Hypertens Pregnancy, 2015; 34(2): 145–152 ! Informa Healthcare USA, Inc. ISSN: 1064-1955 print / 1525-6065 online DOI: 10.3109/10641955.2014.988350
Is the level of maternal serum prohepcidin associated with preeclampsia?
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Candan Iltemir Duvan,1 Serap Simavli,2 Esra Aktepe Keskin,1 Yuksel Onaran,1 Nilgun Ozturk Turhan,3 and Cemile Koca4 1
Department of Obstetrics and Gynecology, Turgut Ozal University, Ankara, Turkey, Department of Obstetrics and Gynecology, Pamukkale University, Denizli, Turkey, 3 Department of Obstetrics and Gynecology, Mugla University, Mugla, Turkey, and 4 Department of Biochemistry, Yildirim Beyazit Universiry, Ankara, Turkey 2
Objective: The objective of the study was to compare pro-hepcidin, hemoglobin (Hb) concentration, hematocrit (Hct), C-reactive protein (CRP), IL-6 and iron status parameters in preeclamptic (PE) and healthy pregnant women, and to examine the relationship between serum pro-hepcidin levels and iron parameters of preeclampsia (PE). Methods: In a prospective controlled study, we collected serum from women with normal pregnancy (n = 37) and from women with PE (n = 30) at the Department of Obstetrics and Gynecology at Turgut Ozal University between February 2010 and January 2013. Pro-hepcidin, hemoglobin (Hb) concentration, hematocrit (Hct), CRP, IL6 and iron status parameters were measured in all patients and compared between groups. Results: Levels of serum prohepcidin in PE and control groups were similar and amount 69.4 ± 19.7 and 71.9 ± 22.1 ng/ml, respectively. The difference was not statistically significant (p: 0.694). On the other hand, the study group had a statistically lower iron binding capacity (IBC), total iron binding capacity, transferin, total protein, albumin levels (p50.05). No significant differences were found among prohepcidin, Hb concentration, Hct, iron, ferritin, IL-6, urea and creatine in both the groups. Conclusion: In pregnancies complicated by PE with normal values of hemoglobin and hematocrit, serum prohepcidin concentrations are similar to those observed in healthy pregnant women. The analysis revealed no significant correlations between prohepcidin level and serum iron, serum ferritin or transferrin in the PE. Keywords Ferritin, preeclampsia, prohepcidin, serum iron
INTRODUCTION Preeclampsia (PE) is a pregnancy-specific syndrome that leading cause of morbidity and mortality for the affected mother and neonate (1,2). PE is best described as blood pressure 140/90 mmHg after 20th week of gestation and proteinuria 300 mg/24 h urine or 2+ dipstick (3). Actually, the etiology and pathogenesis of the PE are not completely understood, but many theories are offered, including; vasoconstriction, endothelial cell injury and systemic inflammation. Although none of these theories have gained universal
Correspondence: Candan Iltemir Duvan, Department of Obstetrics and Gynecology, Turgut Ozal University, Alparslan Tu¨rke¸s Cd No: 57 06560, Ankara/Turkey. Tel: 90 312 203 55 55. E-mail: [email protected]
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acceptance, it is recently widely accepted that vascular endothelial dysfunction is the most important and principal event in the pathophysiology of the disease. Current concepts of the pathogenesis of PE include endothelial dysfunction and oxidative stress (OS) (1,4–6). Iron mediates production of reactive oxygen species (ROS) via the Fenton reaction and induces OS (7). Hydroxyl radical by Fenton reaction can initiate the process of lipid peroxidation, which may result in endothelial cell damage (8–10). OS is important for normal physiological functions and for placental development (11). PE represents a much higher state of OS than normal pregnancies (12). Placental ischemia/hypoxia is considered to play a major role through the induction of OS, which can cause to endothelial cell dysfunction and systemic vasoconstriction (13–15). Increase in the OS is catalyzed in the presence of free transitional metals. Increased OS due to endothelial dysfunction in PE has been well established. When the hypoxic environment of placenta changes into an oxygen-rich environment due to increasing gestational age, this leads to the production of ROS (13,16). These ROS initiate the cellular damage in the presence of transitional metals like iron (17). Disturbances in iron parameters and hematological abnormalities in PE have been reported by some other investigators (18–20). Transferrin, the major iron-transporting protein in plasma, has antioxidant function and thus helps prevent oxidative damage to proteins, lipids and may limit oxidant induction of tissue injury (21,22). Elevated serum ferritin and iron concentration were also observed in PE (19,20). Increased plasma iron concentrations are contrary to the ongoing inflammation in PE. Several findings support the notion that chronic