These authors have equally contributed to this work.
Labile Plasma Iron Generation After Intravenous Iron is Time-dependent and Transitory in Patients Undergoing Chronic Hemodialysis
Article first published online: 18 DEC 2009
© 2009 The Authors. Journal compilation © 2009 International Society for Apheresis
Therapeutic Apheresis and Dialysis
Volume 14, Issue 2, pages 186–192, April 2010
How to Cite
Rangel, É. B., Espósito, B. P., Carneiro, F. D., Mallet, A. C., Matos, A. C. C., Andreoli, M. C. C., Guimarães-Souza, N. K. and Santos, B. F. (2010), Labile Plasma Iron Generation After Intravenous Iron is Time-dependent and Transitory in Patients Undergoing Chronic Hemodialysis. Therapeutic Apheresis and Dialysis, 14: 186–192. doi: 10.1111/j.1744-9987.2009.00786.x
- Issue published online: 26 MAR 2010
- Article first published online: 18 DEC 2009
- Received March 2009; revised June 2009.
- Intravenous iron;
- Labile plasma iron
Iron supplementation in hemodialysis patients is fundamental to erythropoiesis, but may cause harmful effects. We measured oxidative stress using labile plasma iron (LPI) after parenteral iron replacement in chronic hemodialysis patients. Intravenous iron saccharate (100 mg) was administered in patients undergoing chronic hemodialysis (N = 20). LPI was measured by an oxidant-sensitive fluorescent probe at the beginning of dialysis session (T0), at 10 min (T1), 20 min (T2), and 30 min (T3) after the infusion of iron and at the subsequent session; P < 0.05 was significant. The LPI values were significantly raised according to the time of administration and were transitory: −0.02 ± 0.20 µmol/L at the beginning of the first session, 0.01 ± 0.26 µmol/L at T0, 0.03 ± 0.23 µmol/L at T1, 0.09 ± 0.28 µmol/L at T2, 0.18 ± 0.52 µmol/L at T3, and −0.02 ± 0.16 µmol/L (P = 0.001 to 0.041) at the beginning of the second session. The LPI level in patients without iron supplementation was −0.06 ± 0.16 µmol/L. Correlations of LPI according to time were T1, T2, and T3 vs. serum iron (P = 0.01, P = 0.007, and P = 0.0025, respectively), and T2 and T3 vs. transferrin saturation (P = 0.001 and P = 0.0003, respectively). LPI generation after intravenous saccharate administration is time-dependent and transitorily detected during hemodialysis. The LPI increment had a positive correlation to iron and transferrin saturation.
Anemia is a significant problem in patients undergoing chronic hemodialysis treatment in spite of the use of erythropoietin. It is mainly attributed to iron deficiency, which is secondary to decreased iron absorption by the gastrointestinal tract, blood loss into the hemodialysis system or via routine investigations, or to occult gastrointestinal hemorrhage, anticoagulation-related blood loss, and accidental blood loss from arteriovenous fistulas and grafts. On average, maintenance hemodialysis patients are associated with a loss of at least 1–1.5 g of elemental iron each year (1).
Parenteral iron is well known to be superior to oral preparations to replace iron stores for hemodialysis patients when either absolute or functional iron deficiency is present (2). The preparations are complexes of ferric iron with polymeric carbohydrates such as dextran, or sugars such as sucrose or gluconate that form polynuclear complexes with the iron. These iron complexes are thought to be taken up by macrophages and degraded in the reticuloendothelial cells or in the liver, from where the iron is delivered to transferrin and further to the erythroblastic cells of the bone marrow. All cells have transferrin receptors on their surface and almost 80% of them are present in the erythroid marrow.
Under normal conditions, practically all iron in plasma is bound to transferrin, which keeps the iron in a catalytically inactive form and prevents iron-catalyzed hydroxyl-radical generation. However, in iron-overloaded patients, the metal can be bound to other biochemical targets, giving rise to non-transferrin bound iron (NTBI), which comprises chemically heterogeneous forms of iron, both accessible and inaccessible to chelation therapy.
