1. Nutrition 26 (2010) 671–676
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Nutrition
journal homepage: www.nutritionjrnl.com
Basic nutritional investigation
Inhibition of suicidal erythrocyte death by vitamin C
Hasan Mahmud (Doctoral fellow), Syed M. Qadri (Doctoral fellow),
¨
Michael Foller (Dr.med., Dr.rer.nat.), Florian Lang (Prof.Dr.med.) *
¨ ¨
Department of Physiology, University of Tubingen, Tubingen, Germany
a r t i c l e i n f o a b s t r a c t
Article history: Objective: Similar to apoptosis of nucleated cells, suicidal death of erythrocytes is paralleled by cell
Received 3 November 2008 shrinkage and cell membrane disorganization with phosphatidylserine exposure at the erythrocyte
Accepted 18 November 2009 surface. Triggers of suicidal erythrocyte death include cell shrinkage, energy depletion, and
oxidative stress, challenges at least partially effective by increasing the cytosolic Ca2þ concentra-
Keywords: tion. Apoptosis is inhibited by vitamin C. The present study thus explored whether vitamin C
Phosphatidylserine
similarly influences suicidal erythrocyte death.
Cell membrane disorganization
Methods: The cytosolic Ca2þ concentration was estimated from Fluo3 fluorescence, phosphati-
Calcium
Cell volume dylserine exposure from annexin V-binding, and cell volume from forward scatter in fluorescence
Apoptosis activated cell sorting (FACS) analysis.
Results: Energy depletion (48 h glucose removal) increased the cytosolic Ca2þ concentration,
decreased the erythrocytic cell volume, and enhanced annexin V-binding. Similarly, cell shrinkage
by 48 h replacement of extracellular chloride with gluconate and oxidative stress (30 min exposure
to 0.3 mM tert-butylhydroperoxide) triggered suicidal erythrocyte death as evident from enhanced
annexin V-binding. Vitamin C (up to 0.28 mM) did not significantly modify the cytosolic Ca2þ
concentration, annexin V-binding, and cell volume in the absence of stressors stimulating suicidal
erythrocyte death but significantly attenuated the suicidal erythrocyte death following cell
shrinkage, energy depletion, and oxidative stress.
Conclusion: Vitamin C is a potent inhibitor of suicidal erythrocyte death.
Ó 2010 Elsevier Inc. All rights reserved.
Introduction Ca2þ concentration is increased by entry through Ca2þ-
permeable cation channels [5,12–14]. The Ca2þ sensitivity of
Similar to apoptosis of nucleated cells [1], suicidal death of phospholipid disorganization is enhanced by ceramide [15].
erythrocytes [2] is characterized by exposure of phosphati- Besides stimulating cell membrane disorganization, Ca2þ
dylserine at the erythrocyte surface [3–5], which is the result of activates Ca2þ-sensitive Kþ channels [16]. The subsequent Kþ exit
phospholipid disorganization of the cell membrane [6]. Phos- hyperpolarizes the cell membrane driving ClÀ exit. The cellular
phatidylserine-exposing erythrocytes are bound to phosphati- loss of KCl and osmotically obliged water results in cell
dylserine receptors on macrophages [7], which subsequently shrinkage [17].
engulf and degrade the phosphatidylserine-exposing cells [8]. Vitamin C has previously been shown to inhibit suicidal death
Accordingly, dying erythrocytes are rapidly eliminated from or apoptosis of nucleated cells [18–21], an effect which may at
circulating blood [9]. Cell membrane disorganization is triggered least partially be due to its antioxidant activity [22,23].
by increase in cytosolic Ca2þ concentration [3,4], which could The present study thus explored the possibility that vitamin
result from cell shrinkage (chloride replacement by gluconate), C similarly counteracts suicidal erythrocyte death.
oxidative stress [10], and energy depletion [11]. The cytosolic
Materials and methods
¨
Role of each author in the work: Conception and design of the study (M. Foller, Erythrocytes, solutions, chemicals, and vitamin C measurement
F. Lang); generation, collection, assembly, analysis and/or interpretation of data
¨
(H. Mahmud, S. M. Qadri, M. Foller); drafting or revision of the manuscript Experiments were performed at 37 C with erythrocytes from concentrates
¨
(M. Foller, F. Lang); approval of the final version of the manuscript (H. Mahmud, ¨
provided by the blood bank of the University of Tubingen. The study has been
¨
S. M. Qadri, M. Foller, F. Lang). ¨
approved by the ethics committee of the University of Tubingen (184/2003 V).
