation and elongation). The increased oleic acid (18:1 n-9) in the MHS muscle indicated that desaturase activity was elevated in all lipid classes. The results are consistent with the hypothesis that enhanced free radical activity and lipid peroxidation contributes to the abnormalities in Ca2+ homeostasis and polyunsaturated fatty acid metabolism in MH.

The concentrations of thiobarbituric acid reactive substances and conjugated dienes in human plasma are often used as indices of lipid peroxidation. However, concentrations of thiobarbituric acid reactive substances in plasma are markedly affected by the iron content of reagents used in the analysis and by storage of samples at -70 degrees C. The assay also has a large interbatch coefficient of variation (14%). Plasma concentrations of conjugated dienes are not affected by storage and the coefficient of variation is only 4%. However, there is a marked diurnal variation in levels of conjugated dienes which is similar to the changes in concentrations of plasma triglycerides. Precise standardisation of analytical procedures is required before these assays can be reliably used in clinical medicine.

1. Blood antioxidants were measured in venous blood samples from 20 runners and six sedentary individuals. All subjects were male, between 20 and 40 years old, and in steady state with respect to body weight and physical activity patterns. Dietary analysis was undertaken using a 7-day weighed food intake. Correlations were sought between antioxidants in blood and (1) weekly training distance and (2) maximum oxygen uptake. In addition, 12 runners could be classified into two groups undertaking either low (range 16-43 km, n = 6) or high (80-147 km, n = 6) weekly training. 2. Body weight (range 55.3-90.0 kg) and percentage body fat (range 7-19%) both showed negative correlations with the weekly training distance (both P less than 0.001). Energy intake and maximum oxygen uptake were both correlated with the weekly training distance (both P less than 0.001). 3. Plasma creatine kinase activity, an indicator of muscle damage, was significantly correlated with the weekly training distance (P less than 0.01), whereas the plasma concentration of thiobarbituric acid-reactive substances, an indicator of free-radical-mediated lipid peroxidation, decreased with increased maximum oxygen uptake (P less than 0.01). 4. Erythrocyte alpha-tocopherol content was greater in the two running groups (P less than 0.05) compared with the sedentary group, and lymphocyte ascorbic acid concentration was significantly elevated in the high-training group (P less than 0.01) compared with the low-training group. 5. Erythrocyte activities of the antioxidant enzymes, glutathione peroxidase and catalase, were significantly and positively correlated with the weekly training distance (P less than 0.01 and P less than 0.05, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

On incubation with the spin trap alpha-(4-pyridyl-l-oxide)-N-tert-butylnitrone (4-POBN), a characteristic electron spin resonance (ESR) signal was produced at a greater rate in hepatic microsomal fractions from malignant hyprthermia susceptible (MHS) pigs compared with resistant (MHR) pigs. This was accompanied by increased formation of thiobarbituric acid reactive substances (TBARS). Supplementation of diets for six weeks with 235 mg alpha-tocopherol acetate/kg significantly increased microsomal vitamin E content of both pigs types. Moreover, the rate of formation of TBARS and ESR signal height of incubated microsomes from supplemented MHS pigs was decreased to that of MHR pigs. Elevated pyruvate kinase activities and TBARS concentrations in plasma of MHS pigs were also moderated by dietary vitamin E. Vitamin E supplementation may decrease the peroxidative events associated with MH.

NADPH-induced lipid peroxidation of hepatic microsomes from vitamin E-deficient rats has been used to assess the antioxidant effectiveness of dl alpha, d alpha- and gamma-tocopherol. When the tocopherols were added in ethanol to microsomes, the degree of inhibition of formation of thiobarbituric acid reactive substances (TBARS) decreased in the order dl alpha- greater than d alpha- greater than gamma-tocopherol. This reflected the difference in the solubility of the tocopherols in the microsomes, dl alpha-tocopherol being the most soluble and gamma-tocopherol the least. Using inhibition of TBARS produced per tocopherol content in microsome as a measure of antioxidant potency, the effectiveness of the isomers was gamma- greater than d alpha- greater than dl alpha. Despite addition of pharmacological concentrations of the isomers, it was not possible to inhibit lipid peroxidation to the same levels as were found in microsomes from vitamin E sufficient animals. Use of ethanol as a vehicle may not allow optimum orientation of the tocopherols into the lipid bilayer.

