A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells

Summary

Ascorbate plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. Here we describe a medium-throughput, specific and inexpensive microplate assay for the determination of both intra- and extracellular ascorbate in cell culture.

Abstract

Vitamin C (ascorbate) plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. For instance, within the brain, ascorbate acts in a neuroprotective and neuromodulatory manner that involves ascorbate cycling between neurons and vicinal astrocytes - a relationship that appears to be crucial for brain ascorbate homeostasis. Additionally, emerging evidence strongly suggests that ascorbate has a greatly expanded role in regulating cellular and systemic iron metabolism than is classically recognized. The increasing recognition of the integral role of ascorbate in normal and deregulated cellular and organismal physiology demands a range of medium-throughput and high-sensitivity analytic techniques that can be executed without the need for highly expensive specialist equipment. Here we provide explicit instructions for a medium-throughput, specific and relatively inexpensive microplate assay for the determination of both intra- and extracellular ascorbate in cell culture.

Introduction

The discovery of the chemical nature of ascorbic acid (vitamin C), and its identification as the long-sought "anti-scorbutic factor", by Albert Szent-Györgyi and others in papers published from 1928 to 1934¹ were landmark events in the history of biochemistry. Indeed, these discoveries contributed to Szent-Györgyi being awarded the Nobel Prize in Physiology or Medicine in 1937. The ever-expanding suite of roles for ascorbate in animal and plant physiology, as well as human health, continue to be the subjects of active scientific investigation and controversy.

L-Ascorbate is an abundant physiological reductant and enzyme cofactor in mammalian systems, and contributes to numerous well-defined enzymatic reactions involving collagen hydroxylation, carnitine and norepinephrine biosynthesis, tyrosine metabolism and peptide hormone amidation². Intriguingly, mounting evidence suggests that ascorbate plays a role in stimulating other iron-dependent dioxygenases, such as the prolyl and asparaginyl hydroxylases involved in the hydroxylation and targeting of the hypoxia-inducible factors (HIFs) 1α and 2α³. A recent report suggests that ascorbate plays a role in T-cell maturation through affecting chromatin demethylation via its activity in stimulating the nuclear hydroxylases, Jumonji C (JmjC) domain proteins; the latter of which appear to require ascorbate for full activity⁴. Indeed, the stimulation of such enzymes by ascorbate appears to occur by a similar mechanism to the stimulation by ascorbate of the HIF and collagen hydroxylases. Among other classical effects, ascorbate contributes significantly to cellular antioxidation as a water-soluble chain-breaking radical scavenger⁵ and to the recycling of plasma membrane α-tocopherol (vitamin E) via the reduction of the α-tocopheroxyl radical⁶, which is important in protecting against membrane lipid peroxidation⁷. Importantly, although most mammals are capable of de novo hepatic synthesis of ascorbate from D-glucose, higher primates, guinea pigs and some bats depend on dietary sources of the vitamin⁸. This is due to inactivation of the GULO gene, the orthologues of which in unaffected mammals encode the enzyme, γ-gulono-lactone oxidase^9-13. This enzyme is required for the final reaction in ascorbate biosynthesis from glucose¹³.

Following transporter-mediated absorption from the intestinal lumen in humans, ascorbate is distributed throughout the body by the circulatory system. The vitamin is typically found in its reduced form at millimolar concentrations intracellularly (with the notable exception of erythrocytes in which concentrations are typically similar to the prevailing plasma concentration), and at micromolar concentrations (e.g. 50-200 μM) in most extracellular fluids^14,15 .

Under physiological conditions, ascorbate typically undergoes a reversible one-electron oxidation to the ascorbyl free radical (AFR; also known as monodehydroascorbate or semidehydroascorbate). While the AFR is a relatively stable radical¹⁶, in the absence of its rapid one-electron enzymatic reduction back to ascorbate, two AFRs can further dismutate to one ascorbate and one dehydroascorbate (DHA)^9,13,17. Within the interior of the cell, the two-electron oxidation product of ascorbate, DHA, can be rapidly reduced back to ascorbate by glutathione- and NAD(P)H-dependent enzymatic and non-enzymatic reactions¹³.

While it is classically accepted that ascorbate’s only significant role in iron metabolism is to stimulate dietary absorption of non-heme iron¹⁸, we and others have provided evidence strongly suggesting that ascorbate plays a greatly expanded role in the metabolism of this metal. First, ascorbate that is released by ascorbate-replete cells appears to play an important role in modulating the uptake of non-transferrin-bound iron by cells^19,20, and very recent evidence indicates that ascorbate also modulates the uptake of transferrin-bound iron by cells²¹, the latter of which corresponds to a major physiological iron-uptake route²².

