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*
Division of Clinical Nephrology and Hemodialysis, Department of Internal
Medicine, Karl-Franzens University of Graz, Austria.
Department of Laboratory Medicine I, Karl-Franzens University of Graz,
Austria.
Department of Laboratory Medicine II, Karl-Franzens University of Graz,
Austria.
§
Institute of Biochemistry, Karl-Franzens University of Graz,
Austria.
Correspondence to Dr. Brigitte M. Winklhofer-Roob, Institute of Biochemistry, Karl-Franzens University, Schubertstrasse 1, A-8010 Graz, Austria. Phone: +43 316 380 5490; Fax: +43 316 380 9857; E-mail: brigitte.winklhoferoob{at}kfunigraz.ac.at
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-tocopheryl acetate taken 6 h
before the hemodialysis session. Blood was drawn before and 30, 60, 90, 135,
and 180 min after the start of the iron infusion, and areas under the curve
(AUC0-180 min) of ratios of plasma malondialdehyde (MDA) to
cholesterol and plasma total peroxides to cholesterol (two markers of lipid
peroxidation) were determined as the outcome variables. At baseline of the
session without vitamin E supplementation, plasma -tocopherol
concentrations (27.6 ± 1.8 µmol/L) and ratios of -tocopherol
to cholesterol (5.88 ± 1.09 mmol/mol) were normal, plasma MDA
concentrations were above normal (1.20 ± 0.28 µmol/L), and
bleomycin-detectable iron (BDI), indicating the presence of redox-active iron,
was not detectable. Upon iron infusion, BDI and MDA concentrations increased
significantly (P < 0.001). BDI concentrations explained the
increase over baseline in MDA concentrations (MDA = 1.29 + 0.075 x BDI).
Vitamin E supplementation, leading to a 68% increase in plasma
-tocopherol concentrations, significantly reduced the AUC0-180
min of MDA to cholesterol (P = 0.004) and peroxides to
cholesterol (P = 0.002). These data demonstrate that a single oral
dose of vitamin E attenuates lipid peroxidation in patients on hemodialysis
receiving intravenous iron. Given that intravenous iron is applied repeatedly
to patients on hemodialysis, this therapeutic approach may protect against
oxidative stress-related degenerative disease in the long term. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Normally, iron is safely sequestered in transport proteins such as
transferrin and lactoferrin and stored in proteins such as ferritin and
hemosiderin. In healthy subjects, transferrin saturation (TSAT), calculated
from total serum iron and transferrin concentrations, is 
45%
(7). The doses recommended for
iron supplementation in patients on chronic hemodialysis, i.e., 1 to
4 mg iron/kg body weight or 100 to 200 mg iron, lead to an
"oversaturation" of transferrin
(8,9).
Peak serum iron concentrations depend not only on the dose, but also on the
duration of the infusion: The higher the dose and the faster the application,
the higher the peak iron concentrations
(8). However, even infusion
lasting 4 h led to an "oversaturation" of transferrin
(8). High percentage TSAT was
found to be associated with the presence of non-transferrin-bound, potentially
redox-active iron, and iron complexed with citrate or acetate, i.e.,
low molecular weight complexes, was shown to be redox-active
(10,11).
Redox-active iron is a potent pro-oxidant (8,10,11). Hydroxyl radical and lipid alkoxyl radical, formed by the Fenton reaction, represent the reactive oxygen species that trigger iron-induced lipid peroxidation in the presence of hydrogen peroxide or lipid hydroperoxides. These, like any other oxygen free radicals, can initiate the chain reaction of lipid peroxidation by giving rise to the formation of a lipid radical from a polyunsaturated fatty acid (PUFA). In different in vitro models and in the intact animal, iron has been shown to initiate lipid peroxidation (12,13,14,15,16), the consequences of which are disturbances of tissue and organ functions (17,18). Evidence has accumulated that oxidative modification of LDL is causally involved in atherogenesis (19). Vitamin E is a potent antioxidant that terminates the chain reaction of lipid peroxidation (20). It has been demonstrated to inhibit lipid peroxidation in animals and human subjects (21) and to enhance the resistance of LDL to copper(II) ion-induced oxidation both in healthy subjects (22) and patients with impaired vitamin E status (23).
