Willy A. Behrens

A highly sensitive high-performance liquid chromatography method for the estimation of ascorbic and dehydroascorbic acid in tissues, biological fluids, and foods

A highly sensitive procedure for determining ascorbic acid (AA) and dehydroascorbic acid (DHAA) by high-performance liquid chromatography with electrochemical detection in biological fluids, tissues, and foods is described. AA is separated in a C18 reverse-phase column after extraction from the sample with metaphosphoric acid. An aliquot of 20 microliter of diluted extract is injected into the column for the estimation of AA. DHAA is indirectly estimated by converting it to AA after reduction with DL-homocysteine at pH 7.0-7.2 for 30 min at 25 degrees C. After dilution, a 20-microliter aliquot is injected into the column to obtain total vitamin C (AA + DHAA). The concentration of DHAA is calculated by subtraction. AA can be reproducibly quantified at concentrations as low as 50 pg/20 microliter of sample extract. The method described here used a specially designed mobile phase, gave greater stability and a noiseless baseline, and increased substantially the sensitivity and precision. The procedure is rapid, analysis being completed within 10 min after sample preparation, and has been successfully applied to biological fluids, tissues, and foods.

Effects of dietary linseed oil and marine oil on lipid peroxidation in monkey liver in vivo and in vitro

Diets rich in linoleic acid (CO) from corn oil, or in linoleic acid and either α-linolenic acid (LO) based on linseed oil or n−3 fatty acids (MO) from menhaden oil were fed to male and female Cynomolgus monkeys for 15 wk. In the liver a 40% reduction of α-tocopherol occurred in the MO group relative to the CO and LO groups followed by increased formation of lipofuscinin vivo. A four-fold increase of α-tocopherol in the MO diet (MO+E) brought the level in the liver to that found with CO and LO. The increased peroxidation in the MO group in the liver phospholipids was associated with the replacement of 60% of the n−6 fatty acids by n−3 fatty acids from menhaden oil. Similar fatty acid profiles were found in groups fed MO and MO+E, respectively. Compared to the CO fed group, feeding α-linolenic acid only resulted in a slight incorporation of n−3 fatty acids in the liver membranes mainly due to a direct incorporation of α-linolenic acid. However, in monkeys fed menhaden oil more than 30% of the total fatty acids in the liver phospholipids were n−3 fatty acids. The various diets did not influence the activity of liver catalase (EC 1.11.1.6) nor superoxide dismutase (EC 1.15.1.1), but glutathione-peroxidase activity (EC 1.11.1.9) was higher in monkeys fed the MO diet. The catalase activity in females was 20% higher than in males. In anin vitro assay, liver microsomes from monkeys fed the MO diet or the MO diet supplemented with tocopherol produced similar amounts of thiobarbituric acid reactive substances and at a much higher rate than microsomes from the CO and LO groups. It appeared that α-tocopherol did not protect long-chain n−3 C20 and C22 fatty acids as well as n−6 fattya acids against peroxidation. The present data showed that monkeys were not fully able to compensate for increased peroxidative stress but a four-fold supplement of vitamin E to the diets reduced the oxidation.

Chemical and nutritional studies of flaxseed (variety Linott) in rats

Abstract The nutritional effects of flaxseed ( Linum usitatissimum , variety Linott) were studied in the rat. In addition, thermal and storage stabilities of flaxseed were evaluated. Weanling rats were fed diets containing ground flaxseed at levels of 0, 10, 20, or 40% for 90 days. No differences were found in the food intake nor in body and organ weights. Serum triglyceride, total cholesterol, and the low-density lipoprotein (LDL) cholesterol concentrations were significantly lower in the rats fed the 20% and 40% flaxseed diets compared to the 0% flaxseed group. The high-density lipoprotein (HDL) cholesterol concentration and the LDL:HDL-cholesterol ratio were generally lower in the flaxseed-fed rats when compared to the 0% flaxseed group, but a significant lowering occurred only in the 40% flaxseed group. The incorporation of flaxseed in the diet caused significant elevations in the levels of the α-linolenic acid in adipose tissue and in organs. Higher amounts of eicosapentaenoic, docosapentaenoic, and docosahexaenoic acids were observed in the heart and liver of flaxseed-fed rats when compared to the 0% flaxseed group. A significant lowering of tissue vitamin E levels and an elevation of urinary thiobarbituric reacting substances occurred only in the 40% flaxseed group, which suggested that low to moderate intakes of flaxseed did not impart an oxidative stress on rat tissues. Dietary fiber in flaxseed appeared to be largely fermentable and was associated with a large increase in fecal moisture. The phytate in flaxseed had no effect on zinc status. The oil in both the intact and the ground flaxseeds was found to be thermally and oxidatively stable.

Malonaldehyde determination in tissues and biological fluids by ion-pairing high-performance liquid chromatography

A method for the analysis of malonaldehyde by ion pairing high-performance liquid chromatography is described. The method is direct; no thiobarbiturate chromogen formation is required, and sample preparation is simple. After deproteinization with 50% ethanol and removal of particulate by centrifugation samples were passed through a small silica amino column to remove contaminants. Diluted samples (20 μL) were injected onto an octadecylsilane column (25 cm×4.6 mm ID, 5 μm) which is eluted with 30 mM sodium phosphate buffer, pH 6.5 containing 30% ethanol and 1 mM tetradecyltrimethylammonium bromide. Detection was accomplished by monitoring absorbance at 267 nm. The lower limit for reliable quantification was 5 pmol per injection. The method has been successfully applied to the quantification of malonaldehyde present in plasma, urine and tissues of rats kept under different dietary conditions as well as afterin vivo treatment with CCl4 and iron-dextran. The method was also applied to the quantification of malonaldehyde during liver microsomal lipid peroxidation and was compared to the thiobarbituric acid test.

