THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 264, No. 35, Issue of December 15, pp.

21340-21345, 1989
0 1989 by The American Society for Biochemistry and Molecular Biology, Inc Prlnted in U.S.A.

Hyperglycemia Can Cause Membrane Lipid Peroxidation andOsmotic

Fragility in Human Red Blood Cells*
(Received for publication, February 24, 1989)

Sushi1 K. Jain
From the Department of Pediatrics, Louisiana State University School of Medicine, Shreueport, Louisiana 71130

The present study has examined the effect of elevated peroxidation apparentlyinvolves NADPH andcytochrome P -
glucose levels on membrane lipid peroxidation and os- 450-like activity.
motic fragility in human red blood cells (RBC). Defi-
brinated whole blood or RBC were incubated with MATERIALS ANDMETHODS
varying concentrations of glucose at 37 C for 24 h.
Blood fromadultvolunteers was collected into tubes with and
RBC incubated with elevated levelsof glucose showed without EDTA (10.5 mg/7 ml). The blood without EDTA was im-
a significantly increased membrane lipid peroxidation mediately defibrinated by rotating hardwood applicator sticks in the
when compared with control RBC. A significant posi- tubes so that fibrin adhered to the sticks before clotting. This defi-
tive correlation was observed between the extent of brinated blood was used as such in some experiments. Blood with
glucose-induced membrane lipid peroxidation and the EDTA was filtered through cotton wool to remove leukocytes and
osmotic fragilityof treated RBC. Glucose-induced then centrifuged a t 2000 rpm for 7 min in a refrigerated centrifuge.
membrane lipid peroxidation and osmotic fragility The RBC were washed with cold 0.15 M NaCl solution 3 times after
were blocked when RBC were pretreated with fluo- 1:lO dilution.
ride, an inhibitor of glucose metabolism; with vitamin Treatment with Glucose-Defibrinated blood or washed RBC sus-
E, an antioxidant; with para-chloromercurobenzoate pended to 40-45% hematocrit in phosphate-buffered saline (PBS,
made up of 8.1 g of NaCI, 2.302 g of Na2HP04,0.194 g of NaH2P04,
and metyrapone, inhibitors of the cytochrome P-450 pH 7.4) were taken in Erlenmeyer flasks and incubated with varying
system; or with dimethylfurane, diphenylamine, and glucose concentrations in a shaking water bath at 37 C for 24 h.
thiourea, scavengers of oxygen radicals. RBC treated Glucose was estimated in RBC suspensions at 0 h, i.e. immediately
with elevated glucose concentrations also showed an after the addition of stock glucose and again after 24 h of incubation.
increase in NADPH levels. Exogenous addition of Control RBC suspensions contained 5 mM glucose because normal
NADPH to normal RBC lysate induced membrane lipid blood glucose level is 4-6 mM. In the experiments with blood, flasks
peroxidation similar to that observed in the glucose- without exogenous glucose (i.e. those with normal glucose levels) were
treated RBC. These data suggest that elevated glucose used as controls. Glucose levels in flasks containing blood were
slightly different than those in flaskscontaining RBC in buffer.
levels can cause the peroxidation of membrane lipids Flasks containing 35 and 45 mM glucose showed between 1 and 1.5%
in human RBC. hemolysis; other flasks showed less than 0.5% hemolysis at the end
of 24 h of incubation. The extent of hemolysis on glucose treatment
was similar in blood and PBS-RBC. At the end of treatment, RBC
were washed 3 times after 1 : l O dilution with 0.15 M NaCl for bio-
Elevated levels of glucose in themedium or blood are known chemical and osmotic fragility analyses. Because glucose may inter-
fere in the TBA reactivity and osmotic fragility assays, washed RBC
to cause membrane damage and cell death of cultured peri- were measured for glucose to make sure that they were glucose-free.