The relationship between prolonged exercise, oxidative stress, and the protective capacity of the antioxidant defense system has been determined. Venous blood samples were removed from seven trained athletes before and up to 120 h after completion of a half-marathon for measurements of blood antioxidants, antioxidant enzymes, and indices of lipid peroxidation. Plasma creatine kinase (CK) activity, an index of muscle damage, increased (P less than 0.05) to a maximum 24 h after the race but this was not accompanied by changes in conjugated dienes and thiobarbituric acid reactive substances (TBARS), which are indices of lipid peroxidation. An increase (P less than 0.05) in plasma cholesterol concentration (4%) immediately after the race was similar to the change in plasma volume (6%). However, transient increases (P less than 0.05) immediately postrace in the plasma concentrations of uric acid (24%), vitamin A (18%), and vitamin C (34%) were only partly accounted for by the fluid shifts. The immediate postrace increases in alpha- and gamma-tocopherol did not attain statistical significance. Erythrocyte antioxidant enzyme activities were unaffected by the exercise but the alpha- and gamma-tocopherol concentrations progressively increased (P less than 0.001 and P less than 0.05, respectively) up to 48 h postrace. Paradoxically, 24 h after the race erythrocyte susceptibility to in vitro peroxidation was markedly elevated (P less than 0.01). This enhanced susceptibility to peroxidation was maintained even at 120 h postrace and did not correspond to changes in the age of the red cell population. A decrease (P less than 0.001) in total erythrocyte glutathione immediately after the half-marathon was mainly due to a reduction in the reduced form (GSH). The results show that when trained athletes run a comparatively short distance sufficient to result in some degree of muscle damage but which is insufficient to cause elevations in plasma indices of lipid peroxidation, significant alterations in erythrocyte antioxidant status do occur.

Microsomes were prepared from livers of malignant hyperthermia susceptible (MHS) or resistant (MHR) pigs. On incubation with the spin trap alpha-(4-pyridyl-l-oxide)-N-tert-butylnitrone (4-POBN), the microsomes from MHS pigs produced a characteristic electron spin resonance (ESR) signal at a greater rate than those from MHR pigs. Increased formation in the incubations of thiobarbituric acid reactive substances (TBARS) by the microsomes of the MHS pigs indicated an enhanced susceptibility to free radical-mediated lipid peroxidation. These results provide further evidence that MHS pigs have an antioxidant abnormality which may contribute to the fatal MH response. However the nature of the abnormality is unclear. The enhanced formation of unstable free radicals and indices of lipid peroxidation was not due to decreased vitamin E concentration or glutathione peroxidase activity in the microsomes. Furthermore, fatty acid profiles were similar in microsomes from MHS and MHR pigs indicating similar amounts of potential substrate for TBARS formation.

The abilities of pig liver (copper, zinc) metallothionein I and rat liver zinc metallothionein II to modify lipid peroxidation in incubations of liver microsomes have been compared with the activities of reduced glutathione, mannitol, quinacrine, EDTA, dimethyl-pyrroline-N-oxide and phenyl-butyl-nitrone. Lipid peroxidation was determined by assay of thiobarbituric acid reactive substance formation in incubations of microsomes with iron/ADP or a mixture of xanthine and xanthine oxidase. Zinc metallothionein II had no effect on the extent of peroxidation in either system but (copper, zinc) metallothionein I caused a stimulation of peroxidation initiated by xanthine and xanthine oxidase, all other compounds tested were inhibitory. Gel exclusion chromatography of incubations of (copper, zinc) metallothionein I with xanthine and xanthine oxidase revealed aggregation of the metalloprotein. This may have exposed copper in a form capable of initiating peroxidation.

Selenium deficient calves when compared to selenium supplemented calves had increased plasma thyroxine concentrations and decreased plasma tri-iodothyronine concentrations. These changes in the selenium deficient calves were accompanied by significant increases in plasma urea and creatinine concentrations and decreased plasma alkaline phosphatase activity. The demonstration that low selenium status can cause imbalances in thyroid hormone metabolism may provide an explanation for some of the effects of the deficiency.

