Virginia C. Morris

Urinary Malondialdehyde-Equivalents during ingestion of meat cooked at high or low temperatures

Excretion of malondialdehyde (MDA)-generating substances in the urine has been suggested as an indicator ofin vivo lipid peroxidation. However, MDA in the urine also reflects the amount of lipid peroxidation products consumed in the diet. We determined MDA as the thiobarbituric acid (TBA)-MDA complex in urine of 19 healthy adults (10 male and 9 female) fed large quantities (3.6–4.1 g/kg body weight) of ground beef cooked at a low or a high temperature. Subjects are a controlled diet with no alcohol or nutritional supplements. For 7 d they consumed ground beef cooked at 100°C for 20 min (low-temperature meat) followed by 7 d with meat fried at 250°C for 22 min (high-temperature meat). Prior to the study, subjects consumed their normal free choice diet with moderate amounts of meat. The concentration of MDA in urine at baseline was 2.1±0.3 μmol TBA-MDA equivalents/day (mean±SEM). After 7 d of low-temperature meat, urinary TBA-MDA equivalents increased to 23.1±1.4 μmol/d. Urinary TBA-MDA equivalents were consistently lower (6.9–8.0 μmol/d) 1, 2, 3, and 7 d after subjects changed to high-temperature meat. After 7 d of treatment, 97% of the MDA-equivalents in the meat was recovered in 24-h urine samples. The low temperature meat had 3–4 times more MDA than did the high-temperature meat. These data indicate that the amount of meat eaten and the cooking procedures used can dramatically alter urinary MDA. Dietary sources of MDA must be controlled if urinary MDA is to be used as an indicator of oxidative stress.

Rapid determination of glutathione peroxidase and thioredoxin reductase activities using a 96-well microplate format : Comparison to standard cuvette-based assays

Gluthatione peroxidase and thioredoxin reductase are selenocysteine-containing enzymes that are constituents of the cellular antioxidant defense system. Conventional cuvette-based assays for glutathione peroxidase and thioredoxin reductase enzymes are laborious and time consuming. The ability to assay their activities rapidly in multiple samples would aid efforts focused on understanding the impact of these enzymes on the cellular antioxidant defense system. High throughput can be achieved with assays adapted to work in a clinical analyzer but require expensive equipment. Assays designed to work in a 96-well microplate reader provide an alternative methodology for high throughput with reduced instrumentation cost. However, due to differences in the light pathlength when using a 96-well format, the values obtained cannot be compared directly with those obtained using a 1-cm cuvette. Described here are assays for glutathione peroxidase and thioredoxin reductase modified to work in a 96-well format that incorporates light pathlength determinations into the assays. The values obtained using a high throughput 96-well format in conjunction with pathlength determinations are in agreement with those obtained using a standard 1-cm cuvette. While spectrophotometrically derived pathlengths are the most accurate, calculated pathlengths based on assay volume and well size can be used with only a small amount of error introduced. This method can also be applied to many other enzyme assays, thus allowing the rapid analysis of large numbers of samples without the need for expensive equipment.

The urinary excretion of thiobarbituric acid reactive substances and malondialdehyde by normal adult males after consuming a diet containing salmon

In this study we investigated the output of thiobarbituric acid reactive substances (TBARS) and malondialdehyde (MDA), as thiobarbituric acid (TBA)-MDA adduct, in the urine from subjects eating a diet in which the only source of n−3 long-chain, polyunsaturated fatty acids was fresh salmon. Nine healthy men, ages 30–65, were confined in the United States Department of Agriculture Western Human Nutrition Research Center, San Francisco, CA, for 100 d; food intake and exercise levels were controlled. All subjects were placed on a stabilization diet (StD) for 20 d, then six were fed the salmon diet for 40 d. The others remained on the StD. The groups switched diets for the last 40 d. Both diets were isocaloric (16% protein, 54% CHO and 30% fat by energy %). The salmon diet contained 7.5% of calories from n−6 fatty acids (FAs) and 2% from n−3 FAs, primarily eicosapentaenoic acid and docosahexaenoic acid in a 50∶60 ratio, while the StD contained 7.5% from n−6 FAs and <0.3% n−3 FAs (with presumably no significant amounts of C20 or C22 n−3 FAs). Twenty-four hour urinary output was collected, and 2% 3−d pool samples prepared for analysis of urinary TBARS and the TBA-MDA adduct. The total urinary output of each individual varied considerably, and on a daily basis the concentration of autoxidation products in an individuals urine varied also. However, the mean daily output (in μmoles TBA-MDA equivalents/day) at the end of the salmon diet feeding period was significantly greater (7.05±1.33 TBARS,P<0.05; and 7.07±1.73 TBA-MDA adduct,P<0.01) compared to when the subjects were eating the StD (5.65±1.09 TBARS and 4.65±0.76 TBA-MDA adduct). When the TBARS and TBA-MDA adduct values were normalized relative to creatinine output (in nmoles TBA-MDA equivalents/μmole creatinine), the data achieved even greater statistical significance. The mean output of the group eating the salmon diet was 0.478±0.076 for TBARS (P<0.01) and 0.476±0.082 for the TBA-MDA adduct (P<0.001)vs. 0.345±0.059 for TBARS and 0.283±0.041 for the TBA-MDA adduct when the subjects were consuming the StD. Thus, the consumption of cooked fish may increase ones exposure to MDA and other autoxidation products, compounds that may be carcinogenic or mutagenic.

