Variations among cultured cells in glutathione peroxidase activity in response to selenite supplementation.

The aim of this study was to devise conditions for manipulation of the activity of selenium-dependent glutathione peroxidase in cell lines by means of variation in culture medium contents of selenite and fetal calf serum. Nine different cell lines were studied. A low glutathione peroxidase activity was, in most cases, obtained by the use of a medium with a low (2%) serum content. Selenite induced in most of the cell lines an increase in glutathione peroxidase activity, with a plateau ranging from 10 nM to 300-1000 nM. Growth-retarding effects of selenite became apparent at 300-2000 nM, showing a large cell line variation. Supplementation with 50-100 nM selenite for 1 week should generally be suitable for maximal glutathione peroxidase induction. The selenium contents of serum batches were highly variable, pointing to the importance of using only one well-defined, preferably low-selenium, batch. The glutathione peroxidase activities varied considerably between cell lines and the selenite-induced increases ranged from negligible to more than 10-fold. The availability of cell lines with such variable responses should be valuable for experiments aimed at evaluating the importance of glutathione peroxidase and selenium compounds independently of glutathione peroxidase for the protection against oxidative insult.

Effect of dietary fatty acids on the postprandial fatty acid composition of triacylglycerol-rich lipoproteins in healthy male subjects.

OBJECTIVE: The aim of the present study was to investigate the effect of trans-18:1 isomers compared to other fatty acids, especially saturates, on the postprandial fatty acid composition of triacylglycerols (TAG) in chylomicrons and VLDL. DESIGN: A randomised crossover experiment where five interesterified test fats with equal amounts of palmitic acid (P fat), stearic acid (S fat), trans-18:1 isomers (T fat), oleic acid (O fat), or linoleic acid (L fat) were tested. SUBJECTS: A total of 16 healthy, normolipidaemic males (age 23+/-2 y) were recruited. INTERVENTIONS: The participants ingested fat-rich test meals (1 g fat per kg body weight) and the fatty acid profiles of chylomicron and VLDL TAG were followed for 8 h. RESULTS: The postprandial fatty acid composition of chylomicron TAG resembled that of the ingested fats. The fatty acids in chylomicron TAG were randomly distributed among the three positions in accordance with the distributions in test fats. Calculations of postprandial TAG concentrations from fatty acid data revealed increasing amounts up to 4 h but lower response curves (IAUC) for the two saturated fats in accordance with previous published data. The T fat gave results comparable to the O and L fats. The test fatty acids were much less reflected in VLDL TAG and there was no dietary influence on the response curves. CONCLUSIONS: The fatty acid composition in the test fats as well as the positional distributions of these were maintained in the chylomicrons. No specific clearing of chylomicron TAG was observed in relation to time.

New roles for quin2: powerful transition-metal ion chelator that inhibits copper-, but potentiates iron-driven, Fenton-type reactions.

The objective of this study was to investigate whether quin2, through its metal chelating properties, could affect copper- or iron-driven Fenton reactions. Chelation of ferric ion with quin2 uniformly strongly enhanced the formation of oxidizing species, detected with the DMSO and deoxyribose assays, both by H2O2 and a mixture of superoxide/hydrogen peroxide produced by hypoxanthine/xanthine oxidase. Fe(3+)-EDTA gave the same effects, but lacked reactivity with bolus H2O2 as detected with the DMSO assay. Whereas the formation of oxidizing species with Fe(3+)-EDTA and ferric ions alone were strongly inhibited by superoxide dismutase both in the bolus H2O2 and hypoxanthine/xanthine oxidase systems, such formation in the presence of Fe(3+)-quin2 either did not decrease or decreased only moderately. Fe(3+)-quin2 also strongly enhanced plasmid DNA strand breakage in the presence of H2O2. Our findings suggest that quin2 as chelator of ferric ion may be a more powerful enhancer of oxidant formation than other chelators so far tested. The formation of oxidizing species from copper ions and bolus H2O2 was found to be fundamentally dependent on the choice of buffer system. We could only detect significant amounts of oxidants in both assays in Hepes buffer, but not in the phosphate, cacodylate or unbuffered systems, which all gave low reactivity in the DMSO assay compared to the deoxyribose assay. Quin2 chelation of cupric ion effectively inhibited the formation of oxidants as well as plasmid DNA strand breakage.(ABSTRACT TRUNCATED AT 250 WORDS)

H2O2-driven reduction of the Fe3+-quin2 chelate and the subsequent formation of oxidizing species.