All iron agents available show evidence of bioactive iron release either in vitro or in vivo (3,4), making them possible candidates for the generation of NTBI. Plasma NTBI is not observed in healthy individuals, whose transferrin iron-binding capacity (35–55 µM) exceeds that of total plasma iron (8–27 µM) (5). The occurrence of the NTBI in the plasma of patients undergoing hemodialysis treatment has already been reported and it may participate in redox reactions that give rise to reactive oxygen species (6,7). In addition, it was found to induce bacterial growth in vitro (8), disturb neutrophil function (9,10), and increase bacterial infection (11).
Some of the NTBI components may be mobilized by endogenous low molecular weight ligands at physiological levels (e.g. ascorbate), allowing the metal to engage in several redox-active processes that, ultimately, lead to iron toxicity. Labile plasma iron (LPI) represents a component of NTBI that is both redox-active and chelatable, capable of translocating across cell membranes in a non-regulated manner and inducing tissue iron overload. In this way, LPI can be used not merely as a diagnostic marker of iron overload (transfusional and non-transfusional) and cell toxicity, but also as a clinical parameter for assessing the mode and efficacy of iron chelation (12,13). LPI levels correlate significantly with NTBI in thalassemia patients (13), may be a marker of either toxicity in C282Y/C282Y hemochromatosis (14) or mortality in some instances of diabetes (15), and were found to be associated with cognitive impairment in Alzheimer disease (16). Therefore, there is a positive correlation between high LPI levels and high NTBI levels.
However, it is not a consensus if NTBI/LPI induced by iron supplementation is implicated in the generation of oxidative cell injury. In this sense, it was also demonstrated that NTBI generation is transitory after iron administration and is not associated with peroxide generation (17). The most commonly used markers of iron management in hemodialysis patients are transferrin saturation and ferritin values that permit assess to the functional iron supply and iron stores, respectively (18). Because excess iron can act as an oxidant and cause tissue damage, we tested whether LPI was detected when parenteral iron was administered in hemodialysis patients.
The aim of this study was to correlate the levels of LPI, the redox-active and chelatable component of NTBI, to serum iron, transferrin saturation, and ferritin after intravenous ferric saccharate iron administration in patients undergoing chronic hemodialysis.
PATIENTS AND METHODS
Twenty-six patients with ESRD undergoing chronic hemodialysis treatment at the Einstein Dialysis Center, Hospital Israelita Albert Einstein, São Paulo, Brazil, were included. Twenty of them were undergoing parenteral iron treatment; six were not receiving iron and were considered to be the control group. Ferric saccharate III (Noripurum; Altana Pharma Group, Jaguariúna, Brazil) was supplemented to patients according to the National Kidney Foundation Kidney Disease Outcomes Quality Initiative (NKF–K/DOQI) guidelines (18). All subjects were informed about the study protocol and written consent was obtained from all participants. The Brazilian Committee of Ethics and Research approved the study. Another seven patients undergoing dialysis at the Einstein Dialysis Center declined to be included in the study. The exclusion criteria were: blood transfusion within the past three months; recent hospitalization or infection requiring antibiotics within the past three months; evidence of active or occult bleeding; or a history of malignancy, end-stage liver disease or chronic hypoxia.
The median parameters of hemodialysis sessions were: the 4008S machine (Fresenius Medical Care, Bad Homburg, Germany), duration 4 h, blood flux 350 mL/min, dialysate flux 500 mL/min, sodium 138 mEq/L, temperature 35.5°C, bicarbonate 32 mmol/L, and calcium 3.5 mmol/L.
Demographic data analyzed were sex, age, time on dialysis, and etiology of chronic failure. All patients had an arteriovenous fistula and hepatitis serologies were negative.