cytes,endothelial cells, kidney cells, retinal cells, and red Treatment with Malonyldialdehyde (MDA)-RBC suspensions (40-
blood cells (RBC) (1-5). However, the biochemical mecha- 45% hematocrit) in PBS were treated with various concentrations of
nism that results in membrane damage and cell death is not MDA in Erlenmeyer flasks for 24 h in a shaking water bath at 37 C
known. It hasbeen proposed that nonenzymaticglycosylation (12). MDA was freshly prepared by acid hydrolysis of malonaldehyde
of certain proteins results in their irreversible cross-linking, bis(dimethylaceta1) (Aldrich) as described previously (12) and then
neutralized with NaOH tobring its pH to7. MDA-treated RBC were
which may contribute to the loss of elasticity characteristic washed 3 times with cold 0.15 M NaCl before lipid extraction or TBA
of cells exposed to hyperglycemia (6). Membrane lipids are reactivity measurement.
vital for the maintenance of cellular integrity and survival. Measurement of Lipid Peroxidation-Membrane lipid peroxidation
Peroxidation of membrane lipids can result in the inactivation in the glucose-treated RBC was determined by the TBA reactivity of
of enzymes and cross-linkingof membrane lipids and proteins MDA, an end productof fatty acid peroxidation (13).For this purpose,
and in cell death (7-11). By using human RBC, the present 0.2 ml of packed RBC were suspended in 0.8 ml of PBS (made up of
8.1 g of NaCl, 2.302 g of Na2HP04,0.194 g of NaH2P04/liter, pH7.4)
study has documented that elevated glucose levels per se can and 0.025 ml of butylated hydroxytoluene (88 mg/lO ml of alcohol).
cause peroxidation of membrane lipids and increased mem- To this was added 0.5 ml of 30% trichloroacetic acid. Tubes were
brane osmotic fragility. This glucose-induced membrane lipid vortexed, allowed to stand inice for at least 2 h, and thencentrifuged
at 2000 rpm for 15 min. One ml each of the supernatant was trans-
* This study was supported by t,he National American Diabetes ferred into another tube. To this was added 0.075 ml of 0.1 M EDTA
Association, National Institutes of Health Grant lROl HL 30247, and 0.25 ml of 1%TBA in 0.05 N NaOH. Tubes were mixed and kept
and by a grant-in-aid from Hoffmann-La Roche. The costs of publi- in a boiling water bath for 15 min. Absorbance was read at 532 and
cation of this article were defrayed in part by the payment of page 600 nm after tubes were cooled to room temperature ina double beam
charges. This article must therefore be hereby marked aduertise- lambda 3B Perkin-Elmer spectrophotometer. In this study, butylated
ment in accordance with 18 U.S.C. Section 1734 solely to indicate hydroxytoluene was added to RBC to prevent any initiation of mem-
this fact. brane lipid peroxidation during the assay. An addition of butylated
The abbreviations used are: RBC, red blood cell(s);MDA, malon- hydroxytoluene to standardMDA did not affect its color development
yldialdehyde; TBA, thiobarbituric acid PBS, phosphate-buffered sa- with the TBA. Absorbance a t 600 nm was also subtracted from
line; PE, phosphatidylethanolamine: PS, phosphatidylserine; pCMB, absorbance at 532 nm before calculating the TBA reactivity of RBC.
para-chloromercuribenzoate. MDA values in nanomoles/ml RBC or nanomoles/g hemoglobin (Hb)

21340
 Hyperglycemia and Membrane Lipid Peroxidation 21341
were determined using the extinction coefficient of the MDA-TBA
complex at 532 nm = 1.56 X io5 per cm per molar solution. Packed
cell volume was determined by using an Autocrit centrifuge; Hb was
- Blood
A.......a RBC-PBS
determined using the Coulter Counter.
