of hydrogen peroxide and ferrous salts. Haber and Weiss subsequently estab- lished the oxidizing species as H0. (17). The hydroxyl radical formed by the ...
CLIN. CHEM. 41/2, 220-225
(1995)
#{149} Lipids
and Lipoproteins
Susceptibility of Plasma to Ferrous Iron/Hydrogen Peroxide-Mediated Oxidation: Demonstration of a Possible Fenton Reaction Ahmad
Agil,’ Cindy J. Fuller, and Ishwarlal Jialal2
The aim of our study was to evaluate a model system by using iron in the peroxidation of plasma. Lipid peroxidation was monitored by fluorometric measurement of lipid peroxides
(LPO). Plasma
coincubated
with
Fe2
and
H202 had a 268% increase in plasma LPO after 1 h. The optimum concentrations were 0.42 mmol/L Fe2 and 0.73 moVL H202. Coincubation of plasma with these concentrations of Fe2 and H202 separately resulted in no increase in plasma LPO. The increase in plasma LPO after oxidation with Fe2/H2O2 was paralleled by a decrease in plasma polyunsaturated fatty acids, an increase in the relative electrophoretic mobility of low-density lipoprotein (LDL), and decreases in apolipoprotein (apo) B-i 00 and apo A-I immunoreactivity. In vitro oxidation of LDL and high-density lipoprotein separately with this system produced increases of LPO of 246% and 128%, respectively. LPO formation in plasma was inhibited by catalase, desfemoxamine, and mannitol, but not by su-
peroxide dismutase. Hydroxyl radical generation with Fe2/H2O2 was evidenced by fragmentation of deoxyribose. We conclude that the Fe2/H2O2 system, possibly by a Fenton reaction mechanism, resulted in significant plasma oxidation. This model system may be useful for
at isolated LDL oxidation and failed to evaluate the prooxidant and antioxidant factors present in whole plasma, the balance of which could be crucial in determining the susceptibility to lipid peroxidation. Plasma oxidation may be more biologically relevant than LDL oxidation
since
ascorbate, would dants
water-soluble
uric
acid,
antioxidants
and
also be taken into would presumably
other
account be lost during
LDL from plasma. Iron may be an important dation. bound
under
Although virtually to proteins such
normal
modulator
the
as
isolation
of lipid
all of the circulating as transferrin and
conditions,
occur from injured mation, and may
such
plasma constituents (7, 8). These antioxi-
iron release
from
of
peroxi-
iron is ferritin
stores
can
cells in conditions such as inflamstimulate peroxidation (9, 10). In-
creased iron concentrations in plasma have been associated with increased lipid peroxidation, decreased ascorbate concentrations, and reduced superoxide dismutase (SOD; EC 1.15.1.1) activity in aortic occlusive and aneurysmal diseases (11). Iron
may
also
be an important
promoter
examining lipid peroxidation in clinical investigations. Indexing Terms: lipid peroxidation/hydroxyl sis/antioxidants
and high dietary iron intake are associated with an increased risk of acute myocardial infarction in middleaged men. Also, Smith et al. (14) demonstrated that
Lipid
peroxidation
appears
to
radicals/atherosclero-
play
a key
role
in
various degenerative diseases (1). Patients with cardiovascular disease have increased plasma lipid peroxides (LPO) (2 )3 In addition, oxidation-specific epitopes on proteins have been found in carefully extracted material from atherosclerotic plaques (3, 4). Previous
low-density
studies
lipoprotein
have
reported
(LDL)
that
promotes
oxidation
the
of
develop-
of receptor, which may be a key early step in the development of atherosclerosis (5, 6). These studies specifically looked ment of fatty streaks by increasing the oxidized LDL by the macrophage scavenger
uptake
Laboratory of Molecular Pathology & Center for Human Nutrition, Departments of Pathology and Internal Medicine, University of Texas Southwestern Medical Center at Dallas. 1 Visiting scientist from Department of Pharmacology, University of Granada (Spain). Address correspondence to this author at: University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9052. Fax 214-590-2785.
