Easy-to-Use HPLC Method to Measure Intracellular Ascorbic Acid Levels in Human Peripheral Blood Mononuclear Cells and in Plasma

Gwendolyn Van Gorkom,Will Wodzig,Celine Riess,Ahmed El Molla,Erik-Jan Ververs,Birgit Gijsbers,Gerard Bos,Cindy Sarodnik,Catharina Van Elssen

doi:10.3390/antiox11010134

Gwendolyn Van Gorkom, Will Wodzig + Show 7 more

Open Access

https://doi.org/10.3390/antiox11010134

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Journal: Antioxidants	Publication Date: Jan 7, 2022
Citations: 8	License type: CC BY 4.0

Affiliation: Maastricht University

Abstract

Given the growing interest in ascorbic acid (AA), there is a need for a reliable and reproducible method to measure AA status in the human body. Serum AA concentrations do not correlate well with tissue levels, but AA levels in leukocytes do. However, a standard method for clinical application is lacking. This present study describes a method to measure AA in the peripheral blood mononuclear cells (PBMCs) with hydrophilic interaction liquid chromatography (HILIC). The method can also be used in plasma and other leukocyte subsets. The measurements of AA in PBMCs and plasma were performed with HPLC with HILIC separation and UV detection. The sample preparation involved the isolation of PBMCs and lysis and precipitation with acetonitrile. European Medicine Agency guidelines for bioanalytic method validation were followed for the evaluation. A highly precise execution of the method was found with intra- and inter-assay variations at a maximum of 7.8%. In 40 healthy donors, a mean intracellular AA concentration of 7.9 microgram/108 cells was found in PBMCs. A correlation between plasma and PBMC AA concentration was not present (r = 0.22). In conclusion, we developed a convenient, reliable, and reproducible method for the quantitative determination of AA within PBMCs and plasma from human blood.

Highlights

There is an increasing interest in the potential health benefits of vitamin C, or ascorbic acid (AA)
The leukocyte AA level is more stable than serum AA level because it is less affected by dietary changes and is thought to be a more accurate assessment for AA
After re-suspending the peripheral blood mononuclear cells (PBMCs) in ammonium acetate, adding the precipitation reagent, and 10 min incubation, the lysis efficacy was evaluated by microscopy using Trypan blue

Summary

Introduction

There is an increasing interest in the potential health benefits of vitamin C, or ascorbic acid (AA). AA functions as an essential cofactor in numerous enzymatic reactions and as a potent antioxidant, due to its strong reducing potential [2]. Plasma and serum AA levels are very low compared with the rest of the human body and are highly affected by dietary intake, age, gender, and circadian rhythm. After oral intake of AA, the plasma AA increases rapidly, but this effect is only short-lived. These levels are thought to reflect the metabolic turnover rate of the vitamin [5,6]. The leukocyte AA level is more stable than serum AA level because it is less affected by dietary changes and is thought to be a more accurate assessment for AA storage [5]. AA is important for the development, proliferation, and function

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Discussion

Conclusion