Measurement of MDA by the TBA reactivity is the method most
widely used to assess lipid peroxidation (14).Using an additional new
method, peroxidative membrane damage was also assessed by deter-
mining the amount of heterologous phospholipid-MDA adduct sepa-
j
...........{

rated by TLC of RBC lipid extracts (15). The quantitation of phos-

pholipid-MDA adduct onTLC gives the amountof MDA cross-linked
between PE andPS. Details of lipid extraction and TLC are described
in an earlier study (16). Forfurther characterization of the phospho-
lipid-MDA adduct formed in glucose-treated RBC, the lipid was 5 15 25 35
scraped into a tube and eluted with chloroform/methanol (2:l). The
eluate was dried with nitrogen and redissolved into a known amount
of chloroform for quantitation of phosphorus and amino acids. For
amino acid determination, a portion of chloroform extract was dried
in a Teflon-stoppered tube and thenhydrolyzed with 1 ml of 6 N HCI
in an oven at 100 "C for 4 h. The acid hydrolysate was neutralized
with KOH. Control tubes containing standard serine and ethanola-
mine (Sigma) were simultaneously treated with acid. Separation of
amino acid in the extractwas done by thin layer chromatography on
Silica Gel 60 glass plates (Brinkmann) using the solventsystem,
butanol/pyridine/aceticacid/water (303010:10, v/v). Amino acids 5 15 25 35 45
were visualized with ninhydrin spray and quantitated spectrophoto-
metrically as described by Pataki (17).
Treatment withNADPH-The RBC in PBS were lysed by freezing
(1 h, -20 "C) and thawing. The hemolysate was treated with 2 mM
authentic NADPH (Sigma) for 24 h at 37 "C. The membrane lipid
peroxidation was determined as described for RBC.
Glucose utilization in thecell suspension was determined by meas-
uring the decrease in the glucose levels at 0 h and after 24 h of
incubation. Glucose was quantitated using o-toluidine as described in
Sigma kit no. 63. NADPH was measured by the enzymatic cycling
method of Lowry et al. (18).Concentration of NADPH in 20 pl of the
cell suspension or standard NADPH solution was determined by
using a molar extinction coefficient of E s = 6.22.
~ Fluorescence
~ ~ ~
measurements were made in the Perkin-Elmer spectrofluorometer, 5 15 25 35 45
model 650-10. Osmotic fragility was determined by measuring the Glucose, mM
extent of hemolysis in hypotonic NaCl solution (19).Osmotic fragility
of glucose-treated RBCis expressed aspercent of control value FIG. 1. TBA reactivity (MDA) and phospholipid-MDA ad-
because RBC from different individuals are known to have a large duct formation in RBC treated with elevated glucose levels.
variation in osmotic fragility. RBC sorbitol was determined using the Values are mean f S.D. of six experimentseach. PBS-RBCor
sorbitol dehydrogenase method as described by Malone et al. (20) and defibrinated blood was incubated with different concentrations of
Williams-Ashman (21). Mean cell volume and mean cell hemoglobin glucose for 24 h. PL, phospholipid. Note increase in the membrane
of glucose-treated RBC were determined using the Coulter Counter. lipid peroxidation in RBC treated with elevated glucose levels.
All incubations contained 10~1 of pen-strept/ml of cell suspension
to vitiate any microbial growth during overnight incubations. Pen-
strept contained 300 mg of penicillin G and 500 mg of streptomycin change in MDA values in glucose-treated RBC compared with
in 10 ml of distilled water. All biochemicals were purchased from control RBC was similar when values are expressed per g of
Sigma. Data were analyzed using the nonpaired Student's t test. Hb (Fig. 1, lower panel) compared with per ml of RBC (Fig.
1, middle panel).