inflammation decreases iron availability, which result in inflammationinduced anemia (23). As a result decrease in plasma iron concentrations in PE, instead of increased concentrations that have been reported. Released iron species in PE may contribute to endothelial cell injury by decreasing antioxidant system and promotion of OS (24). There is some evidence that, through effects on formation of oxygen free radicals and subsequent lipid peroxidation, iron might be a significant etiologic factor in the endothelial cell damage of PE (20). Hepcidin is a recently discovered low-molecular weight hepatic peptide that plays an important role in iron metabolism (25). Iron is an essential micronutrient indispensable for a multitude of biological processes, including erythropoiesis, oxidative metabolism, cellular immune response and its correct balance is necessary for good health and normal cellular functioning (26,27). Cell and tissue proliferation and immunity are also affected by iron. However, the ability to produce free radicals makes free iron a toxic element (Fenton reaction) (27). Hepcidin is an antimicrobial peptide hormone produced by the liver in response to inflammatory stimuli and iron overload (25,28). Hepcidin regulates intestinal iron absorption, macrophage iron release and the placental passage of iron. Hepcidin is not only an iron-regulatory hormone but also an important link between host defense and iron metabolism. Recently, hepcidin is also reported as a type 2 acute-phase reactant. During infection and inflammation, hepcidin synthesis is markedly increased by a mechanism that is independent of iron status or erythropoietic activity (29–32). This newly
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Is prohepcidin associated with preeclampsia?
discovered peptide has been found to be regulated by inflammation, iron stores, hypoxia and anemia (33). Prohepcidin is a precursor of the active hepcidin. Previous studies have shown that inflammatory stimulation of liver hepcidin synthesis is mediated by macrophage production of cytokines interleukin-6 (IL-6) and IL-1 in human and mice. Urinary hepcidin excretion has also been shown to increase 10–100 fold in patients with infection and inflammation, demonstrating that human hepcidin is a type 2 acute-phase reactant (31,32). It is also reported that serum prohepcidin levels were increased in neonatal sepsis as an acute-phase antimicrobial peptide (34). Hepcidin is essentially a microbicidal peptide and its expression is induced by anemia, inflammation and hypoxia. Inflammatory cytokine IL-6 stimulates synthesis of hepcidin which is responsible for most or all of the features of hepcidin-related disorder (30,31). These studies led us to consider that if serum prohepcidin level is accepted as an acute phase reactant, level of this peptide hormone might be changed in PE. In the literature, there was only one study that investigated the maternal hepcidin concentrations and its association with iron homeostasis in PE (35). The aim of the present study is to investigate maternal serum prohepcidin levels in preeclamptic (PE) women and to give a new insight on the relation between maternal prohepcidin concentration and PE.
MATERIALS AND METHODS This is a prospective controlled study. We enrolled 30 PE and 30 healthy singleton pregnant women in our study. PE was diagnosed according to internationally accepted standard criteria (2). The control group comprised pregnant women matched by gestational age with the PE women. All participants received regular oral iron supplementation in the form of iron (II) sulfate (30 mg/day) as part of routine perinatal care. Informed consent was obtained from all subjects, and our study was reviewed and approved by an independent Ethical Committee of the institution. The study adhered to the tenets of the most recent revision of the Declaration of Helsinki. Levels of prohepcidin were measured by a stable enzyme-linked immunosorbent assay (DRG Instruments, Marburg, Germany) (ng/ml) according to the manufacturer’s instructions. The intra-assay coefficient of variation (CV) was 4.76%, and inter-assay CV was 4.97%. Hemoglobin (g/dl) was analyzed by Beckman Coulter LH 750 hematology analyzer. Serum ferritin (ng/ml) was measured by electrochemiluminesans assay (E170, Roche, Basel, Switzerland). Serum iron (mg/dl), unsaturated iron binding capacity (UIBC) (mg/dl), was measured by Roche, Cobas Integra 800 autoanalyzer. Total iron binding capacity (TIBC) (mg/ dl) values calculated automatically from the sum of serum iron and UIBC, both of which are determined by colorimetric methods. Transferrin saturation calculated directly as (Fe/TIBC) 100 (%). Transferrin and C-reactive protein (CRP) (mg/l) were analyzed by nephelometer (Beckman-Coulter, Immage immunassay) (mg/dl). IL 6 was measured by Siemens Immulite 2000 chemiluminesans immunoassay system (pg/ml).