Plasmodium yoelii: comparative antimalarial activities of dietary fish oils and fish oil concentrates in vitamin E-deficient mice.

Feeding vitamin E-deficient diets containing either fish oils such as menhaden, salmon, or anchovy oil or fish oil concentrates based on n-3 ethyl esters or free fatty acids protected mice against Plasmodium yoelii as indicated by decreased parasitemia and improved survival. The fish oil concentrates depressed plasma tocopherol levels more strongly in vitamin E-supplemented mice than the menhaden oil. The free fatty acid concentrate appeared to suppress parasitemia in vitamin E-deficient mice better than the menhaden oil, although ultimate survival was similar in both groups. Dietary manipulation of host antioxidant status offers promise as a possible means of malaria control.

Characterization of the selenium in rat liver mitochondria as glutathione peroxidase

Summary A large part of the radioselenium in liver mitochondria prepared from rats given 0.1 ppm Se as Na 2 75 SeO 3 in the drinking water can be solubilized by a procedure combining extraction with hypotonic Tris buffer-ethanol and freeze-thawing. The radioselenium in this extract binds to DEAE-Sephadex but can be eluted as a single main radioactive peak with salt solution of relatively low ionic strength. Gel filtration of the radioactive peak fractions from the DEAE-Sephadex column through Sephadex G-150 yielded 2 radioactive peaks, the larger of which was associated with significant glutathione peroxidase activity. These findings are consistent with the concept that most of the selenium in mitochondria exists in the form of glutathione peroxidase.

Effect of dietary selenium on the development of Fusarium-induced tibial dyschondroplasia in broiler chickens.

A trial was conducted to determine the effects of dietary level of selenium on the pathogenesis of Fusarium-induced tibial dyschondroplasia (FITD) in broiler chicks, and to assess the applicability of FITD as an animal model of Kashin-Beck disease of humans. Day-old female broilers were fed diets that were deficient in selenium (0.02 ppm Se), adequate in selenium (0.15 ppm Se), or generous in selenium (0.50 ppm Se). TDP-1, the toxic component of the fungus, was administered to 15 of 26 chicks in each dietary group starting at 1 week of age and continuing until the chicks were killed at 24-30 days of age. Plasma selenium levels and hepatic glutathione peroxidase activity were significantly lower in the selenium-deficient group than in other dietary groups; these parameters were not affected by treatment with TDP-1. The mortality rate of the TDP-1-treated selenium-generous group was significantly less than that in the other TDP-1-treated groups, but there were no differences in the incidence, severity, or character of the FITD lesions among the groups. Thus, the interaction of selenium and TDP-1 did not include an effect on FITD.

MORTALITY IN MICE INFECTED WITH AN AMYOCARDITIC COXSACKIEVIRUS AND GIVEN A SUBACUTE DOSE OF MERCURIC CHLORIDE

An amyocarditic strain of coxsackievirus B3 (CVB3/0) induces heart damage when inoculated into selenium (Se)-deficient mice. Mercury (Hg), an Se antagonist, is known to aggravate viral infections. The experiments reported here assessed the effect of prior Hg treatment in mice subsequently inoculated with an amyocarditic strain of coxsackievirus. A pilot study showed that under our conditions the maximum tolerated dose of HgCl2 in uninfected mice was 6 mg HgCl2/kg body weight. In the main study, doses of 0, 3 or 6 mg HgCl2/kg body weight were administered intraperitoneally (ip) to 7-wk-old male mice fed a standard chow diet. Two hours later, half the mice were inoculated ip with CVB3/0. Ten days postinoculation, no mortality was observed in mice given only virus. In mice not given virus, 10% injected with 6 mg HgCl2/kg body weight died. On the other hand, 64% of the mice given both virus and 6 mg HgCl2/kg body weight died. Fifteen percent of the hearts from virus-infected mice given 3 mg HgCl2/kg body weight and 33% of the hearts from virus-infected mice given 6 mg HgCl2/kg body weight exhibited a higher incidence of lesions than hearts from mice-given virus alone. Moreover, viral heart titers were elevated in infected mice injected with 6 mg HgCl2/kg body weight compared to infected mice receiving no Hg. Thus, an amyocarditic coxsackievirus given to mice after a nonlethal subacute dose of Hg results in mortality, increased incidence of heart lesions, and elevated viral heart titers. These results demonstrate the important role of toxic elements in determining the severity of viral infections.

Nutritional availability to rats of selenium in four seafoods: crab (Callinectes sapidus), oyster (Crassostrea virginica), shrimp (Penaeus duorarum) and Baltic herring (Clupea harengus).