3Nonstandard
abbreviations:
LPO, lipid peroxides;
PUFA,
polyunsaturated fatty acids; LDL, low-density lipoprotein; HDL, high-density lipoprotein; apo, apolipoprotein; SOD, superoxide dismutase; DFO, desferrioxamine; TBA, thiobarbituric acid; and REM, relative electrophoretic mobility. Received April 26, 1994; accepted October 19, 1994. flO
CLINICAL CHEMISTRY, Vol. 41, No. 2, 1995
et al. (13) as serum
reported
of athero-
sclerosis (12). Salonen stored iron (measured
ferritin
that
excess
concentration)
material isolated from atherosclerotic lesions tained catalytic iron detectable by the bleomycin
conassay
and
stimulated the peroxidation of rat liver microThe presence of iron in atherosclerotic plaques has also been documented previously (11). The results of these studies indicate that further investigation is needed into the role of iron in atherogenesis (12, 13). Lipid peroxidation in vitro occurs with irradiation (either ultraviolet or ionizing), in the presence of transition metals such as iron or coincubation with a free-radical initiator. Iron may stimulate peroxidation by hydroxyl radical (HO) formation (10) and may promote decomposition of hydroperoxides (9, 10). Fe2 and H202 can generate H0 by means of the Fenton reaction: somes.
H202 Fenton properties ferrous
+
Fe2
-Fe3
+
OW+HO’
(15, 16) described the powerful oxidizing of the mixture of hydrogen peroxide and salts.
Haber
and
Weiss
subsequently
estab-
lished the oxidizing species as H0 (17). The hydroxyl radical formed by the Fenton reaction can abstract a hydrogen atom from the methylene group of a nonconjugated
polyunsaturated
carbon-centered
radical
fatty
(RC).
acid
The
(PUFA)
to yield
PUFA
undergoes
a
molecular rearrangement to a conjugated double-bond system to partially stabilize RC. The RC can then react with 02 to form a peroxy radical (R00), which can attack an adjacent PUFA to form hydroperoxides
(LOOH) (9, 18) and a new RC. In addition to initiating lipid peroxidation in plasma, ferrous ions may also mediate decomposition of LOOH to peroxyl and alkoxyl radicals, aldehydes, and other reactive products (18, 19). Since data suggest a relation between increasing iron status and atherosclerosis (12, 13), and Fe2 appears to be involved in the Fenton reaction resulting in the production of the highly reactive hydroxyl radicals, we sought to investigate the susceptibility of whole plasma to iron-catalyzed lipid peroxidation by using a Fe2fH202 model system. Iron was chosen as the transition metal instead of copper since more information supports the role of iron in atherogenesis and the Fenton reaction. Materials and Methods Reagents 1,1’,3,3 ‘-Tetramethoxypropane was obtained from Kodak (Rochester, NY), and 1-butanol from Fisher Scientific (Fair Lawn, NJ). All other reagents were obtained from Sigma Chemical Co. (St. Louis, MO).
Plasma Blood samples from healthy normolipidemic volunteers between ages 20 and 50 years were collected after an overnight fast into EDTA (1.5 g/L)-containing tubes. Plasma was separated by centrifugation at 4#{176}C at 2000g for 20 mm.
