Intravenous Iron Gluconate Administration Increases Circulating PAPP-A in Hemodialysis Patients

Abstract

Background. Pregnancy-associated plasma protein-A (PAPP-A) is a proatherosclerotic molecule, interrelated with oxidative stress in hemodialysis (HD) patients. As intravenous (IV) iron might enhance oxidative stress in HD patients, this study investigates circulating PAPP-A during HD session and after IV iron administration. Methods. In 20 HD patients, plasma PAPP-A concentration was assessed immunochemically during 2 HD sessions (prior to HD and at 60, 130, and 240 min of HD session). Sodium ferric gluconate (62.5 mg) was given IV to all patients 65 min after the start of the second HD. Results. Sixty-five min after IV iron application, there was a significant increase in plasma PAPP-A (from 36.0 ± 9.9 to 79.6 ± 28.9 mU/L, p < 0.0001). At the end of this HD session, PAPP-A decreased significantly (p < 0.0001), but still remained 1.5-fold greater compared with predialysis levels (p < 0.0005). Conclusion. IV iron increases circulating PAPP-A, and in this way, it might contribute to more pronounced cardiovascular complications in HD patients.

• atherosclerosis
• hemodialysis
• intravenous iron
• oxidative stress
• pregnancy-associated plasma protein-A (PAPP-A)

Introduction

Patients undergoing chronic hemodialysis (HD) are at increased risk for atherosclerotic complications, cardiovascular disease being the major cause of death in uremia.[1] Cardiovascular mortality in chronic HD patients has been estimated to be approximately 9% per year and is 10 to 20 times greater than in the general population.[2] Apart from traditional risk factors, chronic inflammation is believed to play a central role in atherogenesis in chronic HD patients.[3]

Intravenous (IV) iron administration has become a routine therapeutic procedure, essential for ensuring an adequate response to erythropoietin therapy. However, evidence exists that IV iron administration may enhance oxidative stress in HD patients.[4] IV administered iron is potentially redox active; it can initiate lipid peroxidation and thus contribute to accelerated atherosclerosis in HD patients.[4] Both in vitro[5] and in vivo experiments[6] have shown that iron stimulates neutrophils to increased generation of reactive oxygen species (ROS). However, it is still unclear whether these processes occur in dialysis patients and what is the clinical significance of IV iron therapy in atherogenesis.[7]

Pregnancy-associated plasma protein-A (PAPP-A) is a high-molecular-weight, zinc-binding metalloproteinase, which has more recently been shown to be a specific activator of insulinlike growth factor-I (IGF-I).[8] It is linked to atherosclerosis, and it has been reported as a marker of acute coronary syndrome.[9] More recently, we have shown significant elevation of circulating PAPP-A in HD patients and its close correlation with markers of oxidative stress (advanced oxidation end products [AOPP]) and inflammation (C-reactive protein [CRP]).[10&11]

Because chronic HD patients are at much higher cardiovascular risk than normal population and IV iron therapy might increase this risk by enhancing oxidative stress, in this study we investigate plasma concentrations of PAPP-A (a proatherosclerotic molecule) during HD session and after IV iron administration.

Subjects and Methods

Patients

The study was performed in a group of 20 chronic HD patients (10 males and 10 females), mean age 66 ± 13 years (range, 33–81 years). Etiology of renal failure was as follows: tubulointerstitial nephritis in 6 cases, diabetic (type 2 diabetes mellitus) nephropathy in 4 cases, chronic glomerulonephritis in 2 cases, polycystic kidney disease in 2 cases, and multifactorial or unknown in 6 cases. Mean duration of chronic HD treatment was 38 ± 24 months (range, 9–88 months). All patients were hemodialysed three times weekly, 4 hours per session, via arteriovenous fistula. All HD sessions were carried out with single-use, low-flux polysulphone dialyzers (F6; Fresenius Medical Care, Bad Homburg, Germany), bicarbonate-based dialysate, and all patients received standard IV heparin anticoagulation (54 ± 8 IU/kg of body weight as an IV bolus before the start of the HD session and 27 ± 6 IU/kg of body weight 2 hours after the start of the HD session). Mean ultrafiltration rate was 700 mL/h. The criteria of adequate dialysis (Kt/V>1.2) were fulfilled in all patients, their predialysis serum albumin was 36 ± 4 g/L, their hemoglobin was 103 ± 11 g/L, and their plasma ferritin 201 ± 93 µg/L. All patients received IV erythropoietin therapy (average dose 84 IU/kg of body weight weekly) and IV iron therapy (62.5 mg of sodium ferric gluconate once a week). All patients were in a stable clinical status, without clinical and biochemical signs of acute inflammation. None of the patients received any antioxidants, and they all gave their informed consent prior to entering the study. The study was approved by the Local Institutional Ethical Committee.