Transport of α-and γ-tocopherol in human plasma lipoproteins

Abstract Alpha-and γ-tocopherol and protein were measured in human plasma lipoproteins that were separated by ultracentrifugation followed by column chromatography. Plasma tocopherols were not significantly different in males (n=7) and females (n=7). Low density lipoprotein (LDL) and high density lipoprotein (HDL) were the main carriers of both tocopherols in males and females. More α-tocopherol was found in LDL than in HDL in males but the opposite was true in females; LDL and HDL carried almost the same amounts of γ-tocopherol. Very low density lipoprotein (VLDL) was not a quantitatively important carrier of either of the vitamers. Females had higher protein-HDL than males but males had higher protein-LDL than females. When 800 IU/day of D-α-tocopherol acetate was orally administered to a male volunteer, plasma α-tocopherol values increased almost twofold after 3 days, but γ-tocopherol decreased to half of its original concentration. After 18 and 78 days, plasma α-tocopherol remained high but γ-tocopherol was not detected. These data suggest that the different distribution of α-tocopherol in plasma lipoprotein in males and females could be due to the different levels of proteins in these lipoprotein fractions. It could be postulated that the transport of vitamin E in plasma lipoprotein is an active and selective mechanism for α-tocopherol. Gamma-tocopherol appeared to be transported non-specifically by lipoproteins and its concentration could be determined by the concentration of α-tocopherol.

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.

Interrelationship and competition of α- and λ-tocopherol at the level of intestinal absorption, plasma transport and liver uptake

Abstract α- and λ-Tocopherol (T) were measured in the plasma of 3 groups of rats that were fed a normal or modified AIN-76 diet containing normal (NE), high (HE) or low (LE) vitamin E for 3 months. α-T levels (μg/ml±SD; n=10) were 7.6±1.0 (NE), 19.3±5.1 (HE) and 0.48±0.43 (LE). λ-T levels were 0.32±0.16 (NE), 0.02±0.05 (HE) and 0.20±0.30 (LE). 24 hrs after an oral dose of 50 mg λ-T; α-T levels (n=3) were 7.0±1.2 (NE), 10.7±3.7 (HE) and 1.0±0.3 (LE). λ-T levels were 5.7±2.2 (NE), 0.83±0.46 (HE) and 10.8±3.8 (LE). When 3 rats from groups NE and HE were fed low vitamin E for 3 days prior to the administration of 50 mg λ-T; α-T levels were 4.8±1.3 (NE) and 7.1±1.5 (HE); λ-T levels were 5.9±2.0 (NE) and 4.6±2.6 (HE). When rats in group LE received 50 mg α-T, levels increased to 10.0±0.8 μg α-T/ml and were 8 times higher than those of λ-T when a dose of 50 mg of each of α- and λ-T were fed. None or traces of λ-T were found in a liver cytosol protein (32000 MW) that binds α-T specifically (α-TBP) in all three groups. Small amounts of λ-T were detected in α-TBP in LE rats after they were fed 50 mg of λ-T. These data suggest that the mechanisms for intestinal absorption, plasma transport and liver uptake of vitamin E are specific for α-T. Only when the concentration of α-T is low, can λ-T successfully compete for binding sites at these three levels.

Transfer of α-tocopherol to microsomes mediated by a partially purified liver α-tocopherol binding protein

Abstract A liver α-[ 3 H]-tocopherol binding protein was partially purified using ammonium sulfate fractionation, gel filtration and ion-exchange chromatography from the 100,000 × g liver supernatant of rats fed a low vitamin E diet for 3 months and administered 200 μCi of α-[ 3 H]-tocopherol. The labelled protein was used to study the transfer and binding of α-tocopherol to liver microsomes. In vitro incubations showed that the transfer of α-tocopherol was directly proportional to the amount of α-[ 3 H]-tocopherol binding protein and to the amount of microsomal protein present. Moreover, the transfer of α-tocopherol increased with the temperature of incubation. A 3.8 fold excess of unlabelled α-tocopherol (bound to the same protein) inhibited the transfer of labelled α-tocopherol by 62%. These data suggest that an α-tocopherol binding protein could play a role in the intracellular transfer of α-tocopherol.

Quantitative Analysis of Ascorbic Acid and Isoascorbic Acid in Foods by High-Performance Liquid Chromatography with Electrochemical Detection

Abstract A procedure is presented for the direct and simultaneous determination of ascorbic acid (AA) and isoascorbic acid (IAA) in food products by paired-ion reverse-phase high-performance liquid chromatography. Three Supelcosil C18 columns were used with a pH 5.4 mobile phase containing 0.04 M sodium acetate, 5 mM tetrabutylammonium hydrogen sulfate and 0.015% metaphosphoric acid. Food samples, preserved with metaphosphoric acid and diluted with mobil phase, were injected (20μI) using an autosampler. Detection of AA and IAA was by amperometry using a glassy carbon electrode and Ag/AgCl reference electrode. The applied potential was +0.6 volt and the sensitivity was 20 nA. As little as 0.5 ng of each component could be detected under these conditions. 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 t...