RESULTS
Fig. 2 illustrates TLC of lipids of RBC treated with varying
Fig. 1 shows the effect of elevated levels of glucose on the concentrations of glucose for 24 h. TLC shows the appearance
TBA reactivity and phospholipid-MDA adduct formation in of a new lipid marked as an adduct between PE and PS in
RBC. The lower and middle panels show an increased TBA RBC-PBS treated with elevated glucose concentrations. Co-
reactivity, when the RBC-PBS suspension or defibrinated chromatography ruled out thisnew spot being an intermediate
blood was incubated with elevated levels of glucose,suggesting of glucose metabolism such as glucose-6-P04,fructose-6-P04,
the occurrence of lipid peroxidation in glucose-treated RBC. or glyceraldehyde-3-PO4.This new lipid spot was phosphorus-
Similarly, the upper panel in Fig. 1 shows an increased phos- positive and ninhydrin- and sugar-negative on spraying with
pholipid-MDA adduct formation inRBC treated with elevated specific reagents. Further characterization by acid hydrolysis
glucose levels, which also suggests an increased accumulation suggested that the new phospholipid spot contained PS, PE,
of MDA and lipid peroxidation in these RBC. There was a and MDA. The molar ratio of serinetophosphorus and
significant correlation between the glucose treatment and ethanolamine to phosphorus was 0.40 and 0.47, respectively,
phospholipid-MDA adduct (0.94 for RBC-PBS and 0.91 for and the TBA reactivity to phosphorus in the eluate of phos-
blood) or MDA (TBA reactivity, 0.93 and 0.89 when expressed pholipid adduct was 0.54. It appears that the new lipid spot
per ml of RBC and 0.92 and 0.87 when expressed per gof Hb, in glucose-treated RBC is formed by the cross-linking of PE,
respectively). Mean cell volume (fim3)was 85.8 -+ 0.6, 85.5 k PS, and MDA, but the exact nature of the cross-linking
0.6, 85.1 f 0.7, 85.8 & 0.2, and 86.0 f 0.3, and mean cell between these membrane components is not known and needs
hemoglobin (pg) was 28.4 k 1.1, 28.9 f 0.7, 28.5 5 1.2, 29.1 f further study.
0.8,28.6 f 1.3 (mean f S.D.) of RBC after treatmentof RBC- Fig. 2 also shows that a similar new phospholipid can be
PBS suspension with 5, 15, 25, 35, and 45 mM glucose. This formed when normal RBC are treated in vitro with authentic
shows that elevated glucose leveltreatment does not have any MDA. TBA reactivity of MDA-treated washed RBC was 1.0
effect on MCV and mean cell hemoglobin of RBC. Percentage & 0.3, 2.9 f 0.8, 5.9 f 1.8, 12.2 -C 4.3, 17.7 f 6.1, and 24.1 f
21342 Hyperglycemia andPeroxidation
Membrane
Lipid
120 -

I PE 100 .
Adduct
PS+PI
PC

SM

( I J

5 15 25 35 45 0 515 10 20 25
Glucose,mM Malonyldialdehyde.rnM
Lactate.mM

FIG.2. TLC of lipids of RBC after treatmentfor 24 h with

varying concentrations of glucose or MDA and lactic acid to
RBC-PBS. After the development, the TLC plate was exposed to
iodine vapors for visualization of phospholipid spots. The arrow
indicates the presence of the phospholipid-MDA adduct in glucose-
treated RBC similar to MDA-treated erythrocytes. This phospho-
lipid-MDA adduct spot was phosphorus-positive and ninhydrin- and I . . . . . . . . .
glucose-negative. 0,origin; SM, sphingomyelin; PC, phosphatidyl- o 25s 15 35 45
choline; PI, phosphatidylinositol;Adduct,phospholipid-MDA adduct; GLUCOSE, mM
SF, solvent front.
FIG.3. Osmotic fragility of glucose-treated RBC. Blood or
RBC-PBS were treated with varying concentrations of glucose for 24
9.8 nmol/ml packed cells; phospholipid-MDA adduct forma- h. Values are mean f S.D. of six experiments each.