A sensitive method is described for the determination of Mo in plasma or serum by graphite furnace atomic absorption spectrometry. The method involves extraction of the metal as the 8-hydroxyquinoline complex and is free of the interference effects that prevent the direct analysis of plasma for Mo. Recoveries of internal standards were excellent and results from the analysis of a National Institute of Standards and Technology Standard Reference Material were in good agreement with certified values. The sensitivity of the method, based on the analysis of 1 ml of plasma, is ca. 3 ng ml-1.

The benefits of the inclusion of cobalt and selenium supplements in anthelmintic preparations were demonstrated in a 10 week trial with cobalt- and selenium-deficient blackface wethers. The anthelmintics were based on oxfendazole and on levamisole plus oxyclozanide; three doses provided, in total, 38 mg cobalt and 7.2 or 11.3 mg selenium. Administration of the supplements prevented the weight loss and reduction in food intake observed in unsupplemented animals. Blood glutathione peroxidase activities were restored to normal and increases in serum vitamin B12 levels were observed which were consistent with the prevention of both cobalt and selenium deficiencies.

Effective control of copper poisoning in sheep was obtained by the subcutaneous injection of ammonium tetrathiomolybdate. Three doses, each of 3.4 mg/kg bodyweight, were given on alternate days. This treatment caused a substantial reduction in liver copper content and in liver damage. It also decreased the mortality rate in animals that had developed the haemolytic crisis. The subcutaneous route is as effective as the intravenous route and is more convenient. No adverse side-effects of the treatment were observed.

Selenium (Se) deficiency in rats produced significant increases in the activity of hepatic glutathione S-transferase (GST) with 1-chloro-2,4-dinitrobenzene as substrate and in various GST isoenzymes when determined by radioimmunoassay. These changes is GST activity and concentration were associated with Se deficiency that was severe enough to provoke decreases of over 98% in hepatic Se-containing glutathione peroxidase activity (Se-GSHpx). However, decreases in hepatic Se-GSHpx of 60% induced by copper (Cu) deficiency had no effect on GST activity or concentration. Increased GST activity in Se deficiency has previously been postulated to be a compensatory response to loss of Se-GSHpx, since some GSTs have a non-Se-glutathione peroxidase (non-Se-GSHpx) activity. However, the GST isoenzymes determined in this study, GST Yb1Yb1, GST YcYc and GST YaYa, are known to have up to 30-fold differences in non-Se-GSHpx activity, but they were all significantly increased to a similar extent in the Se-deficient rats.

Selenium (Se) deficiency produced up to a 14-fold decrease in hepatic tri-iodothyronine (T3) production from thyroxine (T4) in vitro. The T3 production rate could not be restored by the addition of a variety of cofactors, nor by the addition of control homogenate. The impairment in hepatic T3 production observed in Se deficiency was reflected in the concentrations of thyroid hormones circulating in plasma, T4 being increased approx. 40% and T3 being decreased by 30%. However, the fall in plasma T3 concentrations was smaller than might be expected in view of the marked decreased in T3 production. Se deficiency had no measurable effect on plasma reverse-tri-iodothyronine concentrations. The data suggest that Se deficiency produces an inhibition of both 5- and 5'-deiodination, consistent with the widely held view that these reactions are catalysed by the same enzyme complex. The mechanism of inhibition appears not be mediated by changes in thiol levels, but a direct role of Se in the activity of the deiodinase complex cannot be excluded.

Copper deficiency in rats increased renal vein and arterial (heart) plasma GSH concentration by approximately 50%. There was no change in plasma GSSG concentration. Renal vein plasma GSSG/GSH ratio was decreased in copper deficiency, which is consistent with previous reports showing a copper-dependent thiol oxidase activity in the renal basement membrane. No change occurred in arterial plasma GSSG/GSH ratio. Hepatic GSH concentrations were also elevated by 50% in copper deficiency, GSSG concentrations were unaffected, but GSSG/GSH ratio was depressed. Renal and cardiac tissue GSH and GSSG were unaffected by copper deficiency. The decreased SOD activity and GSH-Px activity observed in copper deficiency may contribute to increased hepatic and plasma GSH concentrations.