Tissue ascorbic acid, HPLC

Vitamin C Vitamin C activity resides in two naturally occurring compounds ascorbic acid and its oxidation product, dehydroascorbic acid. In human tissues ascorbic acid predominates. Ascorbic acid is labile in most samples, oxidizing to dehydroascorbic acid and then degrading to 2,3-diketogluconic acid. Various reagents can be used to prevent this oxidation in plasma or whole blood samples. Extraction with 5% metaphosphoric or trichloroacetic acid is the usual initial preparation. Only ascorbic acid may be detected by UV spectrophotometry at 245-265 nm, the absorption maxima of dehydroascorbic acid being 210 nm. A similar problem exists with electrochemical detection where ascorbic acid oxidizes at +0.7V with carbon electrodes. Fluorescent derivatives may be formed with 2-4-din-itrophenylhydrazine or o-phenyldiamine. These derivatives can be assayed by reversed-phase HPLC. 

Reaction mixture 25 pi of freshly prepared 2 x reaction buffer, 15 pi of water, and 10 pi of filtered cell or tissue lysate (total volume of 50 pi). The reaction mixture is incubated for 30 min at 37°C in the dark, followed by adding 10 pi of oxidation solution. After oxidation for 30 min in the dark at room temperature, 10 pi of 1% ascorbic acid is added, mixed, and centrifuged for 20 min at 14,000 xg through a Micron 10,000 filter (Millipore, Ultracel YM-10). The filtrate is analyzed by HPLC (ideally only 20 pi of a 1 2 dilution with water are injected into the HPLC system). The starting lysate of 10 pi was diluted sevenfold. 

The assay mixture contained imidazole (pH 7.2), KC1, NADPH, glucose-6-phosphate, DTT, glucose-6-phosphate dehydrogenase, and H2-biopterin. Samples were incubated for 60 minutes with tissue extract and terminated with 2 N TCA. After centrifugation (15,000g for 5 min), the H4-biopterin was oxidized to pterin with iodine, the reaction was terminated with ascorbic acid, and samples were injected onto the HPLC column for analysis. An example of an assay is shown in Figure 9.131, and a composite of the rate of product formation is shown in Figure 9.132. 

Several reports have described the use of HPLC in the analyses of ascorbic acid in foods and vitamin products (71, 72,73,74) and in tissue samples (75). Procedures vary in the type of column, mobile-phase, detection systems and means of stabilization of extracts. Reversed-phased,... 

Figure 2. Analysis of ascorbic acid in a mouse brain tissue extract employing HPLC and an electrochemical detector. (Reproduced, with permission, from Ref. 75. Copyright 1975, Pergamon Press, Inc.)...

A Method for the Determination of Ascorbic Acid in Biological Tissues by HPLC... 

Discussion. The method presented provides a fast and reproducible means of determining the ascorbic acid content of various tissues. In addition to a high rate of sample handling (12 samples/h for complex tissues such as liver, brain, and adrenal gland and 15 samples/h for plasma), the method requires a minimum of sample preparation and is practical for routine analysis of biological samples. Furthermore, the method utilizes equipment available to each laboratory with a rudimentary HPLC system. 

Table IV. Ascorbic Acid Content of Various Tissues from Guinea Pigs Fed Two Different Diets (mg/lOO g or per 100 mL of Tissue) as Determined by HPLC...

Ascorbate Levels in Tissues. Several tissues were dissected from M. sexta and analyzed for L-ascorbic acid by high performance liquid chromatography (HPLC) (Figure 2), paper chromatography, or the dinitrophenylhydrazine method (5). As anticipated, L-ascorbic acid was... 

Figure 2, HPLC of i.-ascorbic acid from insect tissues (65). A, L,-ascor-bic acid, 1.7 fxg B, M. sexta hemolymph (0.01 mL) extract C, M. sexta labial gland extract, 1.4 mg wet weight.

Research Needs. Over the years L-ascorbic acid has been shown to be an essential nutrient for many insects including species of Lepidoptera, Orthoptera, Coleoptera, and Diptera. Others such as cockroaches, houseflies, and mealworms are reared on simple diets without added ascorbic acid. Perhaps those insects require very low levels of vitamin C in their diets. A sensitive analytical method is needed to measure levels of L-ascorbic acid and dehydroascorbic acid in insect tissue and food. Such a method, which is likely to be developed using HPLC with electrochemical detection, could be used to monitor vitamin C levels in feed ingredients as well as in tissues during an insect s life cycle. This information is needed to determine whether ascorbic acid is used to... 

Vitamin C Ascorbic acid Connective tissue Scurvy Photometric, HPLC,... 

Absolute qualitative identification can be assured only if samples are removed and analyzed. Two examples of such a procedure have been reported. The first was an attempt to determine if direct electrical stimulation of the caudate nucleus resulted in the release of dopamine as well as ascorbic acid from that tissue. Micro voltammetric and stimulating electrodes were micromanipulated into excised caudate tissue which was flushed with warmed, oxygenated buffer. Reference and auxiliary electrodes were nearby. Quantitative information was taken, stored, manipulated, and displayed by a minicomputer. Simultaneously a push-pull cannula device sampled the caudate and delivered the perfusate to an iced vial. Changes in the electrochemical signal that followed stimulation were correlated with changes in the dopamine and ascorbic acid content of the perfusate as determined via HPLC with electrochemical detection. It was found that little if any ascorbic acid was released as a result of electrical stimulation in these experiments. Although there is some question concerning the stability of ascorbate in an iced vial, the above example does illustrate this coincident analytical technique. 

Schell, D. A., and Bode, A. M., 1993, Measurement of ascorbic acid and dehydroascorbic acid in mammalian tissue utilizing HPLC and electrochemical detection, Biomed. Chromatog. 7 267-272. 

Capillary electrophoresis (CE) is a technique that combines some characteristics of traditional polyacrylamide gel electrophoresis (PAGE) and modern HPLC. During the last 10 years, the number of CE applications for quantitation of ascorbic acid and related compounds in food, blood and tissue has increased. 

CJ B ates. Use of homocysteine to stabilize ascorbic acid, or to reduce dehydroascor-bic acid, during HPLC separation of large volumes of tissue extracts. Clin Chim Acta 205 249-252, 1992. 

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