tion was 0.1 & 0.1, 0.3 & 0.1, 0.5 f 0.2, 0.7 f 0.2, 1.2 f 0.5,
1.5 f 0.3 (percent of total phospholipid) when RBC were
treated in uitro for 24 h with 0,2.5,5, 10,15,20 mM or pmol/ TABLE I
ml MDA, respectively. The effect of sodium barbital on sorbitol, osmotic fragility, TBA
Glucose treatment for 24 h also caused accumulation of reactivity, and phospholipid (PL)-MDA adduct formation in RBC
lactic acid to a level of20.2 f 0.6 mM in 45 mM glucose- after treatment of RBC-PBS withelevated glucose levels
treated and 12.7 f 0.6 mM in 5 mM glucose-treated (control) Values are mean f S.D. of four experiments. Differences between
cell-PBS suspensions. As illustrated in Fig. 2 (right) and by values of RBC treated with and without sodium barbital at thesame
glucoselevels arenot significant. Negative value is because the
TBA reactivity of treated cells, lactic acid (25 mM) treatment osmotic fragility of this group was always less than control. RBC-
to RBC by itself does not seem to contribute to the lipid PBS suspension was pretreated with sodium barbital for 15 min a t
peroxidation and phospholipid-MDA adduct formation. Glu- 37 "C before glucose additions. RBC-PBS were treated with different
cose treatment also resulted in fall
a in the pH of the medium concentrations of glucose for 24 h. Details of glucose treatment and
from 7.4 to 6.78 in comparison with 6.88 in controls. The osmotic fragility a t 0.55% NaCl and lipid peroxidation measurements
effect of pH in glucose-induced lipid peroxidation was ruled are ~ v e under
n "Materials and Methods."
out because lactic acid treatment also caused a fall in pH to Osmotic
Treatments Sorbitol PL-MDA adduct MDA fragility
6.81 without inducing lipid peroxidation.
Fig. 3 illustrates that the osmotic fragility of treated RBC nmol/rnl % increase
?6 total PL
RBC n y g 1 of control
increased with increasing concentrations of glucose. At similar
5 mM glucose 10.1 f 1.6 0.2 f 0.1 1.1 k 0.2 0
glucose levels, osmotic fragility was higher when RBC were (control)
incubatedin wholeblood in comparison withPBS-buffer. +Sodium bar- 4.7 f 1.1 0.2 f 0.1 1.3 f 0.2 -4f2
This could be due to other factors in plasma formed during bital (6.6
the incubation, such as lysolecithin, which can increase os- mM)
motic fragility of RBC. Asignificant positive correlation 45 mM glucose 18.3 f 2.5 0.9 k 0.2 2.8 f 0.4 65 f 5
between osmotic fragility at 0.55% NaCl and the amount of +Sodium bar- 5.5 f 1.9 0.8f 0.3 2.7 k 0.4 62 f 4
bital
phospholipid-MDA adductformation ( r = 0.89) and TBA
reactivity ( r = 0.91) was observed in RBC buffer treated with
elevated glucose levels. TABLE I1
Treatment of RBC with elevated levels of glucose can
NADPH levels in RBC at varying incubation times after glucose
trigger aldose reductase activity, which results in the accu- treatment of RBC-PBS suspension
mulation of sorbitol (22). Sorbitol does not accumulate in Values are mean f S.D.of four experiments.Note the initial
RBC treated with sodium barbital, an inhibitor of aldose increased NADPH formation and its later disappearance in RBC
reductase (22). Data in Table I show that pretreatment of treated with higher concentrations of glucose.
RBC-PBS with sodium barbital blocked accumulation of sor- Glucose Ih 4h 24 h
bitol; however, there was no effect on osmotic fragility and mM nmollml RBC
lipid peroxidation in RBC treated with elevated glucose levels. 5"0.34 2 0.09 0.49 f 0.07
0.42 f 0.11
This indicates that the increase in the osmotic fragility of 15 0.39 f 0.08 0.39 k 0.10 0.65 f 0.21
glucose-treated RBC may not be due to sorbitol accumulation. 205.76 f O.lOb 0.49 f 0.06 0.61 f 0.16
Sodium barbital did not have any effect on glucose utilization 35
0.79 f 0.15b 0.41 f 0.12 0.60 f 0.11
by RBC. 45
0.91 f 0.14b 0.71 f 0.14b 0.59 f 0.16
Table I1 shows an initial accumulationof NADPH inRBC- a Control.
PBS suspension treated with high glucose. This increase in Significantly different from control values ( p < 0.01).