1. The present study was conducted to determine the biological availability to rats of the selenium in four high-Se seafoods: crab (Callinectes sapidus), oyster (Crassostrea virginica), shrimp (Penaeus duorarum) and Baltic herring (Clupea harengus). 2. Weanling male rats were fed on a Se-deficient Torula yeast diet for 4 weeks followed by either continued depletion or repletion for 4 weeks with 0.05, 0.1 or 0.2 microgram Se as selenite/g, or 0.1 or 0.2 microgram Se as freeze-dried cooked test food/g. Plasma and liver Se levels or glutathione peroxidase (EC 1.11.1.9; GSH-Px) activities were used as indicators of body Se status. 3. Except for oysters, the biological availability of Se in all these seafoods was close to that of selenite (selenite 100%) when the criterion used was either plasma Se level or plasma GSH-Px activity. 4. By the criterion of increased liver Se level of restored hepatic GSH-Px activity, only herring-Se had a biological availability comparable to that of selenite-Se under all conditions tested, whereas crab-Se and oyster-Se were distinctly inferior in this regard. 5. Increasing the amount of crab-Se, oyster-Se or shrimp-Se supplied in the diet from 0.1 to 0.2 microgram/g changed the apparent availability (%) of Se for hepatic GSH-Px restoration from 38 to 78, 22 to 53 and 57 to 90 respectively. 6. The present study demonstrates that the availability of Se in certain foods is a function of the criterion chosen, the level of Se supplied in the diet, and possibly other unknown interacting dietary factors.

Effect of selenium deficiency on liver iron stores in mice

Excess Fe accumulation has been associated with increased risk of chronic disease in humans. Others have shown that rats fed Se deficient diets containing normal Fe levels accumulate excess hepatic Fe. The purpose of this study was to compare the effect of Se deficiency on Fe accumulation in mice fed adequate or high Fe diets. Sixty-four weanling male mice were divided into 2 groups and fed either a Se deficient diet or the same diet supplemented with 0.2 μg Se/g diet added as sodium selenite. Half the mice in each group consumed diets supplemented with adequate (35 μg Fe/g) or high (1050 μg Fe/g) Fe added as ferric citrate. Mice were fed diets over a 4 or 12 week period. Mice fed the high Fe diets had increased liver Fe stores while mice fed the Se deficient diets had decreased liver glutathione peroxidase (GPX1) activity after both 4 and 12 weeks. After 4 weeks, Se deficiency had a significant (P = 0.048) effect on liver Fe stores. Mice fed Se deficient diets had elevated liver Fe concentration compared to mice fed Se adequate diets although differences between individual diets were not significant. After 12 weeks, however, Se deficiency had no effect on liver Fe stores. Mice fed the Se deficient diet containing high Fe had elevated liver TBARS levels compared to mice fed the Se adequate diet containing adequate or high Fe after 4 weeks. Mice fed the Se deficient diet containing high Fe had elevated plasma cholesterol and triglyceride levels compared to mice fed the Se adequate diet containing high Fe after 4 weeks. Mice fed the Se deficient diet containing high Fe had decreased plasma triglyceride levels compared to mice fed the Se adequate diet containing adequate Fe after 12 weeks. Increased oxidative stress, a consequence of decreased Se status may affect liver Fe accumulation as well as plasma cholesterol and triglyceride levels.

Relative nutritional availability to rats of selenium in Finnish spring wheat (Triticum aestivum L.) fertilized or sprayed with sodium selenate and in an American winter bread wheat naturally high in Se

A Finnish national programme to fertilize crops with sodium selenate led us to compare the nutritional availability to rats of selenium in two Finnish spring wheats (Triticum aestivum L.), either fertilized or sprayed with sodium selenate, with that in an American winter bread wheat naturally high in Se. Weanling male rats were given a Se-deficient Torula yeast diet for 4 weeks followed by either continued depletion or repletion for 4 weeks with graded levels of Se as sodium selenite (standard) or wheat (test food). Plasma and liver Se levels and plasma and liver glutathione peroxidase (EC 1.11.1.9; GSH-Px) activities were used as criteria of body Se status. The availability of Se under these conditions was calculated with the point-slope technique at two dietary levels of Se (Expt 1) and with the slope-ratio method (Expt 2). In the point-slope assay, the level of dietary Se fed had a considerable effect on the apparent availability values obtained which made interpretation of the results difficult. In the slope-ratio assay, no difference in the availability of Se from the various wheats was observed when plasma or liver Se levels were used as the response criteria. The Se in the fertilized wheat was somewhat more available than that in the sprayed wheat when plasma or liver GSH-Px activities were the response criteria. Overall, availability values (%) derived by averaging all four response criteria were 86, 77 and 73 for the fertilized and sprayed Finnish wheats and the American wheat respectively (sodium selenite 100). These results show that wheat is a relatively available source of Se to rats regardless of whether its Se content is naturally high or is increased by fertilization or spraying.