ANALYTICAL
BIOCHEMISTRY
204,
1-14
(19%)
REVIEW Ascorbic Acid and Dehydroascorbic Acid Analyses in Biological Samples Philip
W. Washko,
Richard
W. Welch,
Laboratory of Cell Biology and Genetics, National National Institutes of Health, Bethesda, Maryland
Received
March
Kuldeep Institute 20892
R. Dhariwal, of Diabetes,
Yaohui
Digestive,
Wang,
and Kidney
and Mark
Levine
Diseases,
10, 1992
It is axiomatic that accurate measurement precedes understanding. Methods for quantitatively measuring ascorbic acid (vitamin C) and its metabolite dehydroascorbic acid are indispensable for probing the function of these compounds. The stakes are not simply prevention of the deficiency disease scurvy (l), but more importantly determination of optimal requirements of vitamin C (2). Understanding the effect of alteration of the concentration of vitamin C on the biochemical reactions in situ that depend on the vitamin is essential (25). Methods allowing the characterization of reaction kinetics in tissues and organelles are needed (3-5). Although some tissues contain millimolar concentrations of ascorbic acid, the ability to precisely measure picomole quantities of the vitamin is necessary, since sample size from cells and organelles is often limited. In addition, the function of ascorbic acid is not apparent in some tissues. Thus, accurate measurements are valuable not only for characterizing in situ reaction kinetics, but also for discovering new functions of the vitamin. Assays for ascorbic acid have been available since its isolation 60 years ago. However, many ascorbic acid and dehydroascorbic acid assays suffer from the “four-S syndrome,” which results from inattention to stability, sensitivity, specificity, and substance interference. As described here, there are less than a dozen types of assays for ascorbic acid and dehydroascorbic acid. Our purpose is to critically review these categories, particularly with regard to analysis of biological samples. We concentrate on HPLC assays since we believe these to be the best. As a consequence of imperfect assays, one technique may be described in many different reports, each time with a minor modification; in such cases we describe the key articles. Table 1 summarizes the assay types, with special emphasis on the four-S syndrome. 0003.2697/92 $5.00 Copyright Q 1992 by Academic Press, All rights of reproduction in any form
Many assay classes are based upon oxidation of ascorbic acid to dehydroascorbic acid or on the reverse reduction. Ascorbic acid is a physiologic reducing agent. Oxidation occurs at carbons 2 and 3 to form dehydroascorbic acid via the free radical intermediate semidehydroascorbic acid (Fig. 1). For assay purposes, oxidation of ascorbic acid can be accomplished chemically, enzymatically, or electrically. Dehydroascorbic acid can be reduced back to ascorbic acid by many reducing agents; sulfhydryl agents such as dithiothreitol and 2,3dimercapto-1-propanol are commonly used because of their favorable redox potentials. Dehydroascorbic acid also undergoes hydrolysis spontaneously to form 2,3-diketogulonic acid; this step is virtually irreversible. Diketogulonic acid or its derivatives are measured in some assays. The first product formed as ascorbic acid oxidizes is the free radical intermediate semidehydroascorbic acid, also called ascorbate free radical. Compared to other free radicals, semidehydroascorbic acid is relatively stable; nevertheless, its decay has a rate constant of 2.8 X lo5 M-’ s-’ (6). Because of this instability there are only a few assays for the free radical. Most assay techniques address ascorbic acid and/or dehydroascorbic acid, and this is the focus here. Ascorbic acid oxidation as a means of assay is tricky because of spontaneous oxidation of ascorbic acid and hydrolysis of dehydroascorbic acid in solution. Depending on other components in the sample and the assay
conditions,
either substance might be stable for a few
minutes, for several hours, or for months upon storage at -70°C (7). As expected, ascorbic acid oxidation and dehydroascorbic acid hydrolysis are influenced by their concentration in the sample, temperature, light, pH, dissolved oxygen, solvent, ionic strength, and the pres1
Inc. reserved.
WASHKO
ET
TABLE Ascorbic
Assay 2,6-DCPIP” Ferrous chromogenic 2,4-DNPH Fluorometric Gas chromatography Ascorbic acid oxidase Peroxidase HPLC/uv HPLC/EC amperometric HPLC/EC coulometric
Sensitivity (per sample)
Sample
volume (4)
AL. 1
Acid
Assays
Specificity for ascorbic acid
Stability during assays (hr)
a34
nmol
600-4000
No
ND*
a400 33 a-280 317
pm01 nmol pm01 pm01
5-5000 500-4000 420-2000 0.150
No No No Yes
10-400 140 5-450
Yes Yes Yes
33 pm01 330 pm01 a.50 pm01
210
pm01
2-50
Yes
30.1
pm01
5-100
Yes
Storage
stability
Refs.
