Rene Madere

A Procedure for the Separation and Quantitative Analysis of Ascorbic Acid, Dehydroascorbic Acid, Isoascorbic Acid, and Dehydroisoascorbic Acid in Food and Animal Tissue

Abstract A procedure is presented for the direct and simultaneous determination of ascorbic acid (AA) and isoascorbic acid (IAA) in food products and animal tissues by reverse phase high-performance liquid chromatography. Two PLRP-S columns in series were used with a pH 2.2 mobile phase containing 20 mM phosphate buffer and 0.17% metaphosphoric acid. An amperometric detector set at 0.7 volt and 20 mA was used. As little as 0.5 ng of each compound could be detected. When the same samples were incubated with homocysteine to reduce dehydroascorbic acid (DHAA) and dehydroisoascorbic acid (DHIAA) to AA and IAA respectively and reinjected into the system, the values for total AA and IAA were obtained. The concentration of the oxidized forms, DHAA and DHIAA, could then be calculated by substraction.

Effect of Dietary Vitamin E on the Vitamin E Status in the BB Rat during Development and after the Onset of Diabetes

Weanling diabetes-prone BB rats were fed AIN-76 diets containing high (HE, 1 g/kg diet), basal (NE, 0.2 g/kg) or low (LE, trace) vitamin E and were killed at 21, 42 or 60 days of age. Plasma and tissues (adrenals, pancreas, spleen, thymus, liver, brown and white adipose tissue, muscle and testes) were analysed for vitamin E. Vitamin E levels reflected the level in the diet and no diabetic animals were detected at these times. In a second experiment, a total of 90 diabetes-prone BB rats were kept on diets LE and HE for 6 months or until they became diabetic. 11/45 on LE and 5/45 on HE became diabetic. Again, plasma and tissue levels of vitamin E reflected the levels in the diet with the exception of the thymus of diabetic rats fed the high vitamin E diet. Thymus vitamin E levels (microgram/g tissue) were 1.8 and 1.2 in LE-fed diabetics and asymptomatic rats, respectively; and 22.7 and 49.5 in HE-fed diabetics and asymptomatic rats, respectively. The last 2 values were significantly different (p less than 0.005). There were no other differences in plasma or tissue levels of vitamin E in these groups of animals. These findings suggest that high dietary vitamin E may decrease the incidence of diabetes in animals which are able to accumulate sufficient amounts of the vitamin in the thymus. Since the thymus plays a key role in the maturation of T cell populations, which appear to be altered in this disease, it seems possible that the protective effect may be exerted at this level.

Vitamin C and vitamin E status in the spontaneously diabetic BB rat before the onset of diabetes

Ascorbic acid (AA), dehydroascorbic acid (DHAA), and vitamin E were measured in tissues and plasma of 30 control and 30 spontaneously diabetic BioBreeding rats (BBdp) during development and before the onset of diabetes. At weaning, rats were fed an AIN-76 semisynthetic diet for 30, 64, or 113 days, after which plasma and tissues from 10 rats of each group were collected and analysed for AA, DHAA, and vitamin E. AA and DHAA levels were significantly increased in plasma and spleen of the diabetes-prone rats compared with those of the control group at 30 and 64 days, but the difference disappeared by 113 days. No differences were observed in liver, adrenals, thymus, and pancreas at any of the time periods. However, lower levels of vitamin E were observed in adrenal gland, thymus, and pancreas of the diabetes-prone rats. It is concluded that BBdp rats have an altered metabolism of AA, DHAA, and vitamin E, before the onset of diabetes. These changes could be due to genetic and physiological factors operating during development of this rat strain.

Sparing effects of selenium and ascorbic acid on vitamin C and E in guinea pig tissues

BackgroundSelenium (Se), vitamin C and vitamin E function as antioxidants within the body. In this study, we investigated the effects of reduced dietary Se and L-ascorbic acid (AA) on vitamin C and α-tocopherol (AT) status in guinea pig tissues.MethodsMale Hartley guinea pigs were orally dosed with a marginal amount of AA and fed a diet deficient (Se-D/MC), marginal (Se-M/MC) or normal (Se-N/MC) in Se. An additional diet group (Se-N/NC) was fed normal Se and dosed with a normal amount of AA. Guinea pigs were killed after 5 or 12 weeks on the experimental diets at 24 and 48 hours post AA dosing.ResultsLiver Se-dependent glutathione peroxidase activity was decreased (P < 0.05) in guinea pigs fed Se or AA restricted diets. Plasma total glutathione concentrations were unaffected (P > 0.05) by reduction in dietary Se or AA. All tissues examined showed a decrease (P < 0.05) in AA content in Se-N/MC compared to Se-N/NC guinea pigs. Kidney, testis, muscle and spleen showed a decreasing trend (P < 0.05) in AA content with decreasing Se in the diet. Dehydroascorbic acid concentrations were decreased (P < 0.05) in several tissues with reduction in dietary Se (heart and spleen) or AA (liver, heart, kidney, muscle and spleen). At week 12, combined dietary restriction of Se and AA decreased AT concentrations in most tissues. In addition, restriction of Se (liver, heart and spleen) and AA (liver, kidney and spleen) separately also reduced AT in tissues.ConclusionTogether, these data demonstrate sparing effects of Se and AA on vitamin C and AT in guinea pig tissues.