Laboratory parameters and iron administration
The laboratory parameters included hemoglobin, hematocrit, creatinine, urea, iron (normal reference range: 40–160 µg/dL), transferrin saturation (20–50%; calculated as follows: serum iron/total iron-binding capacity [TIBC] × 100; normal reference range for TIBC: 200–400 µg/dL), ferritin (22–322 ng/mL), albumin (3.5–5.5 g/dL), C-reactive protein (0–0.3 mg/dL), ionic calcium (1.14–1.31 mmol/L), phosphorus (2.5–4.8 mg/dL), calcium–phosphorus product, alkaline phosphatase (28–126 U/L), parathyroid hormone (12–65 pg/mL), total cholesterol and fractions, triglyceride, fasting glucose (70–99 mg/dL), pH (7.32–7.43), and bicarbonate (22–29 mmol/L). All samples were collected at the beginning of the first hemodialysis session of the week (Monday or Tuesday) and corresponded to the baseline data.
The urea obtained at the end of the session was used to calculate Kt/V and the urea reduction rate (URR). Weekly doses of parenteral iron and erythropoietin adjusted to body weight were also reported. In all patients, the intravenous iron was administered 30 min before the end of the hemodialysis session. Iron saccharate was injected into the venous dialysis line in a continuous infusion for 5 min. The time points were determined after the end of the infusion.
The target values of hemoglobin and hematocrit were defined by the guidelines of K/DOQI. One milliliter of blood was sampled in an Eppendorf tube during the following times after iron administration, totaling six samples (6.0 mL):
- • At the beginning of the first session of the hemodialysis session in the week, Monday or Tuesday
- • Immediately before the intravenous iron administration, defined as T0
- • After the administration of intravenous iron at 10 min, 20 min, and 30 min, defined as T1, T2, and T3, respectively
- • At the beginning of the second hemodialysis session of the week, Wednesday or Thursday.
To avoid prolonged stays in the dialysis unit, blood samples for the iron parameters were only collected when the patients were on hemodialysis.
Labile plasma iron assay
The LPI assay was performed at the Institute of Chemistry, University of São Paulo, and it is briefly reported. Quadruplicates of 20 µL of serum were transferred to clear-bottomed, 96-well plates (TCC). To two of the wells, 180 µL of iron-free HEPES (N-2 hydroxy-ethylpiperazine-N′-2-ethanesulfonic acid)-buffered saline (HBS) were added, pre-warmed to 37°C, containing 40 µM ascorbate and 50 µM DHR (dihydrorhodamine 123; Biotium, Hayward, CA, USA). To the two other wells, the same solution containing 50 µM L1 (deferiprone; Apotex, Toronto, Canada) was added. Immediately following the addition of reagent, the kinetics of fluorescence increase were followed at 37°C in a FLUOstar OPTIMA Multifunction Microplate Reader (BMG Labtech Instruments, Offenburg, Germany) with a 485/515 nm excitation/emission filter pair, for 40 min, with readings every 2 min. The slopes (m) of change in DHR fluorescence were calculated from measurements between 16 to 40 min. The duplicate values of m in the presence or absence of L1 were averaged and the LPI concentration was determined from Equation 1, where mstd is the slope of the standard curve:
- ((Eqn 1))
In some serum samples, the rate in the presence of chelator exceeds the rate in its absence, yielding a negative value for LPI. These values were considered as zero, which means that LPI was not detected in these samples. The difference in the rate of oxidation of DHR in the presence and absence of the chelator represents the component of plasma non-transferrin-bound iron that is redox active and corresponds to the LPI values.
All results are reported as mean ± SD, unless otherwise indicated. Statistical analyses were performed by SPSS 12.0 (SPSS, Chicago, IL, USA). Fisher's exact test and anova were performed for numerical variables and Pearson's χ2-test was used for categorical variables. Pearson's test was used for correlations. Statistical analysis assumed significance if P < 0.05.