The question of whether redox-active iron occurs as an immediate response to intravenous iron application with a frequently used therapeutic dose and mode of application and what the effects are on in vivo lipid peroxidation have not been addressed before in patients on chronic hemodialysis. Given that redox-active iron causes lipid peroxidation, it could represent the critical link between oversaturation of transferrin and lipid peroxidation. The "bleomycin assay," developed by Gutteridge et al. (24), allows quantification of bleomycin-detectable iron (BDI), a marker of non-transferrin-bound iron that has the potency of becoming redox-active. In contrast, iron bound to proteins is not detected. Among different indexes for assessing in vivo lipid peroxidation in human subjects, plasma malondialdehyde (MDA) concentrations are most frequently used (25). MDA is an end product of nonenzymatic, oxidative degeneration of PUFA containing three or more conjugated double bonds (26,27).
The purpose of this study was to test the hypothesis that a single oral dose of vitamin E taken before intravenous iron application attenuates lipid peroxidation, which occurs in patients receiving iron(III) hydroxide sucrose complex intravenously at a dose of 100 mg during a hemodialysis session. The effect of vitamin E on lipid peroxidation was studied in a two-period cross-over design, using areas under the curve for the 180-min study period (AUC0-180 min) of ratios of plasma MDA to cholesterol and plasma total peroxides to cholesterol as the two outcome variables. This approach was chosen (1) to standardize for plasma volume changes that occur during hemodialysis treatment and (2) because the amount of lipid peroxidation products formed and being present during the entire observation period was considered to be pathophysiologically relevant. The study further aimed to explore the time course of variables of iron status and lipid peroxidation in the absence and presence of iron application and vitamin E supplementation, and analyze relations between these variables.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Study Design
Study Aim A. All patients were investigated twice in a randomized,
two-period cross-over design, 7 d apart with and without supplementation of a
single oral dose of vitamin E 6 h before the start of the hemodialysis
session. All patients received iron(III) hydroxide sucrose complex
intravenously on each of the two occasions. The iron infusion was started 30
min after the hemodialysis session had begun and lasted for 20 min.
Study Aim B. To further explore the effect of iron treatment in the absence and presence of vitamin E supplementation, patients were also investigated during a hemodialysis session without iron application, using the same variables as in the other sessions. This session was performed 1 mo after the randomized cross-over trial, assuming that period effects are negligible.
Treatment
Iron. The iron preparation used (Venofer®, Vifor, Inc., St.
Gallen, Switzerland) was a solution of iron(III) hydroxide sucrose complex of
approximately 43 kD containing 2% iron (20 mg Fe per milliliter of injectable
solution, pH 10.5 to 11.0). A very small proportion (0.14%) of the iron was
found not to be present as a high molecular weight complex when we separated
iron according to molecular weight, using an ultrafiltration membrane
(Vivaspin 500, Vivascience Ltd., Binbrook, Lincoln, United Kingdom) that
discriminates by molecular weight of 10 kD, and determined iron levels in the
ultrafiltrate by the FerroZine method using a Hitachi analyzer. The content of
the ampoules (5 ml) was diluted with sterile 0.9% NaCl solution to give a
total volume of 50 ml that was administered slowly over 20 min by infusion via
the venous line of the extracorporeal circuit, using Pilote C from Fresenius
Vial SA (Brezins, France). A dose of 100 mg was chosen because similar doses
are frequently applied to hemodialysis patients.