 Hyperglycemia and Membrane Lipid Peroxidation 21343
the NADPH was not apparent a t 24 h of incubation. Glycol-
ysis in RBC, even under aerobic conditions, usually termi-
nates in lactate. The netaccumulation of NADPH is a result
of its formation and utilization duringthe incubation of RBC
with glucose. Although maximum NADPH accumulation in
glucose-treated RBC was observed at 1 h, an increase in the
TBA reactivity was observed only at 4 h. MDA levels (TBA
reactivity) in RBC treated with 5 and 45 mM glucose for 4 h
were 1.1 f 0.2 and 1.6 & 0.3 nmol/ml RBC (mean f S.D.),
respectively. Thus, there seems to be a lag between the gen-
eration of maximum NADPH levels and the occurrence of
measurable membrane lipid peroxidation. Phospholipid-MDA
adduct formation in RBC was not observed a t 4 hof treatment
with elevated levels of glucose, which may be because MDA
cross-linking with PE and PS proceeds slowly and is time-
dependent (8). FIG. 4. TLC of lipids of RBC-PBS hemolysates treated with
NADPH for 24 h. A indicates phospholipid-MDA adduct in
The effect of sodium fluoride and vitamin E on the mem- NADPH-treated hemolysates. Abbreviations are as in the legend to
brane lipid peroxidation and osmotic fragility of glucose- Fig. 2.
treated RBC is given in Table 111. Glucose levels were the
same before and afterincubation in flaskscontaining sodium TABLE IV
fluoride (data not given), showing that fluoride blocked glu- Effect ofpCMB and metyrapone on membranelipid peroridation
cose utilization completely. It also blocked the initial increase and osmotic fragilityof RBC after treatment of RBC-PBS
in the NADPH, glucose-induced lipid peroxidation, and os- with elevatedglucose levels
motic fragility. Vitamin E did not have any effect on glucose Values are mean f S.D. of four observations. Values for RBC
utilization (data not given) and NADPH generation but did treated with elevated levels ofglucose and inhibitors are significantly
lower than RBC treated with elevated levels of glucose without any
block the accumulation ofMDA in RBC and its osmotic inhibitor ( p< 0.01).pCMB andmetyrapone were dissolved in alcohol
fragility. This suggests that increased lipid peroxidation in and layered on the bottom of flasks.Alcohol was dried with nitrogen
glucose-treated RBC is associated with the oxidation of glu- before adding the RBC suspension to flasks. RBC were preincubated
cose and accumulation of NADPH. Further,an effect of for 15 min with pCMB and metyrapone before glucose treatment for
antioxidant vitamin E on the glucose-induced changes in TBA 24 h. Osmotic fragility of RBC was measured at 0.55% NaCI. PL,
phospholipid.
reactivity and phospholipid-MDA adduct suggests that lipid
peroxidation does indeed occur in glucose-treated RBC. Treatments PL-MDA adduct MDA Osmotic fragility
To delineate the role of NADPH in glucose-induced lipid
peroxidation, an attemptwas made to load RBC with NADPH
% total PL nmol/rnl RBC ' increase of
control
5 mM glucose (control) 0.2 +. 0.1 0.96 k 0.29
by treating them in vitro with exogenous NADPH. However, +pCMB (0.5mM) 0.3 f 0.11.16 f 0.40 26 f9
this was not successful; it seems that NADPH is not perme- +Metyrapone (0.5mM) 0.2 f 0.1 0.97 f 0.47 14 f 7
able across the RBC membrane. A second attempt was made 45 mM glucose 1.0 f 0.3 2.53 f 0.38 90 f 19
in which RBC were lysed by freezing the thawing and the +pCMB 0.2 f 0.1 1.33 f 0.21 32 f 16
hemolysate treated with authentic NADPH. Fig. 4 shows that +Metyrapone 0.3 f 0.2 1.06 f 0.26 28 f 14
NADPH (2 mM) alone can peroxidize RBC membrane lipids
and form the phospholipid-MDA adduct. Greater lipid per- phospholipids, and TBA reactivity was 7.7 f 2.8 and 12.7 k
oxidation was also confirmed by the increase in TBA reactiv- 6.3 (mean f S.D.) nmol/g Hb in control and NADPH-treated
ity in NADPH-treated hemolysate. The phospholipid-MDA RBC lysates, respectively. TBA reactivity and phospholipid-
adduct was 0.2 f 0.1 and 0.8 k 0.3% (mean f S.D.) of total MDA adduct formation were blocked in thepresence of vita-
min E. This suggests that excess NADPH can induce perox-
TABLE 111 idation of erythrocyte membrane lipids.