The demographic data are described in Table 1. There was no difference in relation to sex, age, time on dialysis, and etiology of chronic renal failure between the groups. In the same way, laboratory parameters and hemodialysis adequacy were comparable between the groups. The mean dose of recombinant erythropoietin in the group with parenteral iron was 127.9 ± 18.7 U/kg/wk in comparison to 67.6 ± 13.3 U/kg/wk in the group without iron replacement (P = 0.07). The mean dose of intravenous iron was 123.7 ± 83.3 mg/wk (median 100 mg/wk).
|All (N = 26)||With iron (N = 20)||Without iron (N = 6)||P|
|Sex (% male)||70.4%||70%||71.4%||P = 0.07|
|Age (years)||67.8 ± 17.8||69.3 ± 18.8||63.3 ± 14.8||P = 0.44|
|Time on dialysis (months)||30.3 ± 21.6||26.6 ± 23.1||40.6 ± 12.1||P = 0.14|
|Etiology of ESRD (% DM)||33.3%||35.0%||28.6%||P = 0.18|
|Hemoglobin (g/dL)||12.1 ± 1.5||12.4 ± 1.4||11.2 ± 1.5||P = 0.09|
|Hematocrit (%)||37.1 ± 4.8||38.0 ± 4.7||34.4 ± 4.3||P = 0.09|
|Iron (µg/dL)||79.4 ± 27.1||76.0 ± 26.4||90.2 ± 14.9||P = 0.27|
|Transferrin saturation (%)||41.3 ± 15.5||39.1 ± 16.1||47.3 ± 12.7||P = 0.23|
|Ferritin (ng/mL)||1067.7 ± 512.6||1055.7 ± 543.2||1100.1 ± 456.0||P = 0.85|
|Creatinine (mg/dL)||8.7 ± 2.5||8.9 ± 2.6||8.2 ± 2.5||P = 0.65|
|Urea before dialysis (mg/dL)||141.3 ± 34.9||145.7 ± 37.3||128.6 ± 24.9||P = 0.37|
|Urea after dialysis (mg/dL)||43.3 ± 11.9||45.1 ± 12.6||38.0 ± 8.1||P = 0.27|
|Urea ration reduction (%)||68.8 ± 7.2||68.4 ± 7.6||70.0 ± 6.5||P = 0.97|
|Weekly Kt/V||4.3 ± 0.8||4.3 ± 0.7||4.2 ± 0.7||P = 0.76|
|Albumin (g/dL)||3.5 ± 0.4||3.5 ± 0.4||3.5 ± 0.4||P = 0.80|
|C-reactive protein (mg/dL)||1.2 ± 1.2||1.4 ± 1.3||0.6 ± 0.4||P = 0.13|
|Ionic calcium (mg/dL)||1.16 ± 0.10||1.16 ± 0.10||1.18 ± 0.05||P = 0.69|
|Phosphorus (mg/dL)||5.5 ± 1.8||5.6 ± 1.9||5.3 ± 1.2||P = 0.75|
|Calcium × phosphorous (mg2/dL2)||51.2 ± 14.4||51.6 ± 15.8||50.2 ± 10.5||P = 0.83|
|Alkaline phosphatase (U/L)||123.6 ± 38.8||127.6 ± 38.2||112.7 ± 41.0||P = 0.39|
|Parathyroid hormone (pg/mL)||299.3 ± 333.1||316.8 ± 389.1||254.3 ± 107.7||P = 0.68|
|Cholesterol (mg/dL)||158.2 ± 33.9||160.3 ± 27.8||152.7 ± 49.2||P = 0.62|
|High density lipoprotein (mg/dL)||37.0 ± 10.8||37.4 ± 8.5||36.0 ± 16.3||P = 0.78|
|Low density lipoprotein (mg/dL)||86.3 ± 27.4||89.5 ± 21.1||77.6 ± 41.0||P = 0.33|
|Triglyceride (mg/dL)||166.9 ± 54.3||160.8 ± 53.6||183.3 ± 56.9||P = 0.36|
|pH||7.3 ± 0.1||7.3 ± 0.1||7.3 ± 0.1||P = 0.67|
|Bicarbonate (mmol/L)||21.1 ± 2.6||21.1 ± 2.7||21.0 ± 2.3||P = 0.92|
|Glucose (mg/dL)||117.8 ± 70.3||113.5 ± 57.1||130.1 ± 104.4||P = 0.60|
|Protein equivalent of nitrogen appearance (g/kg/day)||0.92 ± 0.2||0.93 ± 0.21||0.88 ± 0.17||P = 0.57|
The concentration of LPI ranged according to the time of its administration during the hemodialysis session (Fig. 1). Mean doses of LPI in relation to time were: −0.02 ± 0.20 µmol/L at the beginning of the first session, 0.01 ± 0.26 µmol/L at T0, 0.03 ± 0.23 µmol/L at T1, 0.09 ± 0.28 µmol/L at T2, 0.18 ± 0.52 µmol/L at T3, and −0.02 ± 0.16 µmol/L at the beginning of the second session. Patients that were not supplemented with iron presented with mean values of LPI of −0.06 ± 0.16 µmol/L.