Vitamin E. A single oral dose of 1200 IU of
all-rac--tocopheryl acetate (Vitamin E
"ratiopharm"-Kapseln®, Ratiopharm Arzneimittel Ltd., Vienna,
Austria), i.e., the esterified form of the synthetic allracemic
mixture of -tocopherol, was taken 6 h before the hemodialysis session,
along with a meal for proper absorption. This dose was chosen to achieve high
plasma vitamin E concentrations, while avoiding possible unwanted
gastrointestinal side effects as reported for extremely high doses of vitamin
E (28). The time point was
chosen on the basis of the results of a previous study, showing maximum plasma
-tocopherol concentrations at 6 to 9 h after ingestion of a similar
dose (Winklhofer-Roob et al., unpublished data). This combination
should allow for a maximum protective effect of vitamin E at the time when
intravenous iron is being applied and throughout the observation period.
Blood Sampling and Preparation
Blood was drawn immediately before (0 min, baseline value) and 30, 60, 90,
135, and 180 min after the start of the iron infusion. For determination of
plasma concentrations of MDA, total peroxides, -tocopherol, and
additional antioxidants, blood was drawn on potassium
ethylenediaminetetra-acetic acid (EDTA) (1.6 mg EDTA/ml blood) (S-monovette
KE; Sarstedt, Nümbrecht, Germany) and lithium
heparin (15 IU heparin/ml blood) (S-monovette LH; Sarstedt), respectively, and
centrifuged immediately at 2000 x g at 4°C for 10 min. For
determination of serum concentrations of cholesterol, triglycerides, albumin,
total protein, total iron, transferrin, and BDI serum was obtained by
centrifugation at 2000 x g for 15 min. All samples were kept at
-80°C until analysis. Samples obtained from an individual patient at the
different time points of the hemodialysis sessions with and without vitamin E
supplementation were analyzed in the same run.
Analytical Methods
Clinical routine methods were used for determination of serum iron
(FerroZine method), total protein (Biuret method), cholesterol (CHOD-PAP test
kit), and triglyceride concentrations (GPO-PAP test kit), using reagents from
Boehringer Mannheim (Mannheim, Germany) and a Hitachi analyzer. Serum
transferrin concentrations were measured nephelometrically, using the Behring
nephelometer II (Marburg, Germany), and ferritin concentrations were
determined with the fluorescence polarization immunoassay from Abbott
Laboratories (Santa Clara, CA), using an AxSym analyzer. Plasma concentrations
of - and 
-tocopherol, ß-carotene, lycopene, and retinol
were determined by HPLC (29).
Serum concentrations of BDI were determined by the method of Evans and
Halliwell (30), with a minor
modification concerning the sample volume (we used 5 µl instead of 15
µl). Plastic tubes were acid-washed and checked for possible iron
contamination. Chelex 100 Resin (BioRad Laboratories, Vienna, Austria) was
used as a trace metal chelator. Reagents were from Sigma-Aldrich (Vienna,
Austria). The coefficient of variation was 2.5% within run and 10.2% from run
to run, respectively; the detection limit was 0.5 µmol/L. Plasma MDA
concentrations were measured after derivatization by thiobarbituric acid and
separation on HPLC (31). The
coefficient of variation was 4.4% within run and 6.9% from run to run,
respectively, as reported previously
(32). For determination of
plasma total peroxide concentrations, we used the
"Peroxide-activity" assay (POX ACT) from Tatzber KEG
(Klosterneuburg, Austria), which is based on the reaction of horseradish
peroxidase with plasma peroxides, using tetramethylbenzidine as the chromogen
substrate. EDTA plasma (10 µl) was incubated with the reaction mixture,
consisting of horseradish peroxidase, tetramethylbenzidine, and phosphate
buffer, for 20 min, and absorbances were determined photometrically at 450-nm
wave-length. Using a hydrogen peroxide (H2O2) standard
curve, total peroxide concentrations were calculated and expressed as µmol
H2O2 equivalents per liter plasma. The coefficient of
variation was 5.3% within run and 9.1% from run to run, respectively.
