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Basic nutritional investigation
Inhibition of suicidal erythrocyte death by vitamin C
Hasan Mahmud (Doctoral fellow), Syed M. Qadri (Doctoral fellow),Michael Fo¨ ller (Dr.med., Dr.rer.nat.), Florian Lang (Prof.Dr.med.)
Department of Physiology, University of Tu¨bingen, Tu¨bingen, Germany
a r t i c l e i n f o
Article history:
Received 3 November 2008Accepted 18 November 2009
Keywords:
PhosphatidylserineCell membrane disorganizationCalciumCell volumeApoptosis
a b s t r a c t
Objective:
Similar to apoptosis of nucleated cells, suicidal death of erythrocytes is paralleled by cellshrinkage and cell membrane disorganizationwith phosphatidylserine exposure at the erythrocytesurface. Triggers of suicidal erythrocyte death include cell shrinkage, energy depletion, andoxidative stress, challenges at least partially effective by increasing the cytosolic Ca
2
þ
concentra-tion. Apoptosis is inhibited by vitamin C. The present study thus explored whether vitamin Csimilarly influences suicidal erythrocyte death.
Methods:
The cytosolic Ca
2
þ
concentration was estimated from Fluo3 fluorescence, phosphati-dylserine exposure from annexin V-binding, and cell volume from forward scatter in fluorescenceactivated cell sorting (FACS) analysis.
Results:
Energy depletion (48 h glucose removal) increased the cytosolic Ca
2
þ
concentration,decreased the erythrocytic cell volume, and enhanced annexin V-binding. Similarly, cell shrinkageby 48 h replacement of extracellular chloride with gluconate and oxidative stress (30 min exposureto 0.3 mM tert-butylhydroperoxide) triggered suicidal erythrocyte death as evident from enhancedannexin V-binding. Vitamin C (up to 0.28 mM) did not significantly modify the cytosolic Ca
2
þ
concentration, annexin V-binding, and cell volume in the absence of stressors stimulating suicidalerythrocyte death but significantly attenuated the suicidal erythrocyte death following cellshrinkage, energy depletion, and oxidative stress.
Conclusion:
Vitamin C is a potent inhibitor of suicidal erythrocyte death.
Ó
2010 Elsevier Inc. All rights reserved.
Introduction
Similar to apoptosis of nucleated cells[1], suicidal death of erythrocytes[2]is characterized by exposure of phosphati-dylserine at the erythrocyte surface[3–5],which is the result of
phospholipid disorganization of the cell membrane[6]. Phos-phatidylserine-exposing erythrocytes are bound to phosphati-dylserine receptors on macrophages[7], which subsequentlyengulf and degrade the phosphatidylserine-exposing cells[8].Accordingly, dying erythrocytes are rapidly eliminated fromcirculating blood[9]. Cell membrane disorganization is triggeredby increase in cytosolic Ca
2
þ
concentration[3,4], which couldresult from cell shrinkage (chloride replacement by gluconate),oxidative stress[10], and energy depletion[11]. The cytosolic
Ca
2
þ
concentration is increased by entry through Ca
2
þ
2
þ
sensitivity of phospholipid disorganization is enhanced by ceramide[15].Besides stimulating cell membrane disorganization, Ca
2
þ
activatesCa
2
þ
-sensitiveK
þ
þ
exithyperpolarizes the cell membrane driving Cl
À
exit. The cellularloss of KCl and osmotically obliged water results in cellshrinkage[17].VitaminChaspreviouslybeenshowntoinhibitsuicidaldeathor apoptosis of nucleated cells[18–21], an effect which may atleast partially be due to its antioxidant activity[22,23].The present study thus explored the possibility that vitaminC similarly counteracts suicidal erythrocyte death.
Materials and methods
Erythrocytes, solutions, chemicals, and vitamin C measurement
Experiments were performed at 37
C with erythrocytes from concentratesprovided by the blood bank of the University of Tu¨ bingen. The study has beenapproved by the ethics committee of the University of Tu¨ bingen (184/2003 V).Erythrocytes were incubated at a hematocrit of 0.4% in Ringersolution containing (in mM) 125 NaCl, 5 KCl, 1 MgSO
4
, 32
Role of each author in the work
: Conception and design of the study (M. Fo¨ ller,F. Lang); generation, collection, assembly, analysis and/or interpretation of data(H. Mahmud, S. M. Qadri, M. Fo¨ller); drafting or revision of the manuscript(M. Fo¨ ller, F. Lang); approval of the final version of the manuscript (H. Mahmud,S. M. Qadri, M. Fo¨ ller, F. Lang).
*
Corresponding author. Tel:
þ
49-7071-29-72194; fax:
þ
49-7071-29-5618.