The effectof sodium fluorideand vitamin E on osmotic fragility, Table IV shows the effect of para-chloromercuribenzoate
phospholipid (PL)-MDA adduct, MDA, and NADPH formation in (pCMB) and metyrapone on glucose-induced membrane lipid
RBC after treatmentof RBC-PBS with elevated glucose levels peroxidation and osmotic fragility. pCMB and metyrapone
Values are mean f S.D. of five experiments. Osmotic fragility, are known inhibitors of the cytochrome P-450 system (23,
phospholipid-MDA adduct, and MDA values are after 24 h of incu- 24). Both glucose-induced increased lipid peroxidation and
bation, and the NADPH value is at 1 h of incubation. Differences
between values marked * and y , * and z, # and a, and # and * are osmotic fragility were blocked in the presence of pCMB and
statistically significant ( p < 0.01). Vitamin E and fluoride were metyrapone, which suggests that cytochrome P-450-like ac-
Dreincubated with RBC for 30 min before the elucose addition. tivity may be involved in the glucose-induced lipid peroxida-
PL-MDA MDA NADPH Osmotic tion processes in RBC. Glucose-induced lipid peroxidation
Treatments adduct fragility was also blocked when RBC were preincubated with di-
% increme of methylfurane, diphenylamine, and thiourea (Table V). These
% of total PL nrnollrnl nrnol/rnl
control at agents are known to scavenge singlet oxygen and hydroxyl
RBC RBC
0.55% NaCl
radicals (25,26), thereby preventing oxygen radical attack on
5 mM glucose 0.17 f 0.09# 1.2 f 0.2# 0.38 f 0.12"
membrane lipids and, thus, lipid peroxidation and osmotic
(control)
+NaF (3mM) 0.19 f 0.14 1.2 f 0.2 0.31 +. 0.17 725 fragility. Dimethylfurane, which scavenges both singlet oxy-
+Vitamin E 0.21 f 0.1 1.4 f 0.2 0.45 f 0.11 9f6 gen and hydroxyl radicals, had a greater effect in blocking
(5mM) lipid peroxidation than thiourea, which scavenges only hy-
45 mM glucose 0.71 f 0.18* 2.7f 0.3' 0.85 f 0.12* 95 f 11* droxyl radicals. This suggests that bothhydroxyl radicals and
+NaF 0.20 f 0.17' 1.2 f 0.2' 0.37 f O.0gy 11 f '9 singlet oxygen may be generated in RBC during treatment
+Vitamin E 0.26 f 0.10' 1.5 +. 0.3 0.92 +. 0.19" 38 f 8' with elevated glucose levels.
21344 Peroxidation
Hyperglycemia
Membrane
Lipidand
TABLE V pends upon NADPH-cytochrome P-450 reductase, and it is
Effect of dimethylfurane, thiourea, and diphenylamineon membrane likely to involve reduction of free or chelated ferric to ferrous.
of RBC after treatmentof
lipid peroxidation and osmotic fragility Microsomallipid peroxidationis specific for NADPH, al-
RBC-PES withelevated levels of glucose
though isolated microsomal NADH-cytochrome b6 reductase
Values are mean t- S.D. of four observations. All values of RBC
can also cause peroxidation of liposomes in the presence of
treated with elevated levels of glucose + scavengers are significantly
less than values with elevated levels of glucose without scavenger chelated iron. Other enzymes, such as peroxidases and he-
added ( p < 0.01). ND, not determined because thiourea itself reacts moglobin, can also initiate lipid peroxidation by reducing
with the thiobarbituric acid used to measure MDA. Negative values ferric to ferrous. Recentstudies (32-34) haveshown that
result because the osmotic fragility of these groups was always less oxyhemoglobin in RBC can act like cytochrome P-450 in the
than controls. Diphenylamine was dissolved in alcohol and layered presence of NADPH and exhibit aniline hydroxylase activity.