Statistical Analyses
Study Aim A (Randomized Two-Period Cross-Over Trial). After checking
for the appropriateness of parametric analysis, a paired t test was
applied for analyzing differences in the AUC0-180 min of ratios of
plasma MDA to cholesterol and AUC0-180 min of plasma total
peroxides to cholesterol, respectively, between the sessions with iron
application in the absence and presence of vitamin E supplementation.
Study Aim B (Exploratory Study). For baseline comparisons of
variables of iron, lipid, and antioxidant status, as well as lipid
peroxidation in the sessions with iron application in the absence and presence
of vitamin E supplementation and in the hemodialysis session without
treatment, two-way ANOVA with Tukey multiple comparisons test was applied.
Repeated-measures ANOVA with Tukey multiple comparisons test was used to
analyze changes for the 180-min observation period within treatment sessions
in serum iron and BDI concentrations, TSAT, and ratios of -tocopherol
to cholesterol, MDA to cholesterol, and total peroxides to cholesterol. Linear
regression analysis was applied for studying relations between TSAT and BDI
concentrations as well as between BDI and MDA concentrations at the 30-min
time point, i.e., the time point closest to BDI generation, of the
sessions with iron application in the absence and presence of vitamin E
supplementation. SigmaStat version 2.0 (Jandel Scientific Software, Erkrath,
Germany) was used for all statistical procedures. Data are presented as mean
± SD unless otherwise stated. P < 0.05 was considered
significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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- and -tocopherol
concentrations were well within the normal range
(22,32),
whereas plasma carotenoid (32)
and vitamin C concentrations were low
(33), and retinol
concentrations were elevated
(34), all of which are
frequent findings in hemodialysis patients.
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Study Aim A: Randomized Two-Period Cross-Over Trial
Both the intravenous application of the iron(III) hydroxide sucrose complex
and the oral vitamin E supplementation were well tolerated by all patients
without any overt side effects. As stated above, AUC0-180 min of
ratios of MDA to cholesterol and AUC0-180 min of ratios of total
peroxides to cholesterol have been chosen as the end points for testing the
hypothesis that vitamin E reduces lipid peroxidation in patients receiving
intravenous iron during a hemodialysis session. AUC0-180 min of
ratios of MDA to cholesterol with vitamin E supplementation were significantly
lower than those without (P = 0.004, paired t test); the
mean difference between the two sessions was 10.3 (µmol MDA/mmol
cholesterol) x min (Table
2). AUC0-180 min of ratios of total peroxides to
cholesterol were also significantly smaller with vitamin E supplementation
than without (P = 0.002, paired t test); the mean difference
was 3.18 (mmol H2O2 equivalents/mmol cholesterol)
x min. The response of variables of iron status to the intravenous iron
infusion is shown in Figure 1,
and the corresponding response of vitamin E status to the vitamin E dose is
shown in Figure 2. The time
courses of ratios of MDA to cholesterol and total peroxides to cholesterol are
presented in Figure 3.
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Study Aim B: Exploratory Study
Baseline Comparisons. In Table
3, data obtaine for different biochemical variables at baseline of
the sessions with iron application in the absence and presence of vitamin E
supplementation and of the hemodialysis session without treatment have been
compared with each other using two-way ANOVA with Tukey multiple comparisons
test. Significant differences were found for plasma -tocopherol
concentrations and -tocopherol to cholesterol ratios between the
session with vitamin E supplementation and those without as an effect of the
therapeutic intervention. Significant differences were also found for plasma
MDA concentrations and ratios of MDA to cholesterol between the session
without treatment and the two other sessions and for serum triglyceride
concentrations between the session without treatment and the session with iron
administration in the absence of vitamin E supplementation. Additional
significant differences were observed for other variables. Some of these could
be false-positive significances due to multiple comparisons.