* Corresponding author. Tel: þ49-7071-29-72194; fax: þ49-7071-29-5618. Erythrocytes were incubated at a hematocrit of 0.4% in Ringer
E-mail address: florian.lang@uni-tuebingen.de (F. Lang). solution containing (in mM) 125 NaCl, 5 KCl, 1 MgSO4, 32
0899-9007/$ – see front matter Ó 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.nut.2009.11.025

2. 672 H. Mahmud et al. / Nutrition 26 (2010) 671–676
N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, 5 glucose, 1 CaCl2; pH 7.4 Results
at 37 C for the indicated time periods. Where stated, chloride was replaced by
gluconate, glucose deleted from the medium, or tert-butylhydroperoxide
(0.3 mM; Sigma, Schnelldorf, Germany) added. Removal of ClÀ leads to exit of KCl
To explore the effect of vitamin C on erythrocytic Ca2þ
and osmotically obliged water and thus to cell shrinkage, a well-known trigger of concentration, experiments were performed in erythrocytes
suicidal erythrocyte death. Vitamin C (Sigma) was used at concentrations loaded with the Ca2þ-sensitive dye Fluo3. Exposure of the
between 0.06 and 0.28 mM. erythrocytes to vitamin C (up to 0.28 mM) did not significantly
Vitamin C was determined in the supernatant of erythrocytes incubated in
alter the cytosolic Ca2þ concentration in the presence of glucose
Ringer solution for 48 h (see above) by the laboratory for clinical chemistry
¨
Dr. Gartner (Ravensburg, Germany) using HPLC-UV according to clinical (Fig. 1B). A 48 h glucose deprivation of erythrocytes significantly
standards (DIN EN ISO 15189). increased cytosolic Ca2þ concentration (Fig. 1A and 1B). In the
presence of vitamin C, the increase in the cytosolic Ca2þ
concentration during glucose depletion was significantly
FACS analysis of annexin V-binding and forward scatter
attenuated (Fig. 1A and 1B).
Fluorescence activated cell sorting (FACS) analysis was performed as After a 48 h incubation of erythrocytes in Ringer solution
described [24]. After incubation under the respective experimental condition, originally containing 0.28 mM vitamin C, the concentration of
cells were washed in Ringer solution containing 5 mM CaCl2 and then stained vitamin C dropped to 30.1 Æ 1.7 mM.
with Annexin V-Fluos (Roche, Mannheim, Germany) in this solution for 20 min
under protection from light. In the following, the forward scatter of the cells was
Because an increase in the cytosolic Ca2þ concentration is
determined, and annexin V fluorescence intensity was measured in FL-1 with an expected to trigger cell membrane disorganization, phosphati-
excitation wavelength of 488 nm and an emission wavelength of 530 nm on dylserine exposure at the cell surface was estimated from
a FACS calibur (BD, Heidelberg, Germany). annexin V-binding. In the presence of glucose, annexin
V-binding was low and not significantly modified by vitamin C
Measurement of intracellular Ca2þ (0.11 mM) (Fig. 2B). Following a period of 48 h glucose depletion
annexin V-binding was markedly and significantly increased
After incubation under the respective experimental condition, erythrocytes (Fig. 2A and 2B). In the presence of vitamin C (0.11 mM), the
were washed in Ringer solution and then loaded with Fluo-3/AM (Calbiochem,
stimulating effect of glucose depletion on annexin V-binding was
Bad Soden, Germany) in Ringer solution containing 5 mM CaCl2 and 2 mM Fluo-3/
AM. The cells were incubated at 37 C for 20 min and washed twice in Ringer
significantly attenuated (Fig. 2A and 2B).
solution containing 5 mM CaCl2. The Fluo-3/AM-loaded erythrocytes were As increased Ca2þ concentration is expected to stimulate
2þ
resuspended in 200 mL Ringer. Then, Ca2þ-dependent fluorescence intensity was Ca -sensitive Kþ channels with subsequent cell shrinkage, the
measured in fluorescence channel FL-1 in FACS analysis. cell volume was estimated from forward scatter. As illustrated in
Figure 3A, vitamin C was without significant effect on the
Determination of GSH and GSSG ratio erythrocyte forward scatter in the presence of glucose. Glucose
depletion was followed by a significant decrease of erythrocyte
Human erythrocytes (5% hematocrit) were incubated for 48 h at 37 C in forward scatter, an effect significantly attenuated in the presence
Ringer solution with or without glucose in the presence or absence of 0.28 mM
of vitamin C (0.06 mM) (Fig. 3A and 3B).
vitamin C. Then, the cells were again washed twice in phosphate-buffered saline.