Estrogen administration, postexercise tissue oxidative stress and vitamin C status in male rats

Estrogen can putatively act as an antioxidant and protect tissues from exercise-induced oxidative stress. To test the in vivo efficacy of estrogen, the effects of 2 weeks of daily estrogen (40 microg x kg(-1) body weight beta-estradiol 3-benzoate) injection on indices of immediate postexercise oxidative stress and antioxidant status were determined in adult male rats, with and without 8 weeks of prior dietary vitamin E deprivation. The treadmill running protocol (60 min at 21 m x min(-1), 12% grade) induced significant oxidative stress as indicated by muscle glutathione status. Estrogen administration had little effect on postexercise tissue glutathione status, superoxide dismutase and glutathione peroxidase activity, and vitamin E levels. Estrogen administration induced significant reductions in muscle, liver, and heart vitamin C concentrations following exercise, as well as in unexercised male rats. Tissue vitamin C loss was not directly mediated through liver glycogen or glutathione status. Thus, estrogen administration generally did not appear to influence postexercise tissue indices of oxidative stress or antioxidant status and may have contributed to a decline in overall antioxidant protection by inducing losses in tissue vitamin C content.

The effects of vitamin E and selenium intake on oxidative stress and plasma lipids in hamsters fed fish oil

The aim of the present work was to test the effects of large-dose supplementation of vitamin E (Vit E) and selenium (Se), either singly or in combination, on fish oil (FO)-induced tissue lipid peroxidation and hyperlipidemia. The supplementation of Se has been shown to lower blood cholesterol and increase tissue concentrations of the antioxidant glutathione (GSH); however, the effects of Se supplementation, either alone or in combination with supplemental Vit E, on FO-induced oxidative stress and hyperlipidemia have not been studied. Male Syrian hamsters received FO-based diets that contained 14.3 wt% fat and 0.46 wt% cholesterol supplemented with Vit E (129 IU d-α-tocopheryl acetate/kg diet) and/or Se (3.4 ppm as sodium selenate) or that contained basal requirements of both nutrients. The cardiac tissue of hamsters fed supplemental Se showed increased concentrations of lipid hydroperoxides (LPO) but decreased oxidized glutathione (GSSG) concentrations. The higher concentrations of LPO in the hearts of Se-supplemented hamsters were not lowered with concurrent Vit E supplementation. In the liver, Se supplementation was associated with higher Se-dependent glutathione peroxidase activity and an increase in the GSH/GSSG ratio, whereas a lower hepatic non-Se-dependent glutathione peroxidase activity was seen with Vit E supplementation. Supplemental intake of Se was associated with lower plasma concentrations of total cholesterol and low density lipoprotein cholesterol plus very low density lipoprotein cholesterol. In view of the pro-oxidative effects of Se supplementation on cardiac tissue, a cautionary approach needs to be taken regarding the plasma lipid-lowering properties of supplemental Se.

Improved automated method for determining vitamin C in plasma and tissues

Abstract An improved automated method for determining vitamin C in plasma and tissues is described. Total vitamin C was determined after oxidation to dehydroascorbic acid by reaction with 2,4-dinitrophenylhydrazine. The recovery of ascorbic acid added to plasma was 98 to 101% with a coefficient of variation of 1.8%. Interaction between samples with low, medium, and high concentrations was 1% and the coefficient of variation on 60 replicate analyses of plasma was 2.0%. In the analysis of 16 samples of rat plasma, results obtained by the automated method were essentially the same as those obtained by the original manual method. It was possible to determine vitamin C in 0.15 ml of plasma containing as little as 1.2 μg/ml. Vitamin C was also measured in extracts of rat heart, spleen, kidneys, adrenals, liver, and brain.