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Time Course of Variables of Iron Status. Serum iron concentrations increased rapidly in response to the infusion and peaked within 30 min, as did TSAT (Figure 1). Thirty minutes after the start of the iron infusion, the calculated TSAT was approximately 190%; it decreased thereafter, but still exceeded normal TSAT at 180 min. As depicted in Figure 1, there were no differences between the sessions with and without vitamin E supplementation in the presence of iron infusion, neither in peak values nor in the rate of elimination from serum. In both sessions, BDI was not detectable before the iron infusion, but showed a significant rise in response to intravenous iron (Figure 1). At 30 min, approximately 9% of total serum iron was BDI. Serum BDI concentrations at 30 min were strongly associated with TSAT both in the session without (r = 0.70, P < 0.001) (Figure 4, top panel) and with vitamin E supplementation (r = 0.78, P < 0.001) (Figure 4, bottom panel). In contrast to total serum iron, BDI concentrations did not show rapid elimination from serum from 30 min onward up to the end of the observation period; there were no significant differences between the sessions with and without vitamin E supplementation. No changes in serum iron, TSAT, and BDI concentrations were observed during the 180-min observation period in the session without iron application (Figure 1). BDI concentrations were below the detection limit in all but one patient (this patient's BDI was 0.7 µmol/L and TSAT was 79% at the 0-min time point).
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Effect of Vitamin E Supplementation on Vitamin E Status. When
patients had taken the vitamin E supplement 6 h before the start of
hemodialysis treatment, plasma -tocopherol concentrations and ratios of
plasma -tocopherol to cholesterol at baseline, i.e.,
immediately before the start of the iron infusion, were significantly higher
than those in the sessions without vitamin E supplementation (P <
0.001, two-way ANOVA with Tukey multiple comparisons test)
(Table 2). In individual
patients, plasma -tocopherol concentrations increased up to
approximately 100 µmol/L. Figure
2 shows additional increases in ratios of -tocopherol to
cholesterol during the hemodialysis session with vitamin E supplementation
(P = 0.007, repeated-measures ANOVA with Tukey multiple comparisons
test), but not during those without.
Plasma Volume Changes during Hemodialysis. One of the therapeutic
effects of hemodialysis is ultrafiltration of approximately 2 L per session.
As a consequence, plasma volume is reduced. Between baseline and 180 min,
serum cholesterol, triglyceride, albumin, and total protein concentrations, as
well as plasma -tocopherol (Figure
2), -tocopherol, ß-carotene, lycopene, and retinol
concentrations (data not shown), increased significantly. These changes in the
range of 5 to 15% did not differ between the sessions with and without vitamin
E supplementation except for -tocopherol concentrations, which showed a
more pronounced increase in the session with vitamin E supplementation
(Figure 2). To correct for
plasma volume changes, ratios of MDA to cholesterol and total peroxides to
cholesterol have been chosen as the outcome variables.
Time Course of Variables of Lipid Peroxidation. As shown in Figure 3, plasma MDA concentrations and ratios of MDA to cholesterol increased rapidly upon iron application, reached a maximum within 30 min after the infusion had been started, i.e., 10 min after completion of the infusion, and showed approximately linear elimination from plasma at a rate of 0.16 ± 0.12 µmol/L per h and 0.05 ± 0.05 µmol/mmol per h, respectively, in the session without vitamin E supplementation and 0.19 ± 0.18 µmol/L per h and 0.06 ± 0.06 µmol/mmol per h, respectively, in the session with vitamin E supplementation. There were no significant differences between the two sessions (P = 0.48, paired t test). Ratios of MDA to cholesterol were significantly higher than baseline from 30 to 135 min, in both sessions with iron infusion (P < 0.001, repeated-measures ANOVA with Tukey multiple comparisons test). In the session without iron application, there was no increase in ratios of MDA to cholesterol during the observation period. In contrast, a small but statistically significant decrease was noted (P < 0.001, repeated-measures ANOVA with Tukey multiple comparisons test) (Figure 3, top panel). Ratios of total peroxides to cholesterol were higher than baseline from 30 to 180 min in both sessions with iron application (P < 0.001, repeated-measures ANOVA with Tukey multiple comparisons test) (Figure 3, bottom panel). As depicted in Figure 3, bottom panel, ratios of peroxides to cholesterol did not decrease during the entire observation period. There was no significant increase in the ratios of peroxides to cholesterol in the absence of iron infusion.