All manipulations were then performed on ice. After lysis of 50 mL of the eryth- Because energy depletion of erythrocytes is known to inter-
rocyte pellet in 250 mL distilled water and centrifugation at 14000 rpm, 150 mL fere with the intracellular gluthatione concentration, the GSH
of the supernatant was deproteinated by adding 150 mL metaphosphoric acid and GSSG levels were determined after a 48 h incubation of
(10%). Glutathione (GSSG and GSH) was measured with the Glutathione Assay Kit erythrocytes in Ringer with or without glucose in the absence or
from Cayman Chemicals (Tallinm, Estonia) according to the manufacturer’s
protocol. The GSH and GSSG concentrations refer to the concentrations within
presence of 0.28 mM vitamin C. As shown in Figure 3C, energy
erythrocytes. depletion indeed resulted in a significant reduction of the
intracellular glutathione reduction. However, vitamin C did not
significantly modify total glutathione or GSSH levels in the
Statistics
presence or absence of glucose.
Data are expressed as arithmetic means Æ SEM, and statistical analysis was In a further series of experiments, suicidal erythrocyte death
made using ANOVA with Tukey’s test as posttest, as appropriate. was stimulated by induction of oxidative stress. To this end, the
Fig. 1. Effect of glucose depletion on cytosolic Ca2þ concentration in the presence and absence of vitamin C. (A) Histogram of Fluo3 fluorescence in a representative
experiment of erythrocytes from healthy volunteers incubated for 48 h without glucose in the absence (1, red line) and presence (2, black line) of 0.11 mM vitamin C. (B)
Arithmetic means Æ SEM (n ¼ 15 erythrocyte specimens; each specimen was investigated in duplicates) of the normalized Fluo3 fluorescence in erythrocytes following
incubation for 48 h in the presence (open bars) or absence (closed bars) of glucose in the presence of 0–0.28 mM vitamin C. *** (P 0.001) indicates significant difference
from the presence of glucose. #, ### (P 0.05, P 0.001) indicate significant difference from the absence of vitamin C (ANOVA).

3. H. Mahmud et al. / Nutrition 26 (2010) 671–676 673
Fig. 2. Stimulation of phosphatidylserine exposure by glucose depletion in the presence and absence of vitamin C. (A) Histogram of erythrocyte annexin V-binding in
a representative experiment as in Figure 1. (B) Arithmetic means Æ SEM (n ¼ 5) of the percentage of phosphatidylserine-exposing erythrocytes following incubation as in
Figure 1. *** (P 0.001) indicates significant difference from the presence of glucose. # (P 0.001) indicates significant difference from the absence of vitamin C (ANOVA).
erythrocytes were exposed to 0.3 mM tert-butylhydroperoxide Apparently, vitamin C is not effective by counteracting oxidation.
in Ringer solution for 30 min. As shown in Figure 4, oxidative As shown previously, vitamin C does not act as a prototypic
stress triggered annexin V-binding, an effect significantly antioxidant in erythrocytes subjected to oxidative stress [28].
attenuated in the presence of vitamin C (0.11–0.28 mM). Suicidal erythrocyte death is a physiological mechanism,
Vitamin C similarly interfered with the suicidal erythrocyte which protects against hemolysis [2]. Compromized Naþ/
death following cell shrinkage. As illustrated in Figure 5, KþATPase activity or enhanced leakiness of the cell membrane
removal of extracellular chloride (replacement by gluconate) in defective erythrocytes is followed by cellular gain of Naþ and
was followed by an increase in annexin V-binding, an effect loss of Kþ, deplolarization, and entry of ClÀ [2]. The net gain of
again significantly attenuated in the presence of vitamin C cellular electrolytes with osmotically obliged water leads to cell
(0.06–0.28 mM). swelling [2]. Excessive cell swelling eventually results in rupture
of the cell membrane with release of cellular hemoglobin. The
Discussion released hemoglobin may be filtered in the renal glomerula
thus occluding renal tubules. Phosphatidylserine at the surface
The present study reveals, to our knowledge, a novel effect of of suicidal cells is recognized by macrophages, which clear
vitamin C, i.e. an inhibition of suicidal erythrocyte death. Vitamin affected erythrocytes from the circulating blood prior to
C may be taken up into cells by GLUT1 [25], the major erythro- hemolysis. The activation of Ca2þ-sensitive Kþ channels with
cyte glucose transporter [26]. Owing to rapid cellular uptake, subsequent hyperpolarization and KCl loss delays swelling of
vitamin C may be effective from the intracellular side. Vitamin C the suicidal cells. Suicidal erythrocyte death may be particularly
could in turn inhibit cellular glucose uptake [27]. Vitamin C is at important for the clearance of parasitized erythrocytes in
least partially effective through inhibition of Ca2þ entry. malaria [29–31]. The malaria pathogen Plasmodium falciparum
Fig. 3. Forward scatter and intracellular glutathione concentration prior to and following glucose depletion in the presence and absence of vitamin C. (A) Histogram of
erythrocyte forward scatter in a representative experiment of erythrocytes as in Figure 1. (B) Arithmetic means Æ SEM (n ¼ 13–15 erythrocyte specimens; each specimen was
investigated in duplicates) of the normalized forward scatter of erythrocytes as in Figure 1. *** (P 0.001) indicates significant difference from the presence of glucose, ###
(P 0.001) indicates significant difference from the absence of vitamin C (ANOVA). (C) Arithmetic means Æ SEM (n ¼ 5) of the intracellular concentration of oxidized
glutathione (GSSG, open bars) and of total glutathione (total GSH, closed bars) of erythrocytes following incubation for 48 h in the presence (þglu) or absence (Àglu) of
glucose in the presence of 0 or 0.28 mM vitamin C. *** (P 0.001) indicates significant difference from the presence of glucose.