The objective of this study was to compare effects of quin2 and EDTA in iron-driven Fenton-type reactions. Seven different assays for detection of strong oxidants were used: the DMSO, deoxyribose, benzoate hydroxylation, and plasmid DNA strand breakage assays, detection of 8-oxo-deoxyguanosine in deoxyguanosine mononucleosides and calf thymus DNA, and electron spin resonance with the spin-trap (4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) in the presence of ethanol or DMSO. With H2O2 and Fe3+, quin2 generally strongly increased the formation of reactive species in all assays, whereas with EDTA the results varied between the assays from barely detectable to highly significant increases compared to H2O2 and unchelated Fe3+. We found that the species produced in the reaction between Fe3+-quin2 and H2O2 behaved like the hydroxyl radical in all assays, whereas with Fe3+-EDTA no clear conclusion could be drawn about the nature of the oxidant. The effect of quin2 on the formation of oxidants on Fe2+ autoxidation, varied from generally inhibiting to slightly promoting, depending on the assay used. EDTA had a promoting effect on the amount of oxidant detected by all but one assay. None of the autoxidation systems produced DMSO or ethanol radical adducts with 4-POBN. In the presence of either chelator, H2O2, and Fe2+ DMSO and ethanol radical adducts of 4-POBN were produced. Using the Fe2+ indicator ferrozine, evidence for direct reduction of Fe3+-quin2 by H2O2 was found. Superoxide anion radical appeared to be less efficient than H2O2 as reductant of Fe3+-quin2 as addition of superoxide dismutase in the ferrozine experiments only decreased the amount of Fe2+ available for Fenton reaction by 10-20%. The main conclusions from our study are that the reduction of Fe3+-quin2 can be driven by H2O2 and that Fe2+ in the following oxidation step produces a species indistinguishable from free hydroxyl radical.

Copper supplementation in humans does not affect the susceptibility of low density lipoprotein to in vitro induced oxidation (FOODCUE project).

The oxidative modification of low-density lipoprotein cholesterol (LDL) has been implicated in the pathogenesis of atherosclerosis. Copper (Cu) is essential for antioxidant enzymes in vivo and animal studies show that Cu deficiency is accompanied by increased atherogenesis and LDL susceptibility to oxidation. Nevertheless, Cu has been proposed as a pro-oxidant in vivo and is routinely used to induce lipid peroxidation in vitro. Given the dual role of Cu as an in vivo antioxidant and an in vitro pro-oxidant, a multicenter European study (FOODCUE) was instigated to provide data on the biological effects of increased dietary Cu. Four centers, Northern Ireland (coordinator), England, Denmark, and France, using different experimental protocols, examined the effect of Cu supplementation (3 or 6 mg/d) on top of normal Cu dietary intakes or Cu-controlled diets (0.7/1.6/6.0 mg/d), on Cu-mediated and peroxynitrite-initiated LDL oxidation in apparently healthy volunteers. Each center coordinated its own supplementation regimen and all samples were subsequently transported to Northern Ireland where lipid peroxidation analysis was completed. The results from all centers showed that dietary Cu supplementation had no effect on Cu- or peroxynitrite-induced LDL susceptibility to oxidation. These data show that high intakes (up to 6 mg Cu) for extended periods do not promote LDL susceptibility to in vitro-induced oxidation.

Zinc absorption from composite meals. II. Influence of the main protein source.