Relation between MDA and BDI. At 30 min (i.e., the time point closest to BDI generation) of the session without vitamin E supplementation, MDA was significantly related to BDI concentrations (r = 0.53, P = 0.01, MDA = 1.29 + 0.075 x BDI) (Figure 5, top panel). From the positive slope of the regression line, MDA = 1.29 + 0.075 x BDI, it can be seen that BDI concentrations explained the MDA concentrations. The intercept of 1.29 µmol/L, i.e. the MDA concentration that was not explained by BDI, corresponded well with baseline MDA concentrations of 1.20 ± 0.28 µmol/L, indicating that only the increase over baseline values in MDA concentrations was explained by the increase in BDI concentrations over baseline values; the latter were below the detection limit, as shown in Table 3.
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A significant dependence of MDA on BDI concentrations was no longer observed in the presence of vitamin E supplementation (r = 0.35, P = 0.11, MDA = 1.37 + 0.04 x BDI) (Figure 5, bottom panel).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-tocopherol
concentrations were 1.7-fold higher than without supplementation, allowing us
to study the effect of "above average" vitamin E status. Ratios of
-tocopherol to cholesterol showed a further increase during the
observation period, suggesting that the vitamin E dose could also be taken
more than 6 h before the iron infusion for efficient protection. The
protective effect of vitamin E observed in this study is in agreement with a
study in rats in which iron was injected into brain, and lipid peroxidation
was compared with and without administration of vitamin E
(37). Intravenous iron
application, although associated with marked lipid peroxidation, is essential
for hemodialysis patients on rhEPO therapy. Vitamin E supplementation before
scheduled iron infusions represents a new approach for attenuating this
oxidative stress. Ten minutes after the end of the 20-min infusion of 100 mg of iron(III) hydroxide sucrose complex, serum iron concentrations were more than sixfold higher than at baseline. Values could have peaked before this time point and at a higher level, but this could not be investigated because of limitations of the total amount of blood that could be drawn from the patients. Serum iron concentrations decreased between 30 and 180 min, but did not reach half-maximal concentrations. Healthy volunteers who received a similar dose of the same iron preparation by intravenous injection showed peak serum iron concentrations 10 min after the injection that were more than 10-fold higher than baseline values with a terminal half-life of 5.3 h (9). Ten minutes after the end of the intravenous iron infusion, TSAT in the hemodialysis patients was approximately 190%; 2.5 h after the end of the iron infusion it was still approximately 130%. TSAT is a calculated index that is frequently used in clinical practice. High TSAT values indicate that there is exces iron (expressed as moles of iron) that numerically exceeds the available binding sites according to the moles of transferrin present. The calculation of TSAT is based on the fact that 1 mole of transferrin is able to bind 2 moles of iron and the assumption that transferrin binds iron in the first instance and until saturation of all binding sites; only thereafter would iron be loosely bound to other plasma proteins. TSAT, however, does not indicate whether iron is indeed bound to transferrin or any other plasma proteins or, possibly, is still within the high molecular iron-sucrose complex, if the latter is present in a form that is detected by the method used for determination of serum iron. The high TSAT values found in this study upon iron infusion suggest that the capacity of transferrin to bind and transport the available iron has been exceeded over the entire observation period. As a consequence, high TSAT was associated with the occurrence of BDI. While BDI was not detectable in the serum of the study patients before, it increased rapidly upon infusion of the iron(III) hydroxide sucrose complex. The initial increase in serum BDI concentrations paralleled the increase in serum iron concentrations and TSAT. At 30 min, BDI concentrations correlated with total iron concentrations and TSAT. In the session without iron application, a single patient who showed TSAT of 79% had detectable BDI. This is in agreement with other studies in which healthy subjects did not show BDI (37), but patients with idiopathic hemochromatosis (38) and patients with leukemia undergoing chemotherapy did (39). In these studies, BDI became detectable when total serum iron concentrations exceeded 40 µmol/L (38) and BDI concentrations accounted for up to 32% of total iron concentrations (38). Thirty minutes after the start of the iron infusion, BDI concentrations in our patients were approximately 9% of total iron concentrations, as determined by the FerroZine assay. BDI concentrations did not decline between 30 and 180 min, suggesting that BDI was not handled in the same way as was transferrin-bound iron, which is rapidly transported to its destination, the hematopoietic cells of the bone marrow. In rats, non-transferrin-bound iron was avidly taken up by hepatocytes, whereas transferrin-bound iron was poorly absorbed by the liver (40). Hepatic uptake of non-transferrin-bound iron is thought to contribute to liver injury in chronic iron overload. In our patients, no overt signs of acute toxic effects of BDI and increased lipid peroxidation have been observed. However, this does not rule out that free radical-mediated damage, for instance to DNA and proteins, might have occurred.