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Statistical Analysis Data analysis was performed by using SPSS for Windows, version 11.5 (SPSS Inc., Chicago, IL). Whether the distributions of continuous variables were normally or not were determined by the using Shapiro Wilk test. Data were shown as mean ± standard deviation or median (minimum–maximum), where applicable. While the mean differences between groups were compared by Student’s t-test, otherwise, the Mann–Whitney U test was applied for the comparisons of the median values. Degrees of associations between continuous variables were evaluated by Pearson or Spearman correlation analyses, where applicable. A p value less than 0.05 was considered statistically significant.
RESULTS All included cases and controls were Turkish nationals. Table 1 shows comparisons of the clinical characteristics in the PE and control groups. The mean age and body mass index (BMI) of PE and normal pregnant group showed no statistical difference (Table 1). Our laboratory findings are summarized in Table 2. There were significant statistical differences (p50.05) in the mean values of systolic blood pressure (SBP), diastolic blood pressure and proteinuria between two groups (Table 1). Prohepcidin levels of the study and control groups were 69.4 ± 19.7 and 71.9 ± 22.1 ng/ml, respectively. Mean serum iron concentrations in PE and control pregnant women were 70.2 ± 43.5 and 75.6 ± 40.8, respectively. Similarly, mean serum ferritin concentration in PE and normal pregnant women was 21.9 ± 12.4 and 39.5 ± 43.0, respectively. The difference was not statistically significant (p40.05 all). On the other hand, the study group had a statistically lower IBC, TIBC, transferin, total protein and albumin levels (p50.05). There were significantly high serum ALT and AST concentration in the PE group than in the normal pregnant women. No significant differences were observed in other hematological parameters, including, Hb, Hct, IL-6,
Table 1. Clinical characteristics of healthy pregnant women and PE patients. Healthy pregnancy (n = 30)
Maternal age (years) Gestational age (weeks) SBP (mmHg) Diastolic blood pressure (mmHg) Urine protein (by dipstick) (range) Birth weight (g)
PE (n = 30)
Mean ± SD Median Min–Max Mean ± SD Median Min–Max
p
28.4 ± 5.4
28
NS
39.3 ± 1.1
39.6
99.0 ± 8.1 66.8 ± 10.0 –
3396 ± 241
100 60 0
3300
20–38.5
30.2 ± 4.9
30
22–44
37.3–41.4
35.2 ± 4.1
36.4
26–39.6 50.05
90–120 60–90
148.1 ± 12.1 93.3 ± 4.8
0–0
–
2930–3750 2686 ± 825
140 90 2
2670
140–180 50.05 90–100 50.05 2–3
50.001
780–4190 50.05
Values expressed as mean±SD and median (min-max). PE, preeclampsia; BMI, body mass index; GA, gestational age; NS, not significant. *p50.05 was considered as statistically significant.
Is prohepcidin associated with preeclampsia?
urea and creatine (Table 2). There were also statistically significant differences among gestational age and birth weight of infants (p50.05 all).
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DISCUSSION We found that serum prohepcidin, IL-6, iron and ferritin concentrations were similar in PE and healthy pregnant women. In normal pregnancy, urinary protein excretion increases essentially, due to a combination of increased glomerular filtration rate and increased permeability of the glomerular basement membrane (36). Hence, total protein excretion is considered abnormal in pregnant women when it exceeds 300 mg/ 24 h (37). Although proteinuria is one of the cardinal features of PE, proteinuria may be absent (38). In the recent study, all patients in the PE group had proteinuria 2+ which was detected by dipstick. PE is associated with a substantial elevation in serum indices of iron status. This has been ascribed to increased heme catabolism resulting from mild continuing hemolysis. The increases in serum iron and ferritin are striking and may even have the potential to be used diagnostically to warn of incipient PE (20). It is not known whether the rise in serum iron precedes or contributes to the clinical manifestations of PE, or is solely a result of the disease. There is some evidence to suggest that, through effects on formation of oxygen free radicals and subsequent lipid peroxidation, iron might be a significant etiologic factor in the endothelial cell damage of PE.