The absorption of zinc in man from composite meals of conventional foods with various main protein sources was measured with a radionuclide technique. The meals were extrinsically labeled with 65Zn and the absorption of zinc was determined from a measurement of the whole-body retention of the isotope about 2 weeks after ingestion of the labeled meal. The meals consisted of a meat patty made from either chicken, beef, or soybeans, and included potatoes, tomatoes, and white bread. For meals based on animal protein a positive correlation was found between zinc absorption and zinc content. When 25% of the animal protein in the chicken meal was replaced by defatted soy flour neither the zinc content nor zinc absorption were significantly influenced. The same substitution in a beef meal resulted in a decrease in zinc content and also a lower zinc absorption. The absorption from a soybean meal did not differ from an animal protein meal with the same zinc content. Lower zinc absorption was found when the calcium content of a soybean meal was increased by the addition of milk. For this study, the total zinc content of the meal was the most important factor influencing the amount of zinc absorbed.

Low-fat, high-fiber diet favorably affects several independent risk markers of ischemic heart disease: observations on blood lipids, coagulation, and fibrinolysis from a trial of middle-aged Danes.

We served a low-fat (28% of energy), high-fiber (3.3 g/MJ) diet according to Nordic nutrition recommendations (Rec diet), and a high-fat diet (39% of energy) corresponding to the average Danish diet (Dane diet) for periods of 2 wk in a randomized crossover study of 21 healthy middle-aged individuals. The Rec diet resulted in lower serum concentrations of low-density-lipoprotein (LDL) cholesterol (medians: 2.77 vs 3.04 mmol/L, P < 0.001) and high-density-lipoprotein (HDL) cholesterol (1.08 vs 1.24 mmol/L, P < 0.001), and higher fasting triglycerides (1.11 vs 0.86 mmol/L, P = 0.04) than did the Dane diet. Furthermore, the Rec diet lowered plasma factor VII coagulant activity (FVIIc) (88% vs 96%, P = 0.002) and raised plasma fibrinolytic activity. Our observations indicate that a low-fat, high-fiber diet may not only reduce the atherogenic but also the thrombogenic tendency of an individual compared with a diet corresponding to the average Danish diet.

The effect of individual components of soy formula and cows' milk formula on zinc bioavailability.

Zinc absorption from human milk, cows' milk formulas, and soy formulas was studied in human adults by a radioisotope technique using 65Zn and whole body counting. Individual dietary components were investigated for effects on zinc absorption. Phytate was found to have a strong inhibitory effect on zinc absorption; addition of phytate to cows' milk formula (yielding a phytate concentration similar to that of soy formula) resulted in a decrease in zinc absorption from 31 to 16% similar to the absorption for soy formula (14%). Carbohydrate source, calcium, and zinc levels of the diet did not affect zinc absorption significantly. Iron supplementation of cows' milk formula decreased zinc absorption from 24 to 18% although this decrease was not found to be significant (p less than 0.1). Absorption of zinc from a whey-adjusted cows' milk formula was higher (31%) than from a nonmodified cows' milk formula (22%). Increasing the zinc supplementation level in cows' milk formula but not in soy formula increased zinc absorption to approximate that from breast milk. It is suggested that reduction of phytate content of soy formula may be a more effective avenue of modification than increased level of zinc supplementation.

Fat high in stearic acid favorably affects blood lipids and factor VII coagulant activity in comparison with fats high in palmitic acid or high in myristic and lauric acids.

The effect of fats high in individual, prevalent saturated dietary fatty acids on lipoproteins and hemostatic variables in young healthy subjects was evaluated in a randomized strictly controlled metabolic feeding study. Three experimental diets: shea butter (S; 42% stearic acid), palm oil (P; 43% palmitic palmitic acid), and palm-kernel oil with high-oleic sunflower oil (ML; 10% myristic acid, 30% lauric acid) were served to 15 men for 3 wk each, separated by washout periods. Diet S compared with diet P resulted in significant reduction in plasma cholesterol (22%) LDL cholesterol (26%), apolipoprotein B (18%), HDL cholesterol (12%), apolipoprotein A-I (13%), and a 13% lower factor VII coagulant activity (P = 0.001). Similar differences were observed between diets S and ML. In conclusion, intake of shea butter high in stearic acid favorably affects blood lipids and factor VII coagulant activity in young men, compared with fats high in saturated fatty acids with 12-16 carbons.

The effect of individual dietary components on manganese absorption in humans.