Baseline plasma MDA concentrations were significantly higher in patients on hemodialysis than in healthy subjects. This was true for all three sessions, although significant differences were observed between the two sessions of the randomized trial and a hemodialysis session without treatment 1 mo later. Significant within-subject variation in plasma MDA concentrations over 3-mo periods has been reported in a different clinical setting, where patients showed simultaneous changes in both vitamin C and inflammatory status (33). Changes in these and additional variables did not occur in the present study, but it cannot be ruled out that other as yet unknown variables may have contributed to these differences. That plasma MDA concentrations are increased in patients on hemodialysis has been observed before (35,41) and can be explained by oxidative stress due to uremia (42), hemodialysis treatment (43), and impaired antioxidant status (41,44). In a small group of hemodialysis patients who received iron on a regular basis, both superoxide dismutase activities and PUFA concentrations in erythrocytes were lower, but MDA concentrations did not differ from those in patients not receiving iron or in healthy subjects (44). In our patients, plasma concentrations of vitamin C, a potent antioxidant (45), were extremely low (20.5 ± 17.3 µmol/L) compared with healthy subjects (69.5 ± 16.6 µmol/L) (33). This is a common finding in patients on hemodialysis not taking vitamin C supplements to compensate for losses of vitamin C during hemodialysis treatment (46). Low plasma vitamin C concentrations may have contributed to increased lipid peroxidation in the study patients. Because intake of high doses of vitamin C has been found to be associated with hyperoxalemia (47), vitamin C supplements had not been part of the routine management in our hemodialysis center.
In response to intravenous application of the iron(III) hydroxide sucrose preparation, plasma MDA concentrations increased significantly. MDA concentrations at 30 min were 1.5 times those at baseline. In the absence of vitamin E supplementation, BDI concentrations at 30 min explained the increase over baseline in MDA concentrations. The intercept at the y-axis, 1.29 µmol/L MDA, was in agreement with mean (± SD) MDA concentrations of 1.20 (±0.28) µmol/L at baseline, when BDI was not detectable. This suggests a pathophysiologic relationship between redox-active iron and lipid peroxidation. In the session with vitamin E supplementation, plasma MDA concentrations were not significantly related to BDI concentrations, perhaps as a result of the effect of vitamin E on MDA concentrations.