Ascorbate is essential for normal central nervous system function in mammals^23,24. Together with the adrenal cortex, pituitary gland, thymus, retina and corpus luteum, the brain contains high concentrations of ascorbate relative to other body tissues^23,25-27. Additionally, the exposure of both astrocytes^28,29 and neuron-like cells³⁰ to glutamate is known to trigger the release of ascorbate into the extracellular space, where the ascorbate is thought to help protect neurons against glutamate-induced neuronal dysfunction³¹. While the exact mechanism of glutamate-induced ascorbate release from astrocytes is unknown, we have recently provided evidence indicating the involvement of cell swelling caused by glutamate uptake by the astrocyte glutamate and aspartate transporter (GLAST; also known excitatory amino acid transporter isoform 1 [EAAT1] in humans) and consequent activation of volume-sensitive osmolyte and anion channels (VSOACs) that are permeable to small organic anions such as ascorbate³². The molecular identities of the plasma membrane conduits involved in VSOAC formation remain to be identified^33,34.

Although many assays have been developed for the determination of ascorbate in biological samples, which include spectrophotometric, fluorometric and chromatographic assays^35,36, there is much variability in specificity, sensitivity, interference by chemical contaminants, effective linear range and stability of the endpoint analyte. Additionally, other significant factors that influence the choice of assay are rapidity, ease of use and access to relatively specialized equipment such as a high-performance liquid chromatography (HPLC) apparatus.

Here we present a simple and highly specific colorimetric microplate assay for the determination of intracellular ascorbate in cultured cells, as well as a separate assay for the determination of ascorbate-efflux from cultured cells. The latter assay aims to circumvent the problem of underestimation of ascorbate release from cells due to rapid re-uptake of released ascorbate by sodium-dependent ascorbate transporters (SVCTs). Although both of these methods have appeared in some of our previous publications^{19,20,32,37,38}, this manuscript provides an explicit set of instructions and guidelines for their effective execution.

Protocol

1. Determine Intracellular Ascorbate in Cultured Cells

1. Cell culture and harvesting
   1. Grow suspension (e.g. human erythroleukemia, K562) or adherent cells (e.g. primary astrocytes) using standard culture procedures^19-21,32,38. Note: to ensure cells contain ascorbate, load cultured cells with ascorbate either as ascorbate or DHA^33,39.
2. Create an ascorbate-containing cellular extract
   1. Incubate an appropriate number of phosphate-buffered saline (PBS)-washed suspension or adherent cells with 450 μl of ice-cold Cell Permeabilization Buffer [CPB; 0.1% (w/v) saponin in PBS; 4 °C]. Note: Determine the number of cells required to obtain an absorbance value within the linear range of the assay (see below) empirically by the end-user. Note: tissue extracts may also be used (see Discussion for further details).
   2. Agitate cells on ice for 10 min to ensure thorough cellular lysis.
   3. Create a clarified ascorbate-containing intracellular extract by removing cellular debris by centrifugation of the crude lysate at 16,000 × g for 5 min in a refrigerated microcentrifuge (4 °C).
   4. Carefully remove 4 x 100 μl aliquots from each sample and add to a horizontal sequence of wells in a 96-well flat bottom plate that contains either 25 μl/well of PBS ("-AO") only or 25 μl/well of 45.5 U/ml stock solution of L-ascorbate-oxidase (AO) in PBS ("+AO"). Note: this should be done in parallel with the preparation of the standards (see below).
3. Ascorbate standard-curve construction (must be constructed anew for each assay)
   1. Prepare a stock solution of 10 mM ascorbic acid in ice-cold PBS. Note: Verify the concentration of ascorbate spectrophotometrically using a quartz cuvette with a 1 cm path-length at 265 nm (extinction coefficient = 14.5 mM^-1 cm^-1)⁴⁰.
   2. Carefully prepare a series of ascorbate standards between 0 and 20 μM.
   3. In parallel with step 1.2.4, carefully remove 4 × 100 μl aliquots from each standard and add to a horizontal sequence of wells in a 96-well flat bottom plate that contains 25 μl/well of PBS ("-AO") or a 45.5 U/ml stock solution of AO in PBS ("+AO"). Note: 100 μl of the above ascorbate standards will contain 0-2 nmole of ascorbate. Please note, the purpose of the AO is to control for the contribution of non-ascorbate reductants to ferricyanide reduction.
4. Determine intracellular ascorbate - Step 1: selectively remove ascorbate and quantitatively oxidize ascorbate with ferricyanide
   1. Orbitally mix the 96-well plate at 550 rpm at room temperature for 5 min in the dark by covering the plate in foil. Note: This step should oxidize all ascorbate in the "+ AO" wells to DHA. The "-AO" wells should be unaffected.
   2. Add 50 μl of 3.5 mM potassium ferricyanide (herein referred to as "ferricyanide") in PBS to all wells. The final [ferricyanide] = 1 mM. Note: Use a multipipettor.
   3. Orbitally mix the plate at 550 rpm at room temperature for another 5 min in the dark. Note: This step should result in the reduction of ferricyanide to ferrocyanide by ascorbate in a stoichiometric ratio of 2 molecules of ferricyanide reduced per 1 molecule of ascorbate oxidized.
   4. Immediately add 25 μl of a freshly constructed solution containing 50% (v/v) acetic acid and 30% (w/v) trichloroacetic acid (TCA). Note: Use a multipipettor.
5. Determine intracellular ascorbate - Step 2: quantitate the amount of ferrocyanide formed³⁷
   1. Add 100 μl of ferrocyanide determination solution³⁷ to each well. Note: A working solution should be made immediately before use: 2 ml of 3 M Na-acetate (pH 6.0); 0.5 ml of glacial acetic acid (~17.4 M acetic acid); 2 ml of 0.2 M citric acid; 2 ml of 3.3 mM FeCl₃ in 0.1 M acetic acid; 1 ml of 30 mM ferene-S. The final volume of this working solution should be 7.5 ml.
   2. Orbitally mix the plate in the dark at 550 rpm for 30 min at room temperature.
   3. Read the absorbance values of the wells at 593 nm (i.e. the absorbance maximum of the Fe(II)(ferene-S)₃ complex).
   4. Calculate the amount of intracellular ascorbate as nmoles ascorbate per million cells by initially subtracting the A_{593 nm} values for the '+ AO' wells from the corresponding '- AO' wells for each sample and then interpolating from the ascorbate standard curve (see step 1.3). When constructing the standard curve, plot this "difference value" for each standard against the amount of ascorbate per well.