4. 674 H. Mahmud et al. / Nutrition 26 (2010) 671–676
Fig. 4. Stimulation of phosphatidylserine exposure by oxidative stress in the presence and absence of vitamin C. (A) Histogram of erythrocyte annexin V-binding in
a representative experiment of erythrocytes from healthy volunteers incubated for 30 min in the presence of tert-butylhydroperoxide (0.3 mM) in the absence (1, red line)
and presence (2, black line) of 0.11 mM vitamin C. (B) Arithmetic means Æ SEM (n ¼ 12) of the percentage of annexin V-binding erythrocytes following incubation for 30 min
in the absence (open bars) or presence (closed bars) of tert-butylhydroperoxide (0.3 mM) in the presence of 0–0.28 mM vitamin C. *** (P 0.001) indicates significant
difference from the absence of oxidative stress, # (P 0.05) indicates significant difference from the absence of vitamin C (ANOVA).
induces oxidative stress to the infected erythrocytes, thus trig- Accordingly, suicidal erythrocytes may participate in the vascular
gering suicidal erythrocyte death. The clearance of the suicidal injury of metabolic syndrome [57].
infected erythocytes contributes to the elimination of the At least in theory, the effect of vitamin C on suicidal eryth-
parasite [29–31]. rocyte death may counteract the development of anemia and the
Excessive suicidal erythrocyte death may, however, lead to derangement of microcirculation during the course of the above
anemia. As a matter of fact, in addition to malaria, several anemic diseases or following ingestion of the above substances. As
conditions are secondary to accelerated suicidal erythrocyte a matter of fact, vitamin C administration has proven beneficial
death, such as iron deficiency [9], hemolytic uremic syndrome in sepsis [58–63], in Wilson’s disease [64], and in a variety of
[32], sepsis [33], phosphate depletion [2], and Wilson’s disease further conditions including treatment with antineoplastic drugs
[34]. Moreover, suicidal erythrocyte death is triggered by [19] and radiocontrast agents [65,66].
a variety of substances including IgA antibodies [35], PGE2 [2], In conclusion, vitamin C is a potent inhibitor of suicidal
cordycepin [36], methylglyoxal [37], amyloid peptides [38], erythrocyte death during energy depletion, cell shrinkage, and
lipopeptides [39], retinoic acid [40], paclitaxel [2], amantadine oxidative stress. The antieryptotic effect of vitamin C may
[41], amiodarone [42], anandamide [43], chlorpromazine [2], contribute to its beneficial effect in several diseases and
ciglitazone [44], cyclosporine [2], Bay-5884 [2], curcumin [45], following ingestion of a variety of substances.
valinomycin [46], listeriolysin [47], radiocontrast agents [48],
aluminium [2], lead [2], mercury [2], copper [34], cadmium [49], Acknowledgments
selenium [50], vanadate [51], gold [52], and arsenic [53].
Phosphatidylserine-exposing erythrocytes may adhere to the The authors gratefully acknowledge the meticulous prepara-
vascular wall [54,55] and stimulate the assembly of pro- ¨
tion of the manuscript by Lejla Subasic, Sari Rube, and Tanja Loch
thrombinase and tenase, thus fostering blood clotting [54,56]. and the experimental support by Sergios Gatidis and Vanessa
Fig. 5. Stimulation of phosphatidylserine exposure by chloride removal in the presence and absence of vitamin C. (A) Histogram of erythrocyte annexin V-binding in
a representative experiment of erythrocytes from healthy volunteers incubated for 48 h without chloride in the absence (1, red line) and presence (2, black line) of 0.11 mM
vitamin C. (B) Arithmetic means Æ SEM (n ¼ 7–8) of the percentage of phosphatidylserine-exposing erythrocytes following incubation for 48 h in the presence (open bars) or
absence (closed bars) of chloride in the presence of 0–0.28 mM vitamin C. *** (P 0.001) indicates significant difference from the presence of chloride, ### (P 0.001)
indicates significant difference from the absence of vitamin C (ANOVA).

5. H. Mahmud et al. / Nutrition 26 (2010) 671–676 675
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