16 (RT) ND ND ND
ND*
BIOCHEMISTRY
204,
1-14
(19%)
REVIEW Ascorbic Acid and Dehydroascorbic Acid Analyses in Biological Samples Philip
W. Washko,
Richard
W. Welch,
Laboratory of Cell Biology and Genetics, National National Institutes of Health, Bethesda, Maryland
Received
March
Kuldeep Institute 20892
R. Dhariwal, of Diabetes,
Yaohui
Digestive,
Wang,
and Kidney
and Mark
Levine
Diseases,
10, 1992
It is axiomatic that accurate measurement precedes understanding. Methods for quantitatively measuring ascorbic acid (vitamin C) and its metabolite dehydroascorbic acid are indispensable for probing the function of these compounds. The stakes are not simply prevention of the deficiency disease scurvy (l), but more importantly determination of optimal requirements of vitamin C (2). Understanding the effect of alteration of the concentration of vitamin C on the biochemical reactions in situ that depend on the vitamin is essential (25). Methods allowing the characterization of reaction kinetics in tissues and organelles are needed (3-5). Although some tissues contain millimolar concentrations of ascorbic acid, the ability to precisely measure picomole quantities of the vitamin is necessary, since sample size from cells and organelles is often limited. In addition, the function of ascorbic acid is not apparent in some tissues. Thus, accurate measurements are valuable not only for characterizing in situ reaction kinetics, but also for discovering new functions of the vitamin. Assays for ascorbic acid have been available since its isolation 60 years ago. However, many ascorbic acid and dehydroascorbic acid assays suffer from the “four-S syndrome,” which results from inattention to stability, sensitivity, specificity, and substance interference. As described here, there are less than a dozen types of assays for ascorbic acid and dehydroascorbic acid. Our purpose is to critically review these categories, particularly with regard to analysis of biological samples. We concentrate on HPLC assays since we believe these to be the best. As a consequence of imperfect assays, one technique may be described in many different reports, each time with a minor modification; in such cases we describe the key articles. Table 1 summarizes the assay types, with special emphasis on the four-S syndrome. 0003.2697/92 $5.00 Copyright Q 1992 by Academic Press, All rights of reproduction in any form
Many assay classes are based upon oxidation of ascorbic acid to dehydroascorbic acid or on the reverse reduction. Ascorbic acid is a physiologic reducing agent. Oxidation occurs at carbons 2 and 3 to form dehydroascorbic acid via the free radical intermediate semidehydroascorbic acid (Fig. 1). For assay purposes, oxidation of ascorbic acid can be accomplished chemically, enzymatically, or electrically. Dehydroascorbic acid can be reduced back to ascorbic acid by many reducing agents; sulfhydryl agents such as dithiothreitol and 2,3dimercapto-1-propanol are commonly used because of their favorable redox potentials. Dehydroascorbic acid also undergoes hydrolysis spontaneously to form 2,3-diketogulonic acid; this step is virtually irreversible. Diketogulonic acid or its derivatives are measured in some assays. The first product formed as ascorbic acid oxidizes is the free radical intermediate semidehydroascorbic acid, also called ascorbate free radical. Compared to other free radicals, semidehydroascorbic acid is relatively stable; nevertheless, its decay has a rate constant of 2.8 X lo5 M-’ s-’ (6). Because of this instability there are only a few assays for the free radical. Most assay techniques address ascorbic acid and/or dehydroascorbic acid, and this is the focus here. Ascorbic acid oxidation as a means of assay is tricky because of spontaneous oxidation of ascorbic acid and hydrolysis of dehydroascorbic acid in solution. Depending on other components in the sample and the assay
conditions,
either substance might be stable for a few
minutes, for several hours, or for months upon storage at -70°C (7). As expected, ascorbic acid oxidation and dehydroascorbic acid hydrolysis are influenced by their concentration in the sample, temperature, light, pH, dissolved oxygen, solvent, ionic strength, and the pres1
Inc. reserved.
WASHKO
ET
TABLE Ascorbic
Assay 2,6-DCPIP” Ferrous chromogenic 2,4-DNPH Fluorometric Gas chromatography Ascorbic acid oxidase Peroxidase HPLC/uv HPLC/EC amperometric HPLC/EC coulometric
Sensitivity (per sample)
Sample
volume (4)
AL. 1
Acid
Assays
Specificity for ascorbic acid
Stability during assays (hr)
a34
nmol
600-4000
No
ND*
a400 33 a-280 317
pm01 nmol pm01 pm01
5-5000 500-4000 420-2000 0.150
No No No Yes
10-400 140 5-450
Yes Yes Yes
33 pm01 330 pm01 a.50 pm01
210
pm01
2-50
Yes
30.1
pm01
5-100
Yes
Storage
stability
Refs.
16 (RT) ND ND ND
ND*