Table 2. Laboratory findings. Healthy pregnancy (n = 30) Mean ± SD Prohepcidin 71.9 ± 22.1 (ng/mL) Hemoglobin 12.0 ± 1.1 (g/dl) Serum iron 70.2 ± 43.5 (mg/dl) IBC 468.9 ± 97.5 TIBC 531.5 ± 75.9 Serum ferritin 21.9 ± 12.4 (ng/ml) Transferin 1272.3 ± 1870.9 Transferin sat 13.1 ± 7.4 CRP 26.2 ± 43.7 IL-6 22.5 ± 45.9 Urea 15.1 ± 6.7 Creatinine 0.7 ± 0.6 ALT 7.8 ± 12.3 AST 11.0 ± 11.8 T.Protein 6.3 ± 0.7 Albumine 3.5 ± 0.4
Median Min–Max
PE (n = 30) Mean ± SD
Median Min–Max
p
70.4
34–117
69.4 ± 19.7
66.1
27–105
NS
12.4
9.8–13.5
12.0 ± 0.9
12.1
10–13.7
NS
56
25–214
75.6 ± 40.8
62.5
32–159
NS
523 526.5 20
285–600 368.1 ± 119.5 460–613 424 ± 86.8 7.3–43.4 39.5 ± 43.0
362 435 26.5
135–621 50.05 263–599 50.05 6.6–158 NS
474.5 13.1 9 3.6 13 0.5 5 8 6.2 3.4
339–5334 353.5 ± 84.8 7.9–18.3 20.5 ± 20 1–168 19.1 ± 28.0 1–181 22.0 ± 29.5 3–35 18.6 ± 4.6 0.4–3.2 0.6 ± 0.1 2–57.7 15.7 ± 6.6 6–58.8 2 ± 13.42.5 5–8 5.7 ± 0.9 3–4.6 3.1 ± 0.5
379.5 14.9 7.3 7.8 18 0.6 15 18.9 5.8 3.1
199–469 5.7–84 1–106 1–108 9.9–28 0.4–0.8 6–29 10–71 4.1–7 2.2–3.8
50.05 NS NS NS NS NS 50.05 50.05 50.05 50.05
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Contrary of our study Rayman et al. (20) showed that serum iron concentration, ferritin and percent saturation of transferrin were significantly higher in the PE patients than in control subjects. There were also reported that unsaturated iron-binding capacity and apotransferrin levels were significantly lower. Similarly Siddiqui et al. (39) found that significantly high serum iron and ferritin concentration in the PE group than in the normal pregnant women. No significant differences were observed in other hematological parameters, including hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and MCHC. Same authors suggested that raised serum iron and ferritin may have the potential to be used diagnostically to warn of incipient PE (20,39,40). Another a further possible link between iron status and PE arises from the role that iron plays in the hypoxia-inducible factor transcriptional activation pathway. Several factors regulate hepcidin expression. Its major trigger is the proinflammatory cytokine, IL-6 (30). Casart et al. (41) showed that the serum level of IL-6 was significantly higher in PE women than in normal women as in Greer et al. (42). Elevated IL-6 and higher concentrations of iron are major stimulants of hepcidin expression. Although previous reports (30,39,41,42) represented higher levels of IL-6 and iron, we did not find any difference in PE women which may the reason for normal prohepcidin levels in our study. Laskowska-Klita et al. (43) investigated that maternal serum prohepcidin level in the groups of pregnant women with pregnancy-induced hypertension (PIH) and showed that serum prohepcidin concentrations were similar with healthy pregnant women. Although they investigated serum prohepcidin concentration in the PIH group not in PE women, their results are compatible with our results. Only one study describes the relationship between hepcidin and iron status in PE. This investigation demonstrated that plasma hepcidin, IL-6, iron and ferritin concentrations were increased in PE compared with healthy pregnant women (33). However, these data cannot be compared with our result, as we investigated prohepcidin concentration instead of active hepcidin concentration, this may partly explain why we found no correlations with PE. Our findings indicate that during third trimester pregnancy prohepcidin is detectable in the serum, without any correlation with PE. Prohepcidin may not identify active hepcidin but only the non-functional prohormone. To characterize the role of prohepcidin and active hepcidin in PE further longitudinal studies on larger population are needed.
DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
REFERENCES 1. Roberts JM, Taylor RN, Musci TJ, et al. Preeclampsia: an endothelial cell disorder. Am J Obstet Gynecol 1989;161:1200–4. 2. Brown MA, Lindheimer MD, de Swiet M, et al. The classification and diagnosis of the hypertensive disorders of pregnancy: statement from the International Society for the Study of Hypertension in Pregnancy (ISSHP). Hypertens Pregnancy 2001; 20:IX–XIV.
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Is prohepcidin associated with preeclampsia?