2. Determination of Ascorbate-efflux from Cultured Cells

1. Cell culture and harvesting
   1. Grow suspension or adherent cells as above (see step 1.1). Note: carry out the below assay in a 24-well plat format with suspension and adherent cells for best results.
   2. Resuspend suspension cells, or overlay adherent cells, with 400 μl of pre-warmed HEPES-buffered saline solution, with or without calcium and magnesium, and containing 5 mM D-glucose (HBS/D; pH 7.3, 37 °C) in wells of a 24-well plate. Add the same volume of HBS/D to specified cell-free wells on each 24-well plate to be examined. Note: the latter will serve as cell-free controls for the baseline reaction (see below).

3. Determine the Amount of Ascorbate Released

1. The following stock solutions should be prepared beforehand: 120 U/ml AO in HBS/D (prepare fresh); 2.4 mM Ferene-S in HBS/D; 120 μM FeCl₃ and 600 μM Na-citrate in HBS/D (prepare immediately from more concentrated stock solutions).
2. To start the ferrireduction reaction (final volume per well should be 600 μl), add the following volumes of reagents to individual wells already containing 400 μl of HBS/D:
   1. Add 50 μl of AO (120 U/ml) or HBS/D to paired wells in triplicate. The final AO concentration should be 10 U/ml. Note: Label paired wells as "- AO" and "+ AO".
   2. Mix the plates with gentle orbital mixing for 5 min at 37 °C.
   3. Add 50 μl of 2.4 mM ferene-S to all wells. The final ferene-S concentration should be 200 μM. Mix the plates as above for 5 min at 37 °C.
   4. Add 50 μl of freshly prepared 120 μM ferric citrate. The final iron and citrate concentrations should be 10 μM iron and 50 μM citrate. Mix well.
3. In three triplicate control wells, aspirate the overlying medium and add 600 μl HBS/D containing 0.1% saponin. Note: These will serve as "100% cell-lysis" controls for a lactate dehydrogenase (LDH) release assay to be conducted in parallel (see below).
4. Incubate for 60 min in the dark at 37 °C.
5. At the end of the ascorbate-efflux assay, rapidly aspirate 500 μl from each well and add to appropriately labelled wells in a 24-well plate. Note: for suspension cells, initially remove the cells by centrifugation at 4 °C.
6. Add 300 μl aliquots of the supernatant to a 96-well plate and then read at 593 nm.

