René Madère

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.

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.

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.

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...

Effects of vitamin e status and exercise training on tissue lipid peroxidation based on two methods of assessment

Abstract Effects of dietary vitamin E deprivation, acute exercise and training on tissue lipid peroxidation as determined via a thiobarbituric acid reactive substances (TBARS) assay and an ion-pairing HPLC assay for malondialdehyde are reported. Female rats were fed diets with (+E) or without (-E) vitamin E for 8 weeks, after which subgroups of rats were exercised for 45 min and immediately sacrificed. Remaining animals continued on their diets, were sedentary or trained for 8 additional weeks, and were sacrificed with or without prior exercise. Little malondialdehyde, as determined by HPLC, was found in liver, heart or muscles (with the exception of white vastus) following any condition. Significant levels of TBARS were found in all tissues following all conditions. Significantly higher levels were found in many tissues with the -E diet and acute exercise. TBARS levels did not correlate with HPLC-determined malondialdehyde levels indicating that determination of the presence and degree of exercise induced lipid peroxidation are specific to each assay and conclusions regarding its occurrence must be made with caution.

Ascorbic and dehydroascorbic acid content of infant formula

Abstract A sensitive high-performance liquid chromatography method was used to estimate ascorbic and dehydroascorbic acids in ready-to-feed, liquid-concentrated, and powdered infant formula. The total vitamin C contents in all commercial formula examined were higher than the amounts indicated on the labels. The proportion of dehydroascorbic acid was 9.5% in ready-to-feed, 10.6% in liquid-concentrated, and 20% in the powdered formula.

Ascorbic and dehydroascorbic acid contents of canned food and frozen concentrated orange juice

Abstract A sensitive high-performance liquid chromatography method was used to estimate ascorbic and dehydroascorbic acid in canned foods and in frozen concentrated orange juice. Canned foods were found to be an unreliable and variable source of vitamin C; only orange and fortified apple juices were excellent sources of the vitamin. The proportion of dehydroascorbic acid to ascorbic acid in some products was high but the total vitamin C amount was low. Unlike the canned foods tested, frozen concentrated orange juice was an excellent source of vitamin C and dehydroascorbic acid was very low. These results indicate that the levels of dehydroascorbic acid are low in canned foods, and that these canned foods are not a health concern from the perspective of dehydroascorbic acid intake.

Kinetics of tissue RRR-α-tocopherol depletion and repletion. Effect of cold exposure

Abstract Vitamin E was estimated in plasma and tissues of rats kept for three months on a low vitamin E diet or a high vitamin E diet. Some of the animals from each group were switched to the opposite diet, and the kinetics of uptake and depletion of vitamin E were followed 3, 8, and 15 days after the diet change. Some rats were also submitted to cold exposure (6°C) for three days. During repletion plasma, red blood cells, liver, spleen, and adrenal gland were the only tissues that responded rapidly to the diet change; after three days, their vitamin E levels corresponded to that of the new diet. Heart, brain, lung, muscle, and thymus were slow in reacting to diet change. Fifteen days after the change in diet, white adipose tissue did not respond. The rate of repletion for all tissues was more rapid than the rate of depletion, but liver was the only tissue that after three days had vitamin E levels corresponding to the low-vitamin diet. Cold exposure for three days did not produce any significant change in the vitamin E content of any tissue, indicating that despite high oxygen consumption by the animal, vitamin E was not consumed or mobilized.

Malonaldehyde determination in foods by ion-pairing high-performance liquid chromatography

Abstract A direct method which involved no thiobarbiturate chromogen formation for the analysis of malonaldehyde in foods by ion-pairing high-performance liquid chromatography was developed. After deproteinization and cleaning, diluted samples (20 μl) were injected onto an octadecylsilane column (25 cm × 4·6 mm) i.d., 5 μm) and eluted with 30 m m sodium phosphate buffer, pH 6·5 containing 30% (v/v) ethanol and 0·5 m m tetradecyltrimethylammonium bromide. Detection was accomplished by monitoring absorbance at 267 nm. The lower limit for reliable quantification was 5 pmol per injection. Traces or no malonaldehyde were detected in fresh samples of selected foods. However, after a period of several weeks of refrigerator storage of opened containers, or after chemically induced lipid peroxidation, food samples contained a substantial amount of malonaldehyde.