Increased plasma and tissue levels of vitamin E in the spontaneously diabetic BB rat

Increased plasma and tissue levels of vitamin E were found in spontaneously diabetic BB rats (D) as well as asymptomatic/diabetes-prone BB rats (AD) in comparison to levels in non-diabetic control rats (ND). Treatment of D rats with insulin for 30 days returned plasma and tissue values of vitamin E to control levels. The changes reported here could not be explained solely on the basis of variations in total lipid content of plasma. These data suggest the metabolism of vitamin E is altered in asymptomatic and spontaneously diabetic BB rats and this alteration returns to control values following insulin treatment. Furthermore, it might be speculated that these data indicate a relationship between vitamin E and insulin.

Maternal transfer of vitamin E to fetal and neonatal guinea pigs utilizing a stable isotopic technique

Abstract A vitamin E study was carried out with 12 pregnant guinea pigs utilizing a stable isotopic technique in order to assess the transfer of various forms of vitamin E (natural and synthetic alpha tocopherol) across the placenta and the mammary gland following a continuous oral dosing (60 mg d3-RRR-alpha-tocopheryl acetate/kg + 60 mg d6-all-racemic-alpha-tocopheryl acetate/kg) throughout gestation and lactation with deuterium labeled vitamin E. At late term pregnancy (day 60) and through early lactation (days 1–5), dams and their corresponding fetuses/neonates were sacrificed and various tissues collected for subsequent analysis for levels of the two forms of alpha tocopherol to establish the extent of transfer of vitamin E as well as their relative bioavailability. Vitamin E analysis from fetal and neonatal tissues that included the adrenals, brain, heart, kidneys, liver, lungs, muscle, plasma and spleen indicated a substantial transfer of deuterium labeled alpha tocopherol compounds across the placenta and through the mammary gland. In addition the data showed that in all tissues examined including fetal/neonatal and maternal, that a strong preferential discrimination in favor of the natural form (RRR) of alpha tocopherol as compared to the synthetic form (all-racemic alpha tocopherol) was observed. The relative bioavailability (natural:synthetic alpha tocopherol) across fetal and neonatal tissues was on average 1.85/1, with a range from 1.78/1 to 1.90/1. Maternal tissues on average showed a ratio of 1.82/1, with a range from 1.72/1 to 1.99/1. A higher bioavailability (P ≤ 0.05) was observed with natural than synthetic alpha tocopherol as evidenced by a higher ratio of d3/d6 in all tissues examined. Colostrum contained the highest amount of total vitamin E which followed a gradual decline (P ≥ 0.05) over the remaining 4 day lactation period. The data obtained from this study raise further questions on the current accepted biological potencies of natural: synthetic alpha tocopherol (1.36/1), respectively.

Influence of sources of dietary vitamin E on the maternal transfer of α-tocopherol to fetal and neonatal guinea pigs as determined by a stable isotopic technique

The accepted biological potencies of vitamin E (United States Phamacopeia, 1985) for 1 mg all-rac-alpha-tocopheryl acetate (synthetic form) is 1.00 IU and that of 1 mg (RRR)-alpha-tocopheryl acetate (natural form) is 1.36 IU. In the present study, a stable isotopic (2H) technique was employed to evaluate the bioavailability of natural v. synthetic forms of vitamin E and to determine whether the potency of the forms is the stated relationship of 1.36:1.00 (RRR)-alpha-tocopheryl acetate:all-rac-alpha-tocopheryl acetate. Sixty female in-bred guinea pigs received either 40 or 80 mg vitamin E/kg diet with equal levels of (RRR)-alpha-tocopheryl acetate and all-rac-alpha-tocopheryl acetate throughout gestation and lactation. At late-term pregnancy (day 50 or 60) and during early lactation, dams and their corresponding fetuses or neonates were killed and various tissues collected for subsequent alpha-tocopherol analysis. Vitamin E analysis of fetal and neonatal tissues indicated a substantial transfer of 2H-labelled alpha-tocopherol across the placenta and through the mammary gland. Total alpha-tocopherol concentrations were significantly influenced by tissue type and dose level, but not by stage of gestation or lactation. The relative bioavailability (d3:d6) across fetal and neonatal tissues was on average 1.81:1.00, with a range from 1.62:1.00 to 2.01:1.00. Maternal tissues had a mean ratio of 1.77:1.00. A higher relative bioavailability (P<or=0.05) was observed with natural compared with synthetic alpha-tocopherol as shown by a higher d3:d6 ratio in all tissues examined. Vitamin E was highest in colostrum on day 2 then declined through to day 5. Results from this present experiment further question the accepted biological potencies of natural:synthetic alpha-tocopheryl acetate of 1.36:1.00.