High-performance liquid chromatographic-fluorescence determination of traces of selenium in biological materials.

An improved method for the determination of selenium in biological materials has been developed. This work both extends and validates the procedure of Vézina and Bleau (J. Chromatog. 426, 385-391, 1988) which is based on high-performance liquid chromatographic determination of the fluorophore formed by reaction of Se(IV) with 2,3-diaminonaphthalene. The mass detection limit is 48 pg selenium (3 sigma) and the concentration detection limits are 48 parts per trillion in biological fluids and 120 to 480 parts per trillion in dried biological materials. The linear dynamic range of the method has been extended up to approximately 800 ng. Relative standard deviations of 9.4 to 2.7% were observed in repeated analyses of standards in the range of 0.5 to 500 ng. The proposed method was validated with respect to 23 biological reference materials spanning an 1800-fold range of selenium concentrations and was found to be free of significant constant or proportional biases despite greatly different matrix compositions. This method offers an unsurpassed combination of sensitivity, accuracy, linear dynamic range, and freedom from matrix interferences and may be considered a reference method for the reliable determination of selenium in biological materials.

Vitamin C nutriture has little short-term effect on vitamin E concentrations in healthy women.

To determine whether the postulated sparing effect of vitamin E by ascorbic acid (AA) is important for human nutrition, we studied vitamin E status in 20 healthy pre-menopausal women (age 20-43 y) with high or low vitamin C intakes for 6 wk in a live-in metabolic unit. The experimental diet contained no fruits and vegetables and provided 5 mg/d of AA (Recommended Dietary Allowance = 60 mg/d), 3 mg/d of alpha-tocopherol (RDA = 10 mg/d) and 42 g/d of tocopherol-stripped safflower oil to increase the vitamin E requirement. Half of the subjects revived a daily AA supplement of 495 mg (high AA group). A biochemical ascorbate deficiency was attained in the low AA group as indicated by plasma AA concentrations that reached the lower limit of normal by study d 15. Oral doses (20 mg) of hexadeuterated RRR-alpha-tocopherol acetate (d6-alphaT) were given daily to all subjects on d 15-21. Measures of vitamin E status included d6-alphaT and unlabeled alpha-tocopherol concentrations in plasma, platelets, buccal cells and adipose. The levels of unlabeled alpha-tocopherol decreased over time in plasma and platelets and were unchanged for buccal cells and adipose, but were not significantly affected by AA intake. Likewise, the rise and fall of plasma and platelet d6-alpha T, and measures of lipid peroxidation, were not affected by AA intake. Although vitamin C nutriture did not significantly affect vitamin E status within the 6-wk time period of this experiment, further study of this question is warranted, because some of the present results indicate a trend toward sparing of tissue tocopherol by high AA intake.

High-performance liquid chromatographic determination of selenocysteine with the fluorescent reagent, N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid.

The method described is based on derivatization of selenocysteine with N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid and responds linearly to selenocysteine spiked into plasma. Recovery is insensitive to inter-individual variation or use of serum versus plasma, but is decreased by hemolysis. The derivative is stable for at least three days. The total imprecision of determinations in plasma was 0.8-2.1% (coefficient of variation) over the range of 6-30 microM selenocysteine, with a detection limit of 0.4 microM (3 x S.D.). There was no significant interference from plasma thiols. This appears to be the first report of the selective reaction of free selenocysteine with a fluorescent reagent. This simple method works well in plasma and serum and may be adaptable to other types of samples.

Assay of human transthyretin-bound holo-retinol-binding protein with reversed-phase high-performance liquid chromatography.

We describe a reversed-phase high-performance liquid chromatographic method for the determination of vitamin A-transporting (holo) transthyretin-bound (TTR) retinol-binding protein (RBP) concentrations in serum or plasma. Holo-TTR-RBP and free retinol derived primarily from free RBP are consistently observed with this chromatographic method. Holo-TTR-RBP concentrations determined by this method are highly correlated to holo-TTR-RBP concentrations measured by chromatography. This method has the advantage of using less expensive columns and having peak areas which are more proportional to their true concentrations in plasma, as determined by comparison to purified protein spectrophotometry and radial immunodiffusion. The percentage of RBP circulating as holo-TTR-RBP decreased significantly as the total concentration of RBP or retinol increased. Because purified holo-TTR-RBP did not dissociate under these chromatographic conditions, this suggests that more vitamin A circulates as holo-free RBP or free retinol in the blood of people with high serum RBP.

Liquid-chromatographic assay for free and transthyretin-bound retinol-binding protein in serum from normal humans.