3. North RA, Taylor RS, Schellenberg JC. Evaluation of a definition of pre-eclampsia. Br J Obstet Gynaecol 1999;106:767–73. 4. Hubel CA. Oxidative stress in the pathogenesis of preeclampsia. Proc Soc Exp Biol Med 1999;222:222–35. 5. Roberts JM, Lain KY. Recent Insights into the pathogenesis of pre-eclampsia. Placenta 2002;23:359–72. 6. Challis JR, Lockwood CJ, Myatt L, et al. Inflammation and pregnancy. Reprod Sci 2009;16:206–15. 7. Webster RP, Roberts VH, Myatt L. Protein nitration in placenta – functional significance. Placenta 2008;29:985–94. 8. Hubel CA, Roberts JM, Taylor RN, et al. Lipid peroxidation in pregnancy: new perspectives on preeclampsia. Am J Obstet Gynecol 1989;161:1025–34. 9. Gutteridge JM, Halliwell B. Comments on review of free radicals in biology and medicine, second edition, by Barry Halliwell and John M. C. Gutteridge. Free Radic Biol Med 1992;12:93–5. 10. Halliwell B, Gutteridge JM. Oxygen free radicals and iron in relation to biology and medicine: some problems and concepts. Arch Biochem Biophys 1986;246:501–14. 11. Burton GJ, Jauniaux E. Placental oxidative stress: from miscarriage to preeclampsia. J Soc Gynecol Investig 2004;11:342–52. 12. Redman CW, Sargent IL. Placental stress and pre-eclampsia: a revised view. Placenta 2009;30:S38–42. 13. Myatt L, Cui X. Oxidative stress in the placenta. Histochem Cell Biol 2004;122: 369–82. 14. Reslan OM, Khalil RA. Molecular and vascular targets in the pathogenesis and management of the hypertension associated with preeclampsia. Cardiovasc Hematol Agents Med Chem 2010;8:204–26. 15. Khalil RA, Granger JP. Vascular mechanisms of increased arterial pressure in preeclampsia: lessons from animal models. Am J Physiol Regul Integr Comp Physiol 2002;283:R29–45. 16. Jauniaux E, Watson AL, Hempstock J, et al. Onset of maternal arterial blood flow and placental oxidative stress. A possible factor in human early pregnancy failure. Am J Pathol 2000;157:2111–22. 17. Balla J, Vercellotti GM, Jeney V, et al. Heme, heme oxygenase and ferritin in vascular endothelial cell injury. Mol Nutr Food Res 2005;49:1030–43. 18. Serdar Z, Gur E, Develioglu O. Serum iron and copper status and oxidative stress in severe and mild preeclampsia. Cell Biochem Funct 2006;24:209–15. 19. Basher K, Deb K. Alteration in iron status in pre eclampsia. Mymensingh Med J 2006;15:22–4. 20. Rayman MP, Barlis J, Evans RW, et al. Abnormal iron parameters in the pregnancy syndrome preeclampsia. Am J Obstet Gynecol 2002;187:412–18. 21. Halliwell B, Gutteridge JM. The antioxidants of human extracellular fluids. Arch Biochem Biophys 1990;280:1–8. 22. Jonsson JJ, Johannesson GM, Sigfusson N, et al. Prevalence of iron deficiency and iron overload in the adult Icelandic population. J Clin Epidemiol 1991;44:1289–97. 23. Balla J, Jeney V, Varga Z, et al. Iron homeostasis in chronic inflammation. Acta Physiol Hung 2007;94:95–106. 24. Hubel CA, Kozlov AV, Kagan VE, et al. Decreased transferrin and increased transferrin saturation in sera of women with preeclampsia: implications for oxidative stress. Am J Obstet Gynecol 1996;175:692–700. 25. Krause A, Neitz S, Magert HJ, et al. LEAP-1, a novel highly disulfide-bonded human peptide, exhibits antimicrobial activity. FEBS Lett 2000;480:147–50. 26. Anderson GJ, Frazer DM, McLaren GD. Iron absorption and metabolism. Curr Opin Gastroenterol 2009;25:129–35. 27. Aisen P, Enns C, Wessling-Resnick M. Chemistry and biology of eukaryotic iron metabolism. Int J Biochem Cell Biol 2001;33:940–59. 28. Singh B, Arora S, Agrawal P, et al. Hepcidin: a novel peptide hormone regulating iron metabolism. Clin Chim Acta 2011;412:823–30.