Our knowledge of dietary factors affecting manganese absorption in man is very limited. In this study we used a recently developed radionuclide technique to measure manganese absorption in human adults. Using paired observations, we explored the effects of adding calcium and manganese to human milk on manganese absorption. Furthermore, the effects of adding phytate, phosphate, and ascorbic acid to infant formula as well as iron and magnesium to wheat bread were evaluated. Addition of calcium to human milk resulted in a significant decrease in manganese absorption whereas no significant differences in manganese absorption were observed as a result of the other test meals administered with and without the evaluated dietary component, respectively. Thus, manganese absorption was not significantly affected by most dietary factors evaluated in this study, except for the addition of calcium to human milk.

Biomarkers of habitual fish intake in adipose tissue.

The association between habitual fish and marine n-3 polyunsaturated fatty acid (PUFA) intake, and the fatty acid composition of subcutaneous fat was studied in 24 healthy young volunteers. Habitual dietary intakes were estimated from three 7-d weighted food records made at months 0, 5, and 8 of the 8-mo study period. The adipose tissue fatty acid composition of each individual was determined by gas chromatography as the mean of two gluteal biopsies, obtained in the first and the last month of the study. The daily consumption of fish and of marine n-3 PUFAs in absolute terms (g/d) was significantly associated with adipose tissue docosahexaenoic acid content (DHA; r = 0.55 and 0.58, respectively, P < 0.001), but not with eicosapentaenoic and docosapentaenoic acid contents. Our study indicates that the adipose tissue DHA content is the biomarker of choice for the assessment of long-term habitual dietary intakes of fish and marine n-3 PUFAs.

Effect of 6 dietary fatty acids on the postprandial lipid profile, plasma fatty acids, lipoprotein lipase, and cholesterol ester transfer activities in healthy young men.

BACKGROUND: There is increasing evidence that postprandial triacylglycerol-rich lipoproteins may be related to atherogenic risk. OBJECTIVE: The objective was to investigate the effect of individual fatty acid intakes on postprandial plasma lipoprotein triacylglycerol and cholesterol concentrations, plasma fatty acids, and preheparin lipoprotein lipase and cholesterol ester transfer protein (CETP) activities. DESIGN: Six test fats high (approximately 43% by wt) in stearic acid, palmitic acid, palmitic + myristic acid, oleic acid, elaidic acid (trans 18:1), and linoleic acid were produced by interesterification. After having fasted for 12 h, 16 healthy young men were served the individual test fats incorporated into meals (1 g fat/kg body wt) in random order on different days separated by washout periods. Blood samples were drawn before and 2, 4, 6, and 8 h after the meals. RESULTS: Different responses to the test-fat meals were observed for plasma lipoprotein triacylglycerol and cholesterol concentrations, plasma fatty acid concentrations, and lipoprotein lipase and CETP activities (diet x time interaction: 0.001 < P < 0.05). Intake of the long-chain saturated fatty acids stearic and palmitic acids resulted in a relatively lower lipemic response than did intake of the unsaturated fatty acids, probably because the saturated fatty acids were absorbed less and at a lower rate; therefore, the lipemic response took longer to return to postabsorptive values. CONCLUSIONS: Fatty acid chain length and degree of saturation appear to affect the extent and duration of lipemia and affect hepatic output indirectly. These effects may not be mediated via effects on lipoprotein lipase and CETP activities.

An experimental model for studies of zinc bioavailability from milk and infant formulas using extrinsic labeling.

The ability to assess zinc bioavailability from various diets is essential as the zinc content on many foods can be low or marginal. We have investigated the absorption of zinc from human milk, cows' milk, cows' milk formula (whey-adjusted) and soy protein formula as these fluids can comprise the majority of an infant's diet. Radiozinc was added to the diets in tracer amounts. The extrinsic 65Zn was shown by ultracentrifugation, ultrafiltration, and gel filtration to add to milk fractions and individual binding compounds in a manner analogous to the distribution of native zinc, validating the use of extrinsically labeled milk diets. Labeled diets were fed by intubation to 16-day-old suckling rats. Animals were killed after 4 h and tissues removed and counted. Zinc bioavailability was 28% from human milk, 24% from whey-adjusted cows' milk formula, 15% from cows' milk, and 10% from soy formula. Intubation studies using adult rats showed that zinc absorption was lower from all the diets; however, it was still highest from human milk and cows' milk formula. These results show that the rat pup model may provide a rapid, inexpensive, and sensitive method to assay bioavailability of zinc from infant foods.

