Stanley T. Omaye

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.

Simultaneous Determination of Ascorbic Acid, Isoascorbic Acid (Erythorbic Acid) and Uric Acid in Human Plasma by High-Performance Liquid Chromatography with Amperometric Detection

Abstract A procedure is presented for the direct and simultaneous determination of ascorbic acid (AA), isoascorbic acid (IA), and uric acid (UA) in human plasma by paired-ion reversed-phase high-performance liquid chromatography. An Ultrasphere ODS (C18) column is used with a pH 5.25 mobile phase containing 0.04M sodium acetate, 0.005M tetrabutylammonium phosphate, and 0.2 mg/mL disodium EDTA. Plasma samples preserved with an equal volume of 10% metaphosphoric acid are diluted 10-fold with mobile phase and filtered through 0.2 micron filters. The injection volume is 10 uL. Detection of AA, IA, and UA is by amperometry using a glassy carbon electrode and Ag/AgCl reference electrode. The applied potential is +0.6 volt and the sensitivity setting is 100 nAmps. As little as 0.25 ng of each component can be detected at this setting and the electrode response is linear over the AA, IA, and UA ranges encountered in human plasma.

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.

Experimental vitamin C depletion and supplementation in young men. Nutrient interactions and dental health effects.

Biochemical indices of AA clearly showed that the young men in this study were brought into various states of AA depletion and repletion according to their dietary AA intakes. While previous studies have postulated that supplemental intakes of AA may adversely affect body status of vitamins B6 and B12, we found no changes in the B vitamin status of the young men receiving varying AA intakes. Moderate AA supplementation (605 mg/day) showed no antagonistic effect on markers of vitamins B6 and B12. Blood markers of fat-soluble vitamins A and E and iron status were not affected by AA intakes. The propensity of the gingiva to become inflamed or bleed on probing was reduced after normal (65 mg/day) AA intakes as compared to deficient (5 mg/day) intakes and upon supplementary (605 mg/day) AA intakes as compared to normal intakes. The results suggest that AA status may influence early stages of gingival inflammation and crevicular bleeding, and warrant further study of the relationship between AA and periodontal health.

Simple method for bleeding the unanaesthetized rat by tail venipuncture.

A technique is described for the intermittent collection of blood from the rat tail. By using commonly available equipment, blood samples can easily be obtained from rats without the need for anaesthesia. The development of this technique makes the rat more readily available as an animal model for repeated withdrawals of small blood samples for pharmacokinetic or bioavailability evaluations.

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.

Effect of ascorbic acid on heme metabolism in hepatic microsomes

Abstract Hepatic microsonal cytochrome P-450 levels are significantly decreased (46–68%) in ascorbic acid-deficient guinea pigs. Studies attempting to elucidate the mechanism responsible for decreased cytochrome P-450 demonstrated that ascorbic acid status did not influence the turnover ( t 1 2 ) or the degradation of hepatic cytochrome P-450 heme. Urinary excretion of Δ-aminolevulinic acid (ALA) and coproporphyrin was significantly decreased (30 and 69% respectively) in ascorbic acid-deficient guinea pigs. Injections (ip) of ALA into ascorbic acid-deficient guinea pigs were not effective in returning cytochrome P-450 levels to values found in ascorbic acid-supplemented guinea pigs. In addition, plasma and hepatic iron and blood heme were related directly, while hepatic copper and plasma copper or ceruloplasmin were related inversely, to the ascorbic-acid status of the guinea pig. These data, along with past investigations on heme synthesis in the ascorbic acid-deficient guinea pig, are consistent with mechanisms proposing that ascorbic acid may influence: 1) apocytochrome P-450 synthesis, 2) binding of heme and apo-cytochrome P-450 to form active cytochrome P-450, and/or 3) incorporation of Fe++ into the heme moiety of cytochrome P-450, perhaps via changes in copper metabolism.

Early and delayed biochemical effects of paraquat toxicity on rat lung

Abstract This research was undertaken to study the early and delayed biochemical effects of paraquat dichloride on the lungs of rats. To accomplish this, female rats were given a sublethal dose (18 mg/kg) of paraquat, in a single (ip) injection. At the end of 24 hr, only catalase, β-N-acetylglucosaminidase, and glucose-6-phosphate dehydrogenase (G6PD) activities and nonprotein sulfhydryl (NPSH) levels were significantly (P

Distribution of vitamins A and E in blood and liver of rats depleted of vitamin A or vitamin E.

Young Sprague-Dawley male rats (n=150) were fed a semipurified diet, either without vitamin A (VA), without vitamin E (VE) or supplemented with both vitamins A and E (control). At the end of weeks 0, 1, 3, 5, 6, 7, 8 and 9, groups of rats were anesthetized with methoxyflurane, and blood was collected by cardiac puncture until the rat was exsanguinated. The liver was excised. Whole blood (WB_ from each rat was fractionated into plasma (PLA), leukocytes (LEU), platelets (PLT) and erythrocytes (RBC). Each blood components was extracted with hepatane and livers were extracted with CHCl3/CH3OH (2∶1, v/v). The extracts were analyzed for VA and VE by high performance liquid chromatography. The relationship among blood components in the loss of VA was PLT=LEU>WB>PLA. The relationship among blood components in the loss of VE was PLA>RBC>WB>LEU-PLT. VA and VE levels in other blood components decreased precipitously between weeks 0 and 4 in the animals placed on deficient diets. These results and correlation analyses between vitamin contents of blood components and of livers indicate inadequacies for the use of certain blood components as monitors of lipid-soluble vitamin status in the rat.

Selected pulmonary biochemical and hematological changes produced by prolonged hypoxia in the rat

Rats were exposed to 12% O2 (1 atm) for 48 hr, then 10% O2 for the duration of the exposures. Significant elevations in enzymes of the glutathione peroxidase system were found in the lungs of rats killed 3.5, 7.5, and 12.5 days of exposure compared to air-breathing controls. Superoxide dismutase was also elevated after hypoxia but nonsignificantly. Animals killed 12.5 days after exposures exhibited 75% (P less than 0.05) more thiobarbituric acid reactive products in their lungs compared to controls. These results along with significant increases of lung lipid peroxidation and in an augmentation of protective antiperoxidative lung defense capabilities. Hypoxia also resulted in enzyme elevations of lung phosphofructokinase and pyruvate kinase, which may indicate an adaptive increase in lung glycolytic capabilities which would be helpful in maintaining lung tissue energy requirements during hypoxia. In addition, it was confirmed that red blood cell count, hemoglobin and hematocrit values increased after prolonged hypoxia. The results of this study might also reflect enzyme elevation/induction due to cellular reparative-proliferative processes following hypoxia.