The statistical differences of LPI concentrations in relation to the time of iron administration were the following: at the beginning of the first session vs. T2 (P = 0.005) and vs. T3 (P = 0.004); T0 vs. T3 (P = 0.001) and vs. at the beginning of the second session (P = 0.041); T1 vs. T2 (P = 0.001) and vs. T3 (P = 0.005); and T3 vs. at the beginning of the second session (P = 0.013). The difference of mean values of T2 and T3 was marginal (P = 0.053). It is suggested therefore that LPI concentrations are time-dependent and the labile iron is transitory inasmuch as it is not detected before the second hemodialysis session.
The correlations of LPI concentration with iron and transferrin saturation levels were significantly positive in the following times after intravenous iron administration: T1 and serum iron (P = 0.01); T2 and serum iron (P = 0.007); T2 and transferrin saturation (P = 0.001); T3 and serum iron (P = 0.0025); and T3 and transferrin saturation (P = 0.0003) (Fig. 2A–E). There was no correlation between the LPI concentrations and ferritin levels.
When the laboratory parameters were available, the patients who were not undergoing parenteral iron supplementation continued as such (i.e. were not given any supplementation). On the other hand, in seven out of twenty patients undergoing iron therapy the doses were changed. In four patients the doses were decreased by half and in the remaining three patients the iron was discontinued. It is interesting to note that in two patients in which the iron was discontinued the values of transferrin saturation were 56% and 94%. These values were in accordance to the highest values of LPI concentration found at time T3, that is, 0.92 µmol/L and 2.14 µmol/L, respectively.
In the present study it was found that LPI generation was time-dependent during the hemodialysis session. In addition, LPI generation was a transitory event, inasmuch as its concentration was the same at the beginning of the subsequent hemodialysis session.
In accordance to Sunder-Plassmann and Hörl, 100 mg of iron saccharate could be safe in hemodialysis patients (19). Those authors also demonstrated that serum iron levels and transferrin saturation increased significantly following intravenous injection of iron saccharate with doses ranging from 10 mg to 100 mg over a period of 1 min after the end of the hemodialysis session. Our data showed a positive correlation between LPI generation according to time and serum iron levels (T1–T3) and transferrin saturation (T2–T3), which was also described by others (20). In our study, the highest values of LPI were associated with the more elevated values of transferrin saturation, which lead us to interrupt iron administration in two patients. It is also reported elsewhere that most of the dialysis patients analyzed at least one week after iron supplementation showed no detectable LPI, and those patients who presented a detectable LPI showed higher values of transferrin saturation (21). In this way, LPI detection is in accordance with the transferrin saturation levels suggested by K/DOQI, and could be another tool to adjust the parenteral iron dose in patients undergoing chronic hemodialysis because it permits assessing iron replacement safety.