Manganese retention in man: a method for estimating manganese absorption in man.

Whole-body retention of 54Mn was studied in man by measurements in a sensitive whole-body counter after intake of an extrinsically labeled infant formula. Reproducible retention figures at day 10 were observed after repeated administrations to six subjects; 2.3 +/- 1.1, 3.3 +/- 3.1, and 2.4 +/- 1.4% (means +/- SD) for three separate occasions. Interindividual variation of manganese retention after intake of the same labeled diet was, however, shown to be substantial. Retention at day 10 was 2.9 +/- 1.8% (means +/- SD) and varied from 0.6 to 9.2% when measured in 14 healthy subjects. Large interindividual variations in rate of excretion were observed. The retention measurements for days 10-30, however, could be closely fitted to a single exponential function for each individual. The results regarding intraindividual and interindividual variation in Mn retention indicate that factors influencing Mn absorption can be identified only by repeated administrations using each subject as his/her own control.

Zinc absorption from composite meals. I. The significance of whest extraction rate, zinc, calcium, and protein content in meals based on bread.

The absorption of zinc in man from composite meals based on bread was measured with a radionuclide technique using 65Zn and whole-body counting. Bread was made up from wheat flour of 100 and 72% extraction rate. A lower absolute amount of zinc was absorbed from the white bread compared to the absorption from the same amount of wholemeal bread. When the two types of bread were enriched with zinc chloride the absorption was higher from the white bread than from the wholemeal bread. Addition of calcium in the form of milk products improved the absorption of zinc from a meal with wholemeal bread. A significant positive correlation was found between zinc absorption and the protein content in meals containing milk, cheese, beef, and egg in various combinations with the wholemeal bread.

Competitive inhibition of iron absorption by manganese and zinc in humans.

Zinc and manganese may interfere with iron absorption because of similar physicochemical properties and shared absorptive pathways. The effects of zinc and manganese on iron absorption were studied in human subjects by using paired observations and a dual-radioisotope method (55Fe and 49Fe). Manganese inhibited iron absorption both in solutions and in a hamburger meal. Fractional iron absorption is strongly dose dependent. Adding 2.99 mg Mn to 0.01 mg Fe reduced iron absorption to the same extent as increasing the iron dose 300-fold to 3 mg, strongly indicating a direct competitive inhibition of manganese on iron absorption. In the same experiment with zinc, no inhibitory effect was observed, suggesting different pathways for the absorption of zinc and iron. An intraluminal interaction may occur, because a fivefold excess of zinc to iron (15 mgZn/3 mg Fe) reduced iron absorption by 56% when given in a water solution but not when given with a hamburger meal.

Retention of zinc and calcium from the human colon.

Colonic retention of zinc and calcium was studied after installation during colonoscopy of 30 mumol of zinc and 6.4 mmol of calcium labeled with 65Zn and 47Ca, and measurement of the whole-body retention of the radionuclides. After cecal installation in nine patients, retention (day 13) of zinc was 3.5 +/- 2.1% (mean +/- SD) and of calcium 3.5 +/- 2.7%. The calculated mean absorption was 4.1% for zinc and 14.1% for calcium. Application at the hepatic flexure in four patients resulted in a mean retention (day 13) of 1.2% for zinc and 0.6% for calcium. Under prevailing conditions, colonic absorption of zinc is relatively small, compared to the uptake after oral administration. Colonic absorption of calcium could, however, account for a substantial part of the total calcium uptake.

Effect on blood lipids, coagulation, and fibrinolysis of a fat high in myristic acid and a fat high in palmitic acid.

