Vitamin C in mouse and human red blood cells: an HPLC assay

Abstract

Although vitamin C (ascorbate) is present in whole blood, measurements in red blood cells (RBCs) are problematic because of interference, instability, limited sensitivity, and sample volume requirements. We describe a new technique using HPLC with coulometric electrochemical detection for ascorbate measurement in RBCs of humans, wild-type mice, and mice unable to synthesize ascorbate. Exogenously added ascorbate was fully recovered even when endogenous RBC ascorbate was below the detection threshold (25 nM). Twenty microliters of whole blood or 10 μl of packed RBCs was sufficient for assay. RBC ascorbate was stable for 24h from whole-blood samples at 4°C. Processed, stored samples were stable for >1 month at -80°C. Unlike other tissues, ascorbate concentrations in human and mouse RBCs were linear in relation to plasma concentrations (R=0.8 and 0.9, respectively). In healthy humans, RBC ascorbate concentrations were 9-57 μM, corresponding to ascorbate plasma concentrations of 15-90 μM. Mouse data were similar. In human blood stored as if for transfusion, initial RBC ascorbate concentrations varied approximately sevenfold and decreased 50% after 6 weeks of storage under clinical conditions. With this assay, it becomes possible for the first time to characterize ascorbate function in relation to endogenous concentrations in RBCs.

Published by Elsevier Inc.

Figures

Fig.1

Representative chromatograms of standards (red), RBC samples in 30% methanol/1 mM EDTA (green), and 30% methanol/1 mM EDTA alone (gray). 20 μl of packed mouse red blood cells from (A) wild-type mouse or (B) gulo−/− mouse was prepared as described under Methods. Injection volume was 10 μl. (A) Red, 1 μM ascorbate standard in 30% methanol/1 mM EDTA. 30% methanol/1 mM EDTA alone is shown for comparison. RBC concentration in the chromatogram as shown was 1.5 μM. When dilutions are considered, the final ascorbate concentration in wild-type RBCs was 20.8 μM. (B) Red, 25 nM ascorbate standard in 30% methanol/1 mM EDTA. 30% methanol/1 mM EDTA alone is shown for comparison. RBC concentration in the chromatogram as shown was approximately 26 nM. When dilutions are considered, the final ascorbate concentration in RBCs from this gulo−/− mouse was 370 nM. The gulo−/− mouse was on an ascorbate-free diet for approximately 8 weeks. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig.2

Recovery of ascorbate added to RBCs. RBC ascorbate was measured before and after ascorbate addition to water-lysed RBCs: from (A) wild-type (C57BL/6) mice or mice unable to synthesize vitamin C (gulonolactone oxidase knockout mice, gulo−/−) (inset) and from (B) human subjects. For all samples, the amount of added ascorbate for recovery of 100% would have changed the final internal RBC concentration by 3.6 μM. Internal RBC concentration prior to addition is indicated on the x axis. Packed RBC volumes were 20 or 40 μl, as indicated. Three samples were obtained from each of three wild-type mice, three gulo−/− mice, and four human subjects.

Fig.3

Vitamin C measurements in RBCs: effect of (A, B) various volumes of whole blood drawn initially and (C, D) volume of packed RBCs prepared for assay. Each line indicates that blood was obtained from the same mouse or human. On each line, every symbol represents the mean value ± SD for three individually processed blood samples, with variation of the whole-blood volume used or packed red cell volume used as indicated on the x axis. For all samples, final lysate volume after purified water addition was 100 μl. See Methods for details. (A, B) Volume indicates starting whole blood volume used for vitamin C RBC assay from (A) mice or (B) humans. (C, D) Packed RBC volumes prepared for assay from (C) mice and (D) humans. Packed RBC volumes varied from 1 to 40 μl as indicated. Right axis in (C) represents expanded scale for low ascorbic acid concentrations in RBCs from gulo−/− mice.

Fig.4

Ascorbic acid stability in RBC samples from whole blood, prepared samples in an HPLC autosampler, and samples in frozen storage. For all experiments, 20 μl packed RBCs was prepared as described under Methods; final lysis volume was 100 μl. Each line indicates that blood was obtained from the same mouse or human. On each line, every symbol represents the mean value ± SD for three individually processed blood samples, with variation in time as indicated on the x axis. (A) Mouse whole-blood samples were stored at 4 °C for times indicated before processing. (B) Human whole-blood samples were stored at 4 or 25 °C for the times indicated before processing. (C) Processed mouse or human RBC samples were kept in an autosampler at 4 °C for the times indicated before analyses. (D) Processed mouse or human RBC samples were stored at −80 °C for the weeks indicated before analyses.

Fig.5

RBC ascorbate concentrations as a function of plasma ascorbate concentrations in samples from (A) mouse and (B) human whole blood. (A) Mouse blood. Packed RBC volume was 20 μl, total water lysis volume 100 μl. Each symbol represents the mean ± SD of three separate samples from the same animal. The slope of the line is 0.6 and the y intercept at x = 0 is 1.5 μM. (B) Human blood. Packed RBC volume was 40 μl, total water lysis volume 200 μl. The slope of the line is 0.5 and the y intercept at x = 0 is 6.8 μM.

Fig.6

Ascorbate concentrations in stored human RBCs as a function of storage time. Packed RBCs from 11 healthy donors were prepared by standard transfusion medicine procedures and stored at 4 °C in a dark refrigerator for 6 weeks. Samples were taken at the weeks indicated. Packed RBC volume was 40 μl, total water lysis volume 200 μl. (A) RBC ascorbate concentration as a function of storage time. Each symbol represents the mean ± SD of three separate samples drawn from the same RBC storage bag. Each line and symbol type represents a different donor. (B) Loss of ascorbate in percentage compared to original RBC concentration.