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29. Fleming RE. Iron and inflammation: cross-talk between pathways regulating hepcidin. J Mol Med (Berl) 2008;86:491–4. 30. Ganz T. Hepcidin, a key regulator of iron metabolism and mediator of anemia of inflammation. Blood 2003;102:783–8. 31. Ganz T, Nemeth E. Iron sequestration and anemia of inflammation. Semin Hematol 2009;46:387–93. 32. Nemeth E, Valore EV, Territo M, et al. Hepcidin, a putative mediator of anemia of inflammation, is a type II acute-phase protein. Blood 2003;101:2461–3. 33. Nicolas G, Chauvet C, Viatte L, et al. The gene encoding the iron regulatory peptide hepcidin is regulated by anemia, hypoxia, and inflammation. J Clin Invest 2002; 110:1037–44. 34. Yapakci E, Tarcan A, Celik B, et al. Serum pro-hepcidin levels in term and preterm newborns with sepsis. Pediatr Int 2009;51:289–92. 35. Toldi G, Stenczer B, Molvarec A, et al. Hepcidin concentrations and iron homeostasis in preeclampsia. Clin Chem Lab Med 2010;48:1423–6. 36. Roberts M, Lindheimer MD, Davison JM. Altered glomerular permselectivity to neutral dextrans and heteroporous membrane modeling in human pregnancy. Am J Physiol 1996;270:F338–43. 37. Higby K, Suiter CR, Phelps JY, et al. Normal values of urinary albumin and total protein excretion during pregnancy. Am J Obstet Gynecol 1994;171:984–9. 38. Thornton CE, Makris A, Ogle RF, et al. Role of proteinuria in defining preeclampsia: clinical outcomes for women and babies. Clin Exp Pharmacol Physiol 2010;37:466–70. 39. Siddiqui IA, Jaleel A, Kadri HM, et al. Iron status parameters in preeclamptic women. Arch Gynecol Obstet 2011;284:587–91. 40. Entman SS, Richardson LD. Clinical applications of the altered iron kinetics of toxemia of pregnancy. Am J Obstet Gynecol 1983;146:568–74. 41. Casart YC, Tarrazzi K, Camejo MI. Serum levels of interleukin-6, interleukin-1beta and human chorionic gonadotropin in pre-eclamptic and normal pregnancy. Gynecol Endocrinol 2007;23:300–3. 42. Greer IA, Lyall F, Perera T, et al. Increased concentrations of cytokines interleukin6 and interleukin-1 receptor antagonist in plasma of women with preeclampsia: a mechanism for endothelial dysfunction? Obstet Gynecol 1994;84:937–40. 43. Laskowska-Klita T, Chelchowska M, Blumska-Janiak M, et al. Pro-hepcidin concentration in serum of women in uncomplicated pregnancy, pregnancy complicated by premature rupture of membranes and in pregnancy induced hypertension. Med Wieku Rozwoj 2010;14:374–7.
Is the level of maternal serum prohepcidin associated with preeclampsia?
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Candan Iltemir Duvan,1 Serap Simavli,2 Esra Aktepe Keskin,1 Yuksel Onaran,1 Nilgun Ozturk Turhan,3 and Cemile Koca4 1
Department of Obstetrics and Gynecology, Turgut Ozal University, Ankara, Turkey, Department of Obstetrics and Gynecology, Pamukkale University, Denizli, Turkey, 3 Department of Obstetrics and Gynecology, Mugla University, Mugla, Turkey, and 4 Department of Biochemistry, Yildirim Beyazit Universiry, Ankara, Turkey 2
Objective: The objective of the study was to compare pro-hepcidin, hemoglobin (Hb) concentration, hematocrit (Hct), C-reactive protein (CRP), IL-6 and iron status parameters in preeclamptic (PE) and healthy pregnant women, and to examine the relationship between serum pro-hepcidin levels and iron parameters of preeclampsia (PE). Methods: In a prospective controlled study, we collected serum from women with normal pregnancy (n = 37) and from women with PE (n = 30) at the Department of Obstetrics and Gynecology at Turgut Ozal University between February 2010 and January 2013. Pro-hepcidin, hemoglobin (Hb) concentration, hematocrit (Hct), CRP, IL6 and iron status parameters were measured in all patients and compared between groups. Results: Levels of serum prohepcidin in PE and control groups were similar and amount 69.4 ± 19.7 and 71.9 ± 22.1 ng/ml, respectively. The difference was not statistically significant (p: 0.694). On the other hand, the study group had a statistically lower iron binding capacity (IBC), total iron binding capacity, transferin, total protein, albumin levels (p50.05). No significant differences were found among prohepcidin, Hb concentration, Hct, iron, ferritin, IL-6, urea and creatine in both the groups. Conclusion: In pregnancies complicated by PE with normal values of hemoglobin and hematocrit, serum prohepcidin concentrations are similar to those observed in healthy pregnant women. The analysis revealed no significant correlations between prohepcidin level and serum iron, serum ferritin or transferrin in the PE. Keywords Ferritin, preeclampsia, prohepcidin, serum iron