The hypothesis that myristic acid (C14:0) has a stronger cholesterol-increasing potential than does palmitic acid is based on very few experimental observations. A randomized, strictly controlled dietary study was therefore designed to investigate the effect of a synthetic fat that was high in myristic acid, and palm oil, which is high in palmitic acid, on lipoproteins and hemostatic variables. Twelve men were served two diets (40% of energy as fat) with 41% of fat as myristic (diet M) or palmitic acid (diet P) for 3 wk with 1 mo between the two dietary schedules. Plasma HDL cholesterol was 8% higher with diet M than with diet P: 1.10 +/- 0.06 (mean +/- SEM) vs 1.01 +/- 0.05 mmol/L (P < 0.006). Diet M raised factor VII coagulant (F VIIc) activity to 98% (77-117%) vs 96% (71-109%) (medians and ranges) after diet P (P = 0.02). Total and LDL-cholesterol concentrations did not differ between the diets. In conclusion, the myristic acid test fat was not more cholesterolemic than was palm oil, but it did induce a minor rise in F VIIc activity.

Green tea or rosemary extract added to foods reduces nonheme-iron absorption.

BACKGROUND: Phenolic compounds act as food antioxidants. One of the postulated mechanisms of action is chelation of prooxidant metals, such as iron. Although the antioxidative effect is desirable, this mechanism may impair the utilization of dietary iron. OBJECTIVE: We sought to determine the effect of phenolic-rich extracts obtained from green tea or rosemary on nonheme-iron absorption. DESIGN: Young women aged 19-39 y consumed test meals on 4 separate occasions. The meals were identical except for the absence (meal A) or presence (meal B) of a phenolic-rich extract from green tea (study 1; n = 10) or rosemary (study 2; n = 14). The extracts (0.1 mmol) were added to the meat component of the test meals. The meals were extrinsically labeled with either 55Fe or 59Fe and were consumed on 4 consecutive days in the order ABBA or BAAB. Iron absorption was determined by measuring whole-body retention of 59Fe and the ratio of 55Fe to 59Fe activity in blood samples. RESULTS: The presence of the phenolic-rich extracts resulted in decreased nonheme-iron absorption. Mean (+/-SD) iron absorption decreased from 12.1 +/- 4.5% to 8.9 +/- 5.2% (P < 0.01) in the presence of green tea extract and from 7.5 +/- 4.0% to 6.4 +/- 4.7% (P < 0.05) in the presence of rosemary extract. CONCLUSION: Phenolic-rich extracts used as antioxidants in foods reduce the utilization of dietary iron.

Folic acid enrichment of bread does not appear to affect zinc absorption in young women.

BACKGROUND: In several countries cereals are now enriched with folic acid to reduce the risk of neural tube defects. Human studies suggest that folic acid interferes with zinc absorption. This raises concerns about the zinc status of high-risk groups such as infants, pregnant women, and older persons. OBJECTIVE: We sought to determine the effect of added folic acid on zinc absorption from white bread with high and low zinc contents. DESIGN: Zinc absorption was measured in 15 healthy women (22-33 y), each of whom consumed 4 single meals spaced 2 wk apart in a randomized crossover design. The servings of bread (100 g) differed in zinc and folic acid contents as follows: A, 1.2 mg Zn and 17 microg folic acid; B, 1.2 mg Zn and 144 microg folic acid; C, 3.0 mg Zn and 17 microg folic acid; and D, 2.9 mg Zn and 144 microg folic acid. Meals were extrinsically labeled with 65Zn and absorption was estimated from whole-body retention measurements. Folate status was assessed by measuring plasma and erythrocyte folate and plasma homocysteine concentrations. RESULTS: Mean (+/-SD) zinc absorption did not differ significantly in relation to the folate content of the breads at either the low zinc content (38.8 +/- 13.5% and 40.6 +/- 16.5% for A and B, respectively; P = 0.74) or the high zinc content (26.7 +/- 9.3% and 22.7 +/- 6.6% for C and D, respectively; P = 0.16). There was no significant correlation between folate status and zinc absorption (r < 0.3, P > 0.1). CONCLUSION: Fortification of white bread with a commonly used amount of folic acid did not appear to influence zinc absorption at either a high or a low zinc content.