4. Determination of Extracellular Ascorbate

1. Read the absorbance values of the wells at 593 nm.
2. Calculate the amount of extracellular ascorbate as nmoles ascorbate per mg protein (or per million cells) as described for the determination of intracellular ascorbate in Step 1.5.4. Note: the end user should optimize conditions so that ascorbate release is not limited by intracellular ascorbate.

5. Determination of LDH Release

1. With the remaining 200 μl of extracellular solution from each sample conduct an LDH release assay^32,41 to determine the extent of cellular lysis. Note: calculate as a % of total releasable LDH. Determine releasable LDH from the samples that were treated with 0.1% saponin.

Results

Determination of Intracellular Ascorbate in Cultured Suspension Cells

In the first assay (Figure 1), intracellular ascorbate is determined, following ascorbate-specific (i.e. AO-sensitive) reduction of ferricyanide to ferrocyanide, using the highly sensitive determination of ferrocyanide by a previously published procedure³⁷. The detection of ascorbate is based on the colorimetric chelation of ferrous iron that is generated by the ascorbate-dependent reduction...

Discussion

In this paper we present two rapid, specific and relatively sensitive colorimetric microplate assays for the determination of ascorbate derived from the intra- and extracellular compartments in cultured cells. The assays can be completed with access to standard laboratory equipment and reagents. The only moderately costly reagent required for the assay is AO, which is essential as it imparts a high-degree of analyte specificity toward L-ascorbate. The assays are well suited to either suspen...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Nunc 96-well flat-bottom plates	Thermo	269620	Any flat-bottom 96-well plate can be used
Refrigerated benchtop microcentrifuge	Eppendorf	5415D	A non-refrigerated microcentrifuge that has been equilibrated to temperature in a cold room can also be used
Refrigerated bench-top centrifuge	Eppendorf	5810R	Swing-bucket
Bio-Rad Benchmark Plus Microplate Spectrophotometer	Bio-Rad		Any microplate spectrophotometer capable of reading at 593 nm can be used and is recommended. If a filter-based plate reader is used, choose the closest wavelength possible and use the standard-curve method.
Ependorf MixMate (microplate orbital mixer)	Eppendorf		This is a very versatile and reliable microplate mixer and works very well for these assays
General-purpose buffers
Phosphate-buffered saline (PBS), pH 7.4
MOPS-buffered saline (MBS); 137 mM NaCl, 2.7 mM KCl, 15 mM MOPS-Na+, pH 7.3
MBS + 5 mM D-glucose (MBS/D)
HEPES-buffered saline + 5 mM D-glucose (HBS/D); 137 mM NaCl, 5.2 mM KCl, 1.8 mM CaCl2•2 H2O, 0.8 mM MgSO4•7 H2O, 5 mM D-glucose, 20 mM HEPES-Na+, pH 7.3)
Cell permeabilisation buffer (CPB; 0.1% saponin in PBS)
General chemicals
L-ascorbic acid or sodium L-ascorbate	Sigma-Aldrich		Highest purity preparations should be obtained
Dehydro-L-ascorbic acid (DHA) dimer	Sigma-Aldrich	30790	Aqueous solutions theoretically yield 2 moles of DHA monomer per mole of DHA dimer
Cytochalasin B	Sigma-Aldrich	C6762	Stock solutions prepared in DMSO or ethanol
Ascorbate oxidase (AO)	Sigma-Aldrich	A0157	Stock solutions (120 U/ml) can be prepared in PBS or MBS and then frozen in aliquots
Potassium ferricyanide (FIC)	Sigma-Aldrich	455989	Trihydrate
Ferene-S (3-(2-Pyridyl)-5,6-di(2-furyl)-1,2,4-triazine-5′,5′′-disulfonic acid disodium salt)	Sigma-Aldrich	92940
Sodium L-glutamate	Sigma-Aldrich
L-glutamine	Sigma-Aldrich
Saponin	Sigma-Aldrich	47036	Prepare a 0.1% stock solution
Stock solutions for intracellular ascorbate determination assay
3 M sodium acetate (pH 6.0)
Glacial acetic acid
0.2 M citric acid
3.3 mM FeCl3 in 0.1 M acetic acid
30 mM ferene-S
50% (v/v) acetic acid + 30% (w/v) trichloroacetic acid (TCA)
Stock solutions for ascorbate-efflux assay
AO (120 U/ml)
2.4 mM ferene-S
0.12 mM FeCl3 in 0.6 mM sodium-citrate