E-mail address:
Ó
2010 Elsevier Inc. All rights reserved.doi:10.1016/j.nut.2009.11.025
Contents lists available atScienceDirect
Nutrition
journal homepage:www.nutritionjrnl.com
Nutrition 26 (2010) 671–676
N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, 5 glucose, 1 CaCl
2
; pH 7.4at 37
C for the indicated time periods. Where stated, chloride was replaced bygluconate, glucose deleted from the medium, or tert-butylhydroperoxide(0.3 mM; Sigma,Schnelldorf, Germany)added. Removal of Cl
À
leadstoexitof KCland osmoticallyobligedwaterand thus tocell shrinkage,awell-knowntriggerof suicidal erythrocyte death. Vitamin C (Sigma) was used at concentrationsbetween 0.06 and 0.28 mM.Vitamin C was determined in the supernatant of erythrocytes incubated inRinger solution for 48 h (see above) by the laboratory for clinical chemistryDr. Ga¨ rtner (Ravensburg, Germany) using HPLC-UV according to clinicalstandards (DIN EN ISO 15189).
FACS analysis of annexin V-binding and forward scatter
Fluorescence activated cell sorting (FACS) analysis was performed asdescribed[24].After incubation under the respective experimental condition,
cells were washed in Ringer solution containing 5 mM CaCl
2
and then stainedwith Annexin V-Fluos (Roche, Mannheim, Germany) in this solution for 20 minunder protection from light. In the following, the forward scatter of the cells wasdetermined, and annexin V fluorescence intensity was measured in FL-1 with anexcitation wavelength of 488 nm and an emission wavelength of 530 nm ona FACS calibur (BD, Heidelberg, Germany).
Measurement of intracellular Ca
2
þ
After incubation under the respective experimental condition, erythrocyteswere washed in Ringer solution and then loaded with Fluo-3/AM (Calbiochem,Bad Soden,Germany)inRingersolution containing5mM CaCl
2
and 2
m
MFluo-3/AM. The cells were incubated at 37
C for 20 min and washed twice in Ringersolution containing 5 mM CaCl
2
. The Fluo-3/AM-loaded erythrocytes wereresuspended in 200
m
L Ringer. Then, Ca
2
þ
-dependent fluorescence intensity wasmeasured in fluorescence channel FL-1 in FACS analysis.
Determination of GSH and GSSG ratio
Human erythrocytes (5% hematocrit) were incubated for 48 h at 37
C inRinger solution with or without glucose in the presence or absence of 0.28 mMvitamin C. Then, the cells were againwashed twice inphosphate-buffered saline.All manipulations were then performed on ice. After lysis of 50
m
L of the eryth-rocyte pellet in 250
m
L distilled water and centrifugation at 14000 rpm, 150
m
L of the supernatant was deproteinated by adding 150
m
L metaphosphoric acid(10%).Glutathione (GSSGandGSH) wasmeasuredwiththeGlutathioneAssayKitfrom Cayman Chemicals (Tallinm, Estonia) according to the manufacturer’sprotocol. The GSH and GSSG concentrations refer to the concentrations withinerythrocytes.
Statistics
Data are expressed as arithmetic means
Æ
SEM, and statistical analysis wasmade using ANOVA with Tukey’s test as posttest, as appropriate.
Results
To explore the effect of vitamin C on erythrocytic Ca
2
þ
concentration, experiments were performed in erythrocytesloaded with the Ca
2
þ
-sensitive dye Fluo3. Exposure of theerythrocytes to vitamin C (up to 0.28 mM) did not significantlyalter the cytosolic Ca
2
þ
concentration in the presence of glucose(Fig.1B). A 48 h glucose deprivation of erythrocytes significantlyincreased cytosolic Ca
2
þ
2
þ
concentration during glucose depletion was significantlyattenuated (Fig.1A and1B).
After a 48 h incubation of erythrocytes in Ringer solutionoriginally containing 0.28 mM vitamin C, the concentration of vitamin C dropped to 30.1
Æ
1.7
m
M.Because an increase in the cytosolic Ca
2
þ
concentration isexpected to trigger cell membrane disorganization, phosphati-dylserine exposure at the cell surface was estimated fromannexin V-binding. In the presence of glucose, annexinV-binding was low and not significantly modified by vitamin C(0.11 mM) (Fig. 2B). Following a period of 48 h glucose depletionannexin V-binding was markedly and significantly increased(Fig. 2A and2B). In the presence of vitamin C (0.11 mM), the
stimulatingeffectofglucosedepletiononannexinV-bindingwassignificantly attenuated (Fig. 2A and2B).