INTRODUCTION Preeclampsia (PE) is a pregnancy-specific syndrome that leading cause of morbidity and mortality for the affected mother and neonate (1,2). PE is best described as blood pressure 140/90 mmHg after 20th week of gestation and proteinuria 300 mg/24 h urine or 2+ dipstick (3). Actually, the etiology and pathogenesis of the PE are not completely understood, but many theories are offered, including; vasoconstriction, endothelial cell injury and systemic inflammation. Although none of these theories have gained universal
Correspondence: Candan Iltemir Duvan, Department of Obstetrics and Gynecology, Turgut Ozal University, Alparslan Tu¨rke¸s Cd No: 57 06560, Ankara/Turkey. Tel: 90 312 203 55 55. E-mail: [email protected]
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acceptance, it is recently widely accepted that vascular endothelial dysfunction is the most important and principal event in the pathophysiology of the disease. Current concepts of the pathogenesis of PE include endothelial dysfunction and oxidative stress (OS) (1,4–6). Iron mediates production of reactive oxygen species (ROS) via the Fenton reaction and induces OS (7). Hydroxyl radical by Fenton reaction can initiate the process of lipid peroxidation, which may result in endothelial cell damage (8–10). OS is important for normal physiological functions and for placental development (11). PE represents a much higher state of OS than normal pregnancies (12). Placental ischemia/hypoxia is considered to play a major role through the induction of OS, which can cause to endothelial cell dysfunction and systemic vasoconstriction (13–15). Increase in the OS is catalyzed in the presence of free transitional metals. Increased OS due to endothelial dysfunction in PE has been well established. When the hypoxic environment of placenta changes into an oxygen-rich environment due to increasing gestational age, this leads to the production of ROS (13,16). These ROS initiate the cellular damage in the presence of transitional metals like iron (17). Disturbances in iron parameters and hematological abnormalities in PE have been reported by some other investigators (18–20). Transferrin, the major iron-transporting protein in plasma, has antioxidant function and thus helps prevent oxidative damage to proteins, lipids and may limit oxidant induction of tissue injury (21,22). Elevated serum ferritin and iron concentration were also observed in PE (19,20). Increased plasma iron concentrations are contrary to the ongoing inflammation in PE. Several findings support the notion that chronic inflammation decreases iron availability, which result in inflammationinduced anemia (23). As a result decrease in plasma iron concentrations in PE, instead of increased concentrations that have been reported. Released iron species in PE may contribute to endothelial cell injury by decreasing antioxidant system and promotion of OS (24). There is some evidence that, through effects on formation of oxygen free radicals and subsequent lipid peroxidation, iron might be a significant etiologic factor in the endothelial cell damage of PE (20). Hepcidin is a recently discovered low-molecular weight hepatic peptide that plays an important role in iron metabolism (25). Iron is an essential micronutrient indispensable for a multitude of biological processes, including erythropoiesis, oxidative metabolism, cellular immune response and its correct balance is necessary for good health and normal cellular functioning (26,27). Cell and tissue proliferation and immunity are also affected by iron. However, the ability to produce free radicals makes free iron a toxic element (Fenton reaction) (27). Hepcidin is an antimicrobial peptide hormone produced by the liver in response to inflammatory stimuli and iron overload (25,28). Hepcidin regulates intestinal iron absorption, macrophage iron release and the placental passage of iron. Hepcidin is not only an iron-regulatory hormone but also an important link between host defense and iron metabolism. Recently, hepcidin is also reported as a type 2 acute-phase reactant. During infection and inflammation, hepcidin synthesis is markedly increased by a mechanism that is independent of iron status or erythropoietic activity (29–32). This newly
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Is prohepcidin associated with preeclampsia?