We quantified vitamin A-transporting retinol-binding protein (RBP) in serum or plasma by size-exclusion "high-performance" liquid chromatography, using a TSK 2000 column and fluorescent detection of the bound retinol. Serum or plasma samples filtered through a 0.20-microns (pore size) Millex filter were applied directly to the column. The pH 7.0 mobile phase contained sodium phosphate, disodium EDTA, and mercaptoethanol. Two peaks with RBP immunological activity were eluted: the smaller peak containing at least 86% of the vitamin A, which was identified as transthyretin-bound RBP; the larger peak containing a small amount (less than 14%) of a highly fluorescent vitamin A-containing protein, identified as free RBP. Both free and transthyretin-bound RBP can be quantified by this method.

An internal standard method for the unattended high-performance liquid chromatographic analysis of ascorbic acid in blood components.

A paired-ion, reversed-phase, high-performance liquid chromatography procedure using electrochemical detection and internal standard quantitation based on isoascorbic acid (IA) is described for the determination of ascorbic acid (AA) in blood cells and plasma. By correcting for vial-to-vial variations in the AA oxidation rate, use of IA as an internal standard overcomes a major problem associated with AA instability and eliminates the necessity of assaying samples immediately after they are prepared for analysis. The ion-pairing agent, dodecyltriethylammonium phosphate, gives improved AA-IA resolution over agents with shorter carbon chains and also eliminates the interference of an unidentified substance extracted with platelet AA. Five percent metaphosphoric acid extracts of mononuclear leukocytes (MN), polymorphonuclear leukocytes (PMN), platelets, or plasma were mixed with the IA internal standard and diluted with an EDTA-cysteine solution. The samples were placed in a refrigerated autosampler at 4 degrees C prior to chromatography on a 5-microns octadecylsilyl column. AA concentrations (mean +/- SD) in platelets, MN, and PMN from six healthy volunteers were 0.25 +/- 0.05, 15.2 +/- 6.28, and 2.43 +/- 1.63 micrograms/10(8) cells, respectively; the mean plasma AA concentration was 0.97 +/- 0.34 mg/dl. All are in good agreement with published values. Refrigerated sample extracts containing the internal standard can be reassayed up to 3 weeks later with negligible change in calculated AA concentration. Up to 70 samples can be assayed per day with a detection limit (3 X SD) and minimum quantifiable level (less than 5% coefficient of variation) of 0.02 and 0.2 ng/injection, respectively.

Measurement of vitamin C in blood components by high-performance liquid chromatography. Implication in assessing vitamin C status.

The fact that platelets, PMN leukocytes, and MN leukocytes concentrate ascorbic acid suggests that vitamin C has an important role in their physiological functions. The question still remains as to which one of the cells best reflects vitamin C status. The ascorbic acid content of PMNs and platelets correlates positively with plasma concentration and supplementation with vitamin C, as shown in Evans et al. They also found that MN leukocytes, in contrast, do not show any such relationship; however, MN leukocytes maintain the highest levels of ascorbic acid and play a very important function in immunocompetence. We have found that with a limited number of subjects, ascorbic acid content of MN and PMN leukocytes correlates positively with plasma ascorbic acid, but there was no correlation between platelets and plasma ascorbic acid (unpublished results). Therefore, further work is necessary to evaluate these three blood components for the best cellular marker of vitamin C status. We have developed a reversed-phase HPLC method for ascorbic acid that can be used in conjunction with our cellular differential centrifugation technique for the determination of ascorbic acid in relatively pure blood cell fractions. The chromatographic method is simple, sensitive, and automated. It clearly resolves ascorbic acid, which is the major form of the vitamin found in vivo and is not prone to interference by sugars, carbohydrates, or nucleotides.

A comparison of leukocyte ascorbate levels measured by the 2,4-dinitrophenylhydrazine method with high-performance liquid chromatography using electrochemical detection.

In leukocytes a dynamic relationship between the reduced form of ascorbic acid (AA) and its oxidized product dehydro-AA has been described. It is therefore important to know which form of the vitamin predominates when choosing a methodology. The purpose of this study was to find out if the majority of ascorbate in human leukocytes isolated by centrifugation through Percoll is in the reduced AA form by measuring reduced AA by HPLC and comparing the values to those obtained by using the 2,4-dinitrophenylhydrazine (DNPH) method which measures total ascorbate, and quantifying the reduced and oxidized forms of the vitamin in leukocytes using a modification of the DNPH method. There was no significant difference (P greater than .05) between the HPLC and DNPH values for 12 individuals and 87% of the AA was found to be in the reduced form. These results support the assumption that the majority of AA found in a mixed leukocyte population isolated through Percoll is in the reduced form and that both methods can be used for AA measurements.