tubes containing EDTA (1 g/L). LDL (d = 1.0191.063 kgfL) and HDL (d = 1.063-1.21 kgfL) were isolated by sequential ultracentrifugation, as previously described (20). The isolated LDL and HDL were dialyzed against 150 mmol/L NaC1 and 1 mmol/L EDTA (pH 7.4), filtered through a 0.45-sm filter, and stored at 4#{176}C under N2. Protein was measured by the method of Lowry et al. (21), with bovine serum albumin as calibrator. into
Oxidation of Lipoproteins dialyzed against 1 L of phos(0.01 mol/L phosphate, 0.15 mol/L NaC1, pH 7.4) at 4#{176}C in the dark. LDL and HDL (200 mg/L protein) were then incubated with 0.42 mmol/L Fe2 and 0.73 mol/L H2O2 in the buffer in a total volume of 1 mL for 1 h at 37#{176}C. Oxidation was stopped with butylated hydroxytoluene and refrigeration as in plasma experiments. LDL
and HDL
phate-buffered
were
saline
Analysis of Oxidation Plasma LPO formed after Fe2/H2O2 oxidation was assayed by a modification of the thiobarbituric acid (TBA)-reactive substances method of Yagi (22). Briefly, three modifications were made to improve the specificity of the plasma LPO measurement. First, watersoluble substances, which also react with TBA to yield the same
product
as LPO,
were
removed
by including
a
phosphotungstic acid (0.1 gfL)-sulfl.iric acid (35 mmol/L) step to precipitate lipids with the proteins. The supernate was discarded. Second, interference from bilirubin was prevented by reading the fluorescence at 553 nm rather than at 532 nm. Finally, sialic acid interference was eliminated by adding acetic acid Plasma Oxidation Systems in the TBA reagent to adjust the reaction mixture pH Lipid peroxidation was initiated by incubating to 3.0. The fluorescence was read at 553 nm with 515 plasma (500 L) with 50 L of various concentrations nm excitation with a Perkin-Elmer (Norwalk, CT) of ferrous sulfate (0.21-1.67 mmoIIL), with or without LM-5 spectrofluorometer. The amount of LPO was 50 p.L of various concentrations of H202 (0.18-0.73 expressed in terms of malondialdehyde equivalents mol/L). The samples were incubated for 1 h at 37#{176}C. (moI/L of plasma), as calibrated with freshly diluted The length of incubation corresponded to the steady 1,1’,3,3’-tetramethoxypropane. The intra- and interasstate of maximum plasma oxidation, as determined in say CVs of this assay were 4.2% and 4.0%, respectively, preliminary time-course experiments (data not shown). and the results correlated highly with LPO analysis by In some experiments, oxidation was performed in the HPLC (23). LPO concentrations of LDL and HDL were presence or absence of catalase (EC 1.11.1.6; 50 and measured as described above for plasma, except that 100 mgfL) to determine the importance of H202; SOD pretreatment with phosphotungstic acid/sulfuric acid (50 and 100 mg/L), to determine whether 02. is inwas eliminated and the TBA reaction was carried out volved in plasma oxidation; desferrioxamine (DFO, 1 directly on 50 L of sample mixed with 0.1 gIL phosand 2 mmoIJL), to investigate the importance of iron in photungstic acid and 3.5 mL of water (24). oxidation; and mannitol (100 and 200 mmol/L), to The fatty acid composition before and after plasma determine the role of H0. All of the inhibitors were oxidation was analyzed by the direct transesterificaadded before the addition of Fe2 and H202 to test for tion method of Lepage and Roy (25). Plasma (100 FL), inhibition of oxidation. All experimental points were pre- and postoxidation, was extracted with methanol: performed in triplicate. Oxidation was stopped by adbenzene (4:1 by vol) and transesterified with 200 L of dition of 40 prnol/L butylated hydroxytoluene and reacetyl chloride for 1 h at 100#{176}C. The sample was then frigeration. neutralized with 5 mL of K2CO3, and 2 1.iL of the benzene phase was injected into the GC.100A Lipoprotein Preparation (Hewlett-Packard, Avondale, PA) 5880A gas chromatoPlasma for LDL and high-density lipoprotein (HDL) graph with a 30 m X 0.75 mm (i.d.) SP-2330 capillary were collected from healthy fasting human volunteers column (0.2-nm film thickness; Supelco, Bellefont, PA) CLINICAL CHEMISTRY, Vol. 41, No. 2, 1995 1