MDA concentrations did not increase in the absence of iron application but showed a significant decrease at an approximate rate of 0.03 (µmol/L) per h from 0 to 180 min. Because the same was true for ratios of MDA to cholesterol, this decrease was considered to not result from plasma volume changes but rather from elimination of MDA by hemodialysis treatment. When patients had received iron, MDA elimination from plasma was linear from 30 min onward at an approximate rate of 0.16 (µmol/L) per h. Elimination may have started even before the 30-min time point, but this has not been investigated. Free MDA, due to its relatively low molecular mass, can be removed by hemodialysis. We recently detected small quantities in the dialysate (Roob et al., unpublished data). Although an end product of lipid peroxidation, MDA is further metabolized. In rats, approximately 70% of an oral dose of 14C-labeled MDA was expired as 14CO2 within 12 h (48). In rodents, MDA became rapidly distributed throughout all major organs, and acid-labile MDA metabolites, with very little free MDA, were excreted in urine (49). In rats receiving 100 mg of iron dextran intravenously, MDA concentrations increased in plasma, spleen, and heart 3 h after the injection. The MDA concentrations correlated with the respective iron concentrations and persisted in liver and spleen until day 28 (14). MDA is not only an indicator of in vivo lipid peroxidation, but has cytotoxic properties and, for instance, the ability to derivatize apoB-100, the protein moiety of LDL, thereby producing chemical adducts that are potent immunogens considered to play a role in atherogenesis (19).
While ratios of plasma total peroxides to cholesterol did not change in the absence of iron(III) hydroxide sucrose infusion, they increased rapidly upon iron application, reached a plateau at 30 min, and did not decline during the entire observation period. With the assay used, plasma peroxides of the general chemical structure ROOH have been detected. Because their chemical nature is not further defined, it is difficult to speculate about their metabolism. However, the time profiles indicate that these peroxides were not efficiently eliminated from plasma. Persistence of elevated peroxide concentrations, in the presence of transition-metal ions, might facilitate the generation of highly reactive oxygen species from ROOH. This underscores the possible benefit of reduction of plasma peroxide concentrations by vitamin E.
In summary, a single oral dose of 1200 IU of vitamin E efficiently reduces
oxidative stress in patients receiving 100 mg of iron(III) hydroxide sucrose
intravenously during a hemodialysis session, when taken 6 before the
hemodialysis session. This beneficial effect was demonstrated in patients on
hemodialysis with normal vitamin E status before supplementation, indicating a
protective effect of high plasma vitamin E concentrations. Close correlations
between calculated TSAT and BDI concentrations on one hand and BDI and MDA
concentrations on the other suggest that lower doses of the iron(III)
hydroxide sucrose complex may limit the extent of BDI and subsequent MDA
formation in the first instance. Indeed, a recent abstract reported BDI only
in two of 15 patients receiving doses 50 mg
(50). It remains to be
investigated whether iron doses lower than those applied in the present study
in combination with vitamin E supplements could fully prevent iron-induced
lipid peroxidation. Also, correction of impaired vitamin C status may
potentiate the vitamin E effect through regeneration of vitamin E from the
vitamin E radical formed during the antioxidant action of vitamin E. The
effect on lipid peroxidation of intravenous iron application not in
association with or at a later time point of a hemodialysis session could
prove to be different owing to possible removal of lipid peroxidation products
by hemodialysis. Finally, different iron preparations may behave differently,
as suggested by higher rates of adverse events reported for instance for the
iron dextrans (6). The final
goal of antioxidant supplementation in this clinical setting is to prevent
long-term side effects associated with increased lipid peroxidation as a
result of repeated exposure to iron-induced oxidative stress.
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Acknowledgments |
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This work was supported by grants from the Austrian Science Foundation (P8612-MED and P11690-MED). We are indebted to Dr. Richard Salkeld (Vitamin Research Department of F. Hoffmann-La Roche, Basel, Switzerland) for determinations of plasma antioxidants, Dr. Martin A. van't Hof (Medical Statistics Department, University of Nijmegen, The Netherlands) for expert statistical advice, and Andreas Meinitzer (Department of Laboratory Medicine I, Karl-Franzens University of Graz) and Jutta Winkler (Institute of Biochemistry, Karl-Franzens University of Graz) for excellent technical assistance.
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Footnotes |
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-Tocopherol. In: Handbook of
Antioxidants, edited by Cadenas E, Packer L, New York, Basel, Hong
Kong, Marcel Dekker, 1996, pp3
-25
-tocopherol on the vitamin E
content of human low density lipoproteins and resistance to oxidation.J Lipid Res
32:1325
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