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

Authors :
Gwendolyn van Gorkom
Birgit Gijsbers
Erik-Jan Ververs
Ahmed El Molla
Cindy Sarodnik
Celine Riess
Will Wodzig
Gerard Bos
Catharina Van Elssen
RS: GROW - R3 - Innovative Cancer Diagnostics & Therapy
Interne Geneeskunde
MUMC+: MA Hematologie (9)
RS: NUTRIM - R3 - Respiratory & Age-related Health
MUMC+: MA Med Staf Artsass Interne Geneeskunde (9)
Source :
Antioxidants, Antioxidants, Vol 11, Iss 134, p 134 (2022), Antioxidants; Volume 11; Issue 1; Pages: 134, Antioxidants, 11(1):134. Multidisciplinary Digital Publishing Institute (MDPI)
Publication Year :
2021

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.

Details

ISSN :
20763921
Volume :
11
Issue :
1
Database :
OpenAIRE
Journal :
Antioxidants (Basel, Switzerland)
Accession number :
edsair.doi.dedup.....fd334a6baf32a3f7c65c001db311c94e