discovered peptide has been found to be regulated by inflammation, iron stores, hypoxia and anemia (33). Prohepcidin is a precursor of the active hepcidin. Previous studies have shown that inflammatory stimulation of liver hepcidin synthesis is mediated by macrophage production of cytokines interleukin-6 (IL-6) and IL-1 in human and mice. Urinary hepcidin excretion has also been shown to increase 10–100 fold in patients with infection and inflammation, demonstrating that human hepcidin is a type 2 acute-phase reactant (31,32). It is also reported that serum prohepcidin levels were increased in neonatal sepsis as an acute-phase antimicrobial peptide (34). Hepcidin is essentially a microbicidal peptide and its expression is induced by anemia, inflammation and hypoxia. Inflammatory cytokine IL-6 stimulates synthesis of hepcidin which is responsible for most or all of the features of hepcidin-related disorder (30,31). These studies led us to consider that if serum prohepcidin level is accepted as an acute phase reactant, level of this peptide hormone might be changed in PE. In the literature, there was only one study that investigated the maternal hepcidin concentrations and its association with iron homeostasis in PE (35). The aim of the present study is to investigate maternal serum prohepcidin levels in preeclamptic (PE) women and to give a new insight on the relation between maternal prohepcidin concentration and PE.
MATERIALS AND METHODS This is a prospective controlled study. We enrolled 30 PE and 30 healthy singleton pregnant women in our study. PE was diagnosed according to internationally accepted standard criteria (2). The control group comprised pregnant women matched by gestational age with the PE women. All participants received regular oral iron supplementation in the form of iron (II) sulfate (30 mg/day) as part of routine perinatal care. Informed consent was obtained from all subjects, and our study was reviewed and approved by an independent Ethical Committee of the institution. The study adhered to the tenets of the most recent revision of the Declaration of Helsinki. Levels of prohepcidin were measured by a stable enzyme-linked immunosorbent assay (DRG Instruments, Marburg, Germany) (ng/ml) according to the manufacturer’s instructions. The intra-assay coefficient of variation (CV) was 4.76%, and inter-assay CV was 4.97%. Hemoglobin (g/dl) was analyzed by Beckman Coulter LH 750 hematology analyzer. Serum ferritin (ng/ml) was measured by electrochemiluminesans assay (E170, Roche, Basel, Switzerland). Serum iron (mg/dl), unsaturated iron binding capacity (UIBC) (mg/dl), was measured by Roche, Cobas Integra 800 autoanalyzer. Total iron binding capacity (TIBC) (mg/ dl) values calculated automatically from the sum of serum iron and UIBC, both of which are determined by colorimetric methods. Transferrin saturation calculated directly as (Fe/TIBC) 100 (%). Transferrin and C-reactive protein (CRP) (mg/l) were analyzed by nephelometer (Beckman-Coulter, Immage immunassay) (mg/dl). IL 6 was measured by Siemens Immulite 2000 chemiluminesans immunoassay system (pg/ml).
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Statistical Analysis Data analysis was performed by using SPSS for Windows, version 11.5 (SPSS Inc., Chicago, IL). Whether the distributions of continuous variables were normally or not were determined by the using Shapiro Wilk test. Data were shown as mean ± standard deviation or median (minimum–maximum), where applicable. While the mean differences between groups were compared by Student’s t-test, otherwise, the Mann–Whitney U test was applied for the comparisons of the median values. Degrees of associations between continuous variables were evaluated by Pearson or Spearman correlation analyses, where applicable. A p value less than 0.05 was considered statistically significant.
RESULTS All included cases and controls were Turkish nationals. Table 1 shows comparisons of the clinical characteristics in the PE and control groups. The mean age and body mass index (BMI) of PE and normal pregnant group showed no statistical difference (Table 1). Our laboratory findings are summarized in Table 2. There were significant statistical differences (p50.05) in the mean values of systolic blood pressure (SBP), diastolic blood pressure and proteinuria between two groups (Table 1). Prohepcidin levels of the study and control groups were 69.4 ± 19.7 and 71.9 ± 22.1 ng/ml, respectively. Mean serum iron concentrations in PE and control pregnant women were 70.2 ± 43.5 and 75.6 ± 40.8, respectively. Similarly, mean serum ferritin concentration in PE and normal pregnant women was 21.9 ± 12.4 and 39.5 ± 43.0, respectively. The difference was not statistically significant (p40.05 all). On the other hand, the study group had a statistically lower IBC, TIBC, transferin, total protein and albumin levels (p50.05). There were significantly high serum ALT and AST concentration in the PE group than in the normal pregnant women. No significant differences were observed in other hematological parameters, including, Hb, Hct, IL-6,
Table 1. Clinical characteristics of healthy pregnant women and PE patients. Healthy pregnancy (n = 30)
Maternal age (years) Gestational age (weeks) SBP (mmHg) Diastolic blood pressure (mmHg) Urine protein (by dipstick) (range) Birth weight (g)
PE (n = 30)
Mean ± SD Median Min–Max Mean ± SD Median Min–Max
p
28.4 ± 