Study Design and Blood Sampling

The study was performed within 2 HD sessions. The first HD session was carried out without parenteral iron administration. During the second HD session, 62.5 mg of sodium ferric gluconate was administered as an IV bolus to each patient in the 65th minute of the HD session. Blood samples were taken from each patient via the arteriovenous fistula immediately prior to starting the HD session and at 60, 130, and 240 min after the start of the session. Blood was centrifuged at 1450 g (4°C) for 10 min. Plasma was stored at − 20°C and processed within 30 days. PAPP-A and total protein concentration were assessed in each sample.

Laboratory Assays

PAPP-A was measured immunochemically, using the TRACE (time resolved amplified cryptate emission) technology, based on nonradiating energy transfer. Commercial kit KRYPTOR-PAPP-A (Brahms, Germany and Cezanne, France) contains two different monoclonal antibodies—one is conjugated with europium cryptate and another one with fluorescent agent XL 665. The antigens (PAPP-A) present in plasma samples are sandwiched between the two conjugates. The fluorescent signal measured during the formation of the antigen-antibody complex by the KRYPTOR analyzer (Cezanne, France) is proportional to the antigen concentration (mU/L). Plasma PAPP-A concentration was divided by plasma total protein concentration for correction for changes in blood volume during HD session.

Serum albumin, plasma total protein, and plasma ferritin concentration were assessed by routine laboratory methods recommended by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC).

Statistical Analysis

Nonparametric analysis of variance (ANOVA) repeated measures test (Friedman ANOVA) and nonparametric Wilcoxon matched pairs test were used for analysis of the data (Statistica 6.1®, Stat Soft, Inc., USA). The results are expressed as mean ± standard deviation (SD). The results were considered statistically significant at p < 0.05.

Results

During the first HD session, performed without IV iron administration, there was a significant elevation of circulating PAPP-A 60 min after the start of the HD session (p < 0.05), which persisted until 130 min after the start of the HD session. At the end of the HD session, plasma PAPP-A concentration decreased significantly (p < 0.005) to predialysis values. After correction for changes in blood volume we obtained the same results, except for postdialysis serum PAPP-A concentration: it was significantly less than the plasma PAPP-A concentration before the HD session (p < 0.05) (Table 1).

Table 1. Circulating PAPP-A during two HD sessions and effect of IV iron administration (N = 20)

	PAPP-A (mU/L)		PAPP-A/prot. (mU/g prot)
Time of HD session (min)	Without IV Fe	With IV Fe 65 min after start of HD session	Without IV Fe	With IV Fe 65 min after start of HD session
0	28.9 ± 13.3	29.0 ± 11.2	0.44 ± 0.22	0.43 ± 0.18
60	34.1 ± 10.7	36.0 ± 9.9**,	0.51 ± 0.19	0.53 ± 0.19,
130	37.7 ± 15.2,,	79.6 ± 28.9***,†††	0.54 ± 0.24,††,	1.14 ± 0.46***,†††
240	28.5 ± 12.4	47.4 ± 13.5***	0.39 ± 0.18,	0.64 ± 0.20***

Plasma PAPP-A concentration was corrected for total plasma protein concentration (PAPP-A/prot., the results are expressed as PAPP-A concentration per gram of protein). All results are expressed as mean ± standard deviation.

*p < 0.05 vs. time 0.

**p < 0.005 vs. time 0.

^▵p < 0.0001 vs. time 130.

^†p < 0.005 vs. time 240.

^•p < 0.0001 vs. time 130 of HD session with IV Fe administration.

***p < 0.0005 vs. time 0.

^†††p < 0.0001 vs. time 240.

^††p < 0.001 vs. time 240.

^○p < 0.0001 vs. time 240 of HD session with IV Fe administration.

