A simple and sensitive assay for ascorbate using a plate reader

Abstract

We have developed a rapid, inexpensive, and reliable assay for the determination of ascorbate using a plate reader. In this assay, ascorbic acid is oxidized to dehydroascorbic acid using Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy) and then reacted with o-phenylenediamine to form the condensation product, 3-(dihydroxyethyl)furo[3,4-b]quinoxaline-1-one. The rate of appearance of this product is monitored over time using fluorescence. With this method, it is possible to analyze 96 wells in less than 10min. This permits the analysis of 20 samples with a full set of standards and blanks, all in triplicate. The assay is robust for a variety of samples, including orange juice, swine plasma, dog plasma, and cultured cells. To demonstrate the usefulness of the assay for the rapid determination of experimental parameters, we investigated the uptake of ascorbate and two different ascorbate derivatives in U937 cells. We found similar plateau levels of intracellular ascorbate at 24h for ascorbate and ascorbate phosphate. However, the intracellular accumulation of ascorbate via the phosphate ester had an initial rate that was three to five times slower than that via the palmitate ester. Only lower concentrations of the palmitate ester could be examined because the ethanol needed as solvent decreased cell viability; it behaved similarly to the other two compounds at lower concentrations. To come to these conclusions, only nine plates needed to be analyzed to provide us with the end result after only 7h of analysis. This clearly demonstrates the strength of the plate reader assay, which allows the analysis of large-sample sets in a fraction of the time required for the methods that are most commonly used today. The assay is quick, is very economical, and provides results with uncertainties on the order of only 5%.

Figure 1. Overview of assay

A. The reaction of DHA with OPDA produces a fluorescent product that is monitored over time, as shown in this 150 μM concentration standard. The initial rate of formation of DHA-OPDA is dependent on the [DHA] in the sample, which is proportional to the total AscH⁻ in the original sample. B. The initial rates from a set of concentration standards are used to plot a standard curve. From this linear plot of slope vs. standard concentration, the [AscH⁻] of unknown samples is determined. Data are means, n=3; error bars represent S.E.

Figure 2. Methanol slows chromophore formation

Solutions of ascorbate (25 μM) were prepared using different concentrations of MeOH (0–90% v/v). The apparent concentrations of ascorbate were determined relative to standards containing 60% MeOH. Data are means, n=6; error bars represent S.E.

Figure 3. Ascorbate and ascorbate phosphate uptake are comparable in U937 cells

U937 cells were exposed for 24 hours to different concentrations of ascorbate (10–1000 μM), ascorbate phosphate (10–1000 μM), or ascorbate palmitate (10–50 μM) in full medium. Palmitate was dissolved in 100% ethanol. Concentrations of ascorbate palmitate higher than 50 μM decreased cell viability thus higher concentrations were not studied. All experiments were done at least 3 times. In each experiment each point is the result of triplicate samples. Data are means; error bars represent S.E.

Figure 4. Ascorbate phosphate results in a slower intracellular accumulation of ascorbate compared to non-derivatized ascorbate

U937 cells were incubated in full media and exposed to ascorbate (50 or 1000 μM) or ascorbate phosphate (50 or 1000 μM). Cells were harvested at different time points (1, 2, 4, 8, 16, 20, and 24 h). All experiments were done at least 3 times. In each experiment each point is the result of triplicate samples. Data are means; error bars represent S.E.