for fatty acid analysis. The chromatographic conditions were: injector temperature, 225#{176}C; detector temperature, 230#{176}C. The column temperature program was as follows: initial oven temperature 150#{176}C for 5 mm, increasing by 8#{176}C/mm to a final temperature of 220#{176}C, held for 15 mm. The carrier gas was nitrogen, at a flow rate of 30 mL/min. The fatty acid calibrator was obtained from Nuchek Prep (Elysian, MN). The fatty acid was expressed as a percentage: fatty acid area/total fatty acid area (unsaturated and saturated). Seven fatty acids were incorporated in the total fatty acids: 14:0, 16:0, 18:0, 18:1, 18:2, 18:3, and 20:4. Plasma lipoprotein electrophoresis was performed at pH 8.6 in 0.05 mol/L barbital buffer on 0.05% agarose gels as described previously (26). Relative electrophoretic mobility (REM) was measured by comparing the mobility of LDL in oxidized plasma with that for LDL from untreated plasma. Similar experiments were conducted with LDL and HDL in vitro. Apolipoprotein
(apo)
A-I and
apo B immunoreactivity
before and after plasma oxidation were measured by iminunonephelometry (Behring Diagnostics, Somerville, NJ). Hydroxyl Radical Generation (Fenton Reaction) The presence of H0 radicals in the Fe2t’H202 system was assessed by the degradation of deoxyribose as described by Gutteridge (27). Reaction tubes contained 0.73 molJL H2O2 and 0.8 mmol/L deoxyribose in 10 mmol/L phosphate buffer, pH 7.4. The final volume was 1.0 mL. The reaction was initiated by adding 0.42 mmol of FeSO4, and the mixture was incubated for 10 mm at 37#{176}C. The reaction was carried out in triplicate. Solutions
of iron
and
deoxyribose
were
made
up just
before use. Deoxyribose degradation by generated hydroxyl radical in the presence or absence of mannitol (100 mmolJL) was measured by the TBA-reactive substances assay (28). The samples were heated for 10 mm at 100#{176}C, cooled in ice, and mixed with catalase (15 gIL) to prevent the effect of H2O2 in the TBA reaction (29). The absorbance was read in a spectrophotometer at 532
nm
against
a blank
(deoxyribose
in
phosphate
buffer).
Table 1. Effect of Fe2
or H202 on plasma oxidation.
Incubation conditlonV Plasma 1 + Fe504, mmoVL
LPO, gAmoUL”
0.21 0.42
± 0.06
2.11
±
0.07
2.12 ± 2.16 ± 2.60 ± 2.75 ±
0.83
1.67 Plasma
2.08
2
0.02
0.08 0.20 0.24
+ H202, mol/L
0.18 0.37 0.73
2.88 ± 0.34 2.67 ± 0.15 2.76 ± 0.28
500 L
at 37’C for I h in the presence or
of plasma was incubated
absence of 50 p1 of FeSO4 or H202 at venous concentrations. #{176} Mean ± SEM of three separate
experiments.
Table 2. Effect of coincubation with both Fe2’ and H202 on plasma LPO. incubation condltlonV FeSO4 (0.42 mmoVL) + H202,
LPO, pmol/IP
+ plasma A
2.22 ± 0.18
moVL
2.57 ± 0.22 4.40 ± 8.00 ± 052d
0.18
0.37 0.73 H202 (0.73
0d
moVL) +
plasma B
2.28
±
0.18
+ Fe2, mmoVL
0.21
4.44 ± 0.21
0.42
8.38 ± O.26c 79.56 ± 137d 162.92 ± 3.02c
0.84
1.67
Plasma (500 p1) was incubated at 37CC for 1 h in the presence of 50 p1 of various concentrations of FeSO4 and H202. Mean ± SEM of three separate experiments. Thresholds for statistical significance, using the Bonferroni correction: P = 0.0125 (‘P