Following IV iron administration at 65 min after the start of the HD session, there was a significant increase in plasma PAPP-A concentration 130 min after the start of the HD session (from 36.0 ± 9.9 to 79.6 ± 28.9 mU/L, p < 0.0001). At the end of the HD session, plasma PAPP-A concentration decreased significantly (p < 0.0001), but it still remained 1.5-fold greater compared to predialysis plasma PAPP-A concentration (p < 0.0005). We obtained the same results after correction of plasma PAPP-A for changes in blood volume (Table 1).

Discussion

We have observed a significant increase in circulating PAPP-A during HD session and its decrease to predialysis values at the end of the session. After IV iron administration, there was more than a 2-fold increase in plasma PAPP-A concentration, followed by a significant decrease of circulating PAPP-A at the end of the session, with plasma PAPP-A concentration remaining 1.5-fold higher when compared with predialysis values.

PAPP-A, a member of metzincin superfamily of metalloproteinases, is an IGF binding protein-4 (IGFBP-4) protease, produced by human fibroblasts.[8] It was originally identified in human pregnancy serum, and it is now used for screening of Down syndrome in the first trimester of pregnancy.[12] It is responsible for proteolytic cleavage of IGFBP-4 and thus it acts as a positive regulator of IGF bioavailability. As shown by more recent studies, IGF-I enhances inflammatory responses in endothelial cells,[13] and IGF-I, IGF-II, and their regulatory proteins are growth promoters for arterial cells and mediators of cardiovascular disease.[14] As a positive regulator of IGF bioavailability, PAPP-A is supposed to play an important role in local proliferative responses such as wound healing, bone remodeling, and atherosclerosis.[8] Apart form pregnancy, PAPP-A is also present in the serum of nonpregnant subjects (the mean values determined by sensitive immunochemical methods are 8.33 mU/L in nonpregnant women and 10 mU/L in men.[15] Kalousova et al. recently reported elevation of circulating PAPP-A in HD patients when compared with healthy controls (25.8 ± 15.5 mU/L in HD patients vs. 8.75 ± 2.42 mU/L in healthy subjects, p < 0.05) and a close correlation of plasma PAPP-A concentration with AOPP and CRP[10], [16] More recently, abundant expression of PAPP-A was found in both ruptured and eroded atherosclerotic plaques, and elevated serum PAPP-A was suggested to be a valuable marker of acute coronary syndromes[9] and a strong independent predictor of ischemic cardiac events.[17] We have also observed association between higher PAPP-A levels and higher mortality rate in HD patients.[18]

The results of this study show a significant elevation of plasma PAPP-A concentration 60 min after the start of the HD session, which persists until the end of the HD session, when circulating PAPP-A returns to predialysis values. This observation is in accordance with Kalousova et al.,[19] although the study was performed with different types of dialysis membranes. After IV iron administration, we observed a significant (more than two-fold) increase in PAPP-A concentration. Again, at the end of the HD session, PAPP-A levels decreased but remained higher than at the start of the session. The initial increase in plasma PAPP-A concentration could also be caused by the punction of the arteriovenous fistula. We can speculate that the increase of PAPP-A after the start of the HD session and after IV iron administration could be caused by acute oxidative vessel wall injury, with the release of PAPP-A from the damaged vessel wall into the circulation. It could also be explained as increased PAPP-A synthesis in response to bioincompatibility reactions on the dialysis membranes, similar to those previously shown in other metalloproteinases.[20] One possible source of increased PAPP-A production could be by activation of monocytes, which are known to produce matrix metalloproteinases, involved in the process of atherosclerosis.[20] IV iron administration may be responsible for acute oxidative stress and acute phase reaction,[21&22] associated with acute vascular damage and release of PAPP-A into the circulation. We can also speculate that IV iron could cause further activation of monocytes with increased PAPP-A production. However, other sources of PAPP-A cannot be excluded. Possible interpretation of the decrease in plasma PAPP-A concentration at the end of the HD session could be PAPP-A metabolic degradation, clearance of PAPP-A free fragments, adsorption of PAPP-A to the dialysis membrane, and possibly other mechanisms.

We can conclude that IV iron administration increases circulating levels of PAPP-A. In this way, this proatherosclerotic molecule related to oxidative stress might contribute to more pronounced cardiovascular complications in HD patients.

Acknowledgments

This study was supported by grant IGA MH CZ NR/8094-3. The authors are thankful to Mrs. Soukupova and Mrs. Kremenova for technical assistance.