The efficacy of parenteral iron formulations in supporting erythropoiesis is unquestionable. Besides, adverse drug events are extremely rare (0.000196%), and are more related to higher molecular weight iron dextran formulation (20). However, it is a matter of discussion if iron supplementation in dialysis patients is really safe either in the short or long term. It is well known that intravenous iron administration transiently increases LPI generation (4,7,21,22). Although the iron preparations contain 2–6% low molecular weight and redox-active iron, most of it is scavenged by transferrin within less than one hour after its administration (7). In addition, other authors showed that serum iron, transferrin saturation and NTBI, assessed by high-performance liquid chromatography, significantly increased after 100 mg iron saccharate administration either in 60 min or 6 min (21). The transitory effect of LPI detection could explain at least in part the lack of its association with peroxide generation (17) and with the impairment of vascular reactivity in patients on regular hemodialysis (22).
On the other hand, the long-term effect (10 weeks) of intravenous iron saccharate administration is associated with oxidative DNA injury evaluated by serum 8-hydroxy-2′-deoxyguanosine generation, which is attributed to higher values of iron storage and suggests that ferritin levels could be a surrogate marker of oxidative injury (23). Other studies linked intravenous iron supplementation during a follow-up of 12 to 24 months to oxidative stress, assessed by advanced oxidation protein products (AOPP) generation, as well as with the evidence of early atherosclerosis defined by the increased carotid artery intima-media thickness (24,25). In addition, increased levels of serum ferritin were correlated to the intravenous iron dose received during the period preceding both studies, and subsequently to AOPP generation and to atherosclerosis. In our study, we did not find a correlation between LPI generation and ferritin levels that could be attributed to the fact that both groups presented similar values of serum ferritin, as well as C-reactive protein levels. However, our data should be analyzed with caution because we did not evaluate the effects of iron supplementation during a long-term follow-up. Furthermore, in chronic hemodialysis patients it is reported that high serum ferritin levels (≥500 ng/mL), in conjunction with low transferrin saturation (<25%), are associated with inflammation (C-reactive protein ≥ 10 mg/L) (26). In other words, high ferritin values are mainly attributed to inflammation. In our patients receiving parenteral iron, we did not find a correlation between ferritin and C-reactive protein (data not shown), which could be at least in part explained by the small number of patients analyzed in the study. However, in extreme abnormal situations of iron imbalance, such as thalassemia and hemochromatosis, the association between ferritin and LPI seems to be stronger (13,14).
On the other hand, the degree of in vitro iron donation directly to transferrin is not only concentration-dependent, but also variable according to the intravenous iron agent employed (3). Labile iron or biologically available iron occurs in low levels and represents 2.5–5.8% of total iron administered and varies according to the sequence: ferric gluconate > iron sucrose ≥ iron polymaltose. Ascorbic acid in vitro increases intracellular labile iron and the mobilization of iron to transferrin, which is mainly observed with iron sucrose but not with ferric gluconate or iron dextran (27). Ascorbic acid can also significantly decrease serum soluble transferrin receptors and increase the percentage of transferrin saturation, which independently occurs from the levels of serum ferritin and is explained by alterations in intracellular iron metabolism (28).
Labile iron pools include either intracellular or extracellular compartments; when extracellular, the iron is bound to ligands other than transferrin and may be associated with pathological conditions in which there is iron overload (reviewed in (29)). The detection of both pools requires the development of distinct probes with specific characteristics. In the present study, we detected LPI by using an oxidative-sensitive fluorescent probe (dihydrorhodamine 123), in which the redox-active and chelator-accessible iron catalyses the auto-oxidation of ascorbate, present in natural levels. It is a sensitivity method and permits assessing the role of LPI in iron regulation, the oxidative stress response, the modulation of expression of iron-storage proteins in response to stimuli, cellular iron transport, and cellular iron overload, deprivation or chelation. Other methods, such as spectroscopy and high-performance liquid chromatography, are less robust and could be associated with disruption and destruction of the biological sample.
The onset of LPI generation after intravenous saccharate iron administration is time-dependent and transitorily detected in patients undergoing chronic hemodialysis treatment. It may be used as a useful tool in the assessment of iron replacement safety.
Acknowledgments: We acknowledge financial support from CNPq and FAPESP (Brazilian funding agencies), as well as technical support from the Instituto de Ensino e Pesquisa, Hospital Israelita Albert Einstein.