As increased Ca
2
þ
concentration is expected to stimulateCa
2
þ
-sensitive K
þ
channels with subsequent cell shrinkage, thecell volume was estimated from forward scatter. As illustrated inFigure 3A, vitamin C was without significant effect on theerythrocyte forward scatter in the presence of glucose. Glucosedepletion was followed by a significant decrease of erythrocyteforwardscatter,aneffect significantlyattenuatedin thepresenceof vitamin C (0.06 mM) (Fig. 3A and3B).
Because energy depletion of erythrocytes is known to inter-fere with the intracellular gluthatione concentration, the GSHand GSSG levels were determined after a 48 h incubation of erythrocytes in Ringer with or without glucose in the absence orpresence of 0.28 mM vitamin C. As shown inFigure 3C, energydepletion indeed resulted in a significant reduction of theintracellular glutathione reduction. However, vitamin C did notsignificantly modify total glutathione or GSSH levels in thepresence or absence of glucose.In a further series of experiments, suicidal erythrocyte deathwas stimulated by induction of oxidative stress. To this end, the
Fig. 1.
Effect of glucose depletion on cytosolic Ca
2
þ
concentration in the presence and absence of vitamin C. (A) Histogram of Fluo3 fluorescence in a representativeexperiment 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 followingincubation 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 differencefrom the presence of glucose. #, ### (
P
<
0.05,
P
<
0.001) indicate significant difference from the absence of vitamin C (ANOVA).
H. Mahmud et al. / Nutrition 26 (2010) 671–676
672
erythrocytes were exposed to 0.3 mM tert-butylhydroperoxidein Ringer solution for 30 min. As shown inFigure 4, oxidativestress triggered annexin V-binding, an effect significantlyattenuated in the presence of vitamin C (0.11–0.28 mM).Vitamin C similarly interfered with the suicidal erythrocytedeath following cell shrinkage. As illustrated inFigure 5,removal of extracellular chloride (replacement by gluconate)was followed by an increase in annexin V-binding, an effectagain significantly attenuated in the presence of vitamin C(0.06–0.28 mM).
Discussion
The present study reveals, to our knowledge, a novel effect of vitaminC,i.e.aninhibitionofsuicidalerythrocytedeath.VitaminC may be taken up into cells by GLUT1[25], the major erythro-cyte glucose transporter[26]. Owing to rapid cellular uptake,vitamin C may be effective from the intracellular side. Vitamin Ccould in turn inhibit cellular glucose uptake[27]. Vitamin C is atleast partially effective through inhibition of Ca
2
þ
entry.Apparently, vitamin C is noteffective bycounteractingoxidation.As shown previously, vitamin C does not act as a prototypicantioxidant in erythrocytes subjected to oxidative stress[28].Suicidal erythrocyte death is a physiological mechanism,which protects against hemolysis[2]. Compromized Na
þ
/K
þ
ATPase activity or enhanced leakiness of the cell membranein defective erythrocytes is followed by cellular gain of Na
þ
andloss of K
þ
, deplolarization, and entry of Cl
À
[2]. The net gain of cellular electrolytes with osmotically obliged water leads to cellswelling[2]. Excessive cell swelling eventually results in ruptureof the cell membrane with release of cellular hemoglobin. Thereleased hemoglobin may be filtered in the renal glomerulathus occluding renal tubules. Phosphatidylserine at the surfaceof suicidal cells is recognized by macrophages, which clearaffected erythrocytes from the circulating blood prior tohemolysis. The activation of Ca
2
þ
-sensitive K
þ
channels withsubsequent hyperpolarization and KCl loss delays swelling of the suicidal cells. Suicidal erythrocyte death may be particularlyimportant for the clearance of parasitized erythrocytes inmalaria[29–31]. The malaria pathogen
Plasmodium falciparum
Fig. 2.
Stimulation of phosphatidylserine exposure by glucose depletion in the presence and absence of vitamin C. (A) Histogram of erythrocyte annexin V-binding ina representative experiment as inFigure 1.(B) Arithmetic means
Æ
SEM (
n
¼
5) of the percentage of phosphatidylserine-exposing erythrocytes following incubation as inFigure 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).
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 inFigure 1.(B) Arithmetic means
Æ
SEM (
n
¼
13–15 erythrocyte specimens; each specimen wasinvestigated in duplicates) of the normalized forward scatter of erythrocytes as inFigure 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 oxidizedglutathione (GSSG, open bars) and of total glutathione (total GSH, closed bars) of erythrocytes following incubation for 48 h in the presence (
þ
glu) or absence (
À
glu) of glucose in the presence of 0 or 0.28 mM vitamin C.
***
(
P
<
0.001) indicates significant difference from the presence of glucose.
H. Mahmud et al. / Nutrition 26 (2010) 671–676
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