on thebottom of flasks. Alcohol was dried with nitrogen before adding The present study hasshown that in vitro treatment of RBC
RBC suspension. Cells were preincubated with scavengers for 15 min
before the start of glucose treatment for 24 h. Osmotic fragility of hemolysates with standard NADPH can result in the forma-
RBC was measured at 0.55% NaCl. PL, phospholipid. tion of phospholipid-MDA adduct and TBA reactivity. Also,
Treatments
PL-MDA
adduct MDA Osmotic
fragility
glucose-induced peroxidationinRBC was blocked inthe
presence of para-chloromercuribenzoate and metyrapone, in-
% total PL nmollrnl RBC % Of hibitors of cytochrome P-450. This may suggest the presence
control
5 mM glucose (con- 0.2 & 0.1
1.18 t- 0.23 of a NADPH-dependent cytochrome P-450-like activity and
trol) its involvement in the glucose-induced lipid peroxidation in
+Thiourea (2 mM) 0.1 -t 0.1 ND -4 t- 8 the human RBC. The effect of carboxyhemoglobin in drug
+Dimethylfurane 0.2 t- 0.1 1.03 f 0.30 8t-5 metabolism or glucose-inducedlipid peroxidationisnot
(2 mM) known. An increase in the cytochrome P-450 activity has been
+Diphenylamine 0.2 t- 0.1 1.07 t- 0.27 -9 +- 6 reported in certain tissuesof diabetic animals (35).
(1mM) Apparently in hyperglycemia greater NADPH formation
45 mM glucose 0.9 f 0.3 2.18 f 0.34 96 t- 26
+Thiourea 0.5 k 0.2 ND 21 +. 16 stimulates the NADPH-dependent cytochrome P-450 system-
+Dimethylfurane 0.3 k 0.1 1.31 k 0.38 13 t- 9 like activity of the hemoglobin, which may form oxygen
+Diphenylamine 0.3 k 0.2 1.49 k 0.28 16 t- 11 radicals and result in the membrane lipidperoxidation of
RBC observed in the present study.
A significant positive correlation between the glucose-in-
DISCUSSION
duced membrane lipid peroxidation and the increased osmotic
This study has demonstrated that elevated levels of glucose fragility of RBC shows that the observedglucose-induced
can result in the peroxidation of membrane lipids in RBC. lipid peroxidative damagecan cause changes in the properties
Glucose-induced lipid peroxidation does not seem to be due of the RBC membrane.
to the accumulation of lactic acid or to a change in p H over Previous studies have reportedelevated levels of lipid per-
the course of incubation, because exogenous addition of lactic oxidation products in RBC, plasma, and retina (4, 36-42) of
acid alone to the extent formed in glucose treatment did not diabetic patients and animals. The present findings suggest
have any effect on lipid peroxidation in RBC. that membranelipid peroxidation induced by elevated glucose
Fluoride is known classically to shut down glycolysis by levels mayhave a role in the increasedlipid peroxidation
inhibiting enolase. The inhibitoryeffect of fluoride on glucose products found in diabetes. This study alsomay be the basis
oxidase is also known (27). In the present study, there was for abiochemical mechanism by which hyperglycemia induces
complete inhibition of glucose metabolism by fluoride, also cellular injury known to occur in various tissues in diabetics.
suggesting that fluoride can inhibit enzymes other than eno-
lase. Inhibition of glucose utilization by fluoride could also be Acknowledgments-I am gratefulto Chinwe Izundu and John Duett
due to decreased ATP due to theeffect of fluoride on glycol- for technical assistance and Barbara MacRoberts for editing this
ysis. Both dimethylfurane and thiourea blocked glucose-in- manuscript.
duced lipid peroxidation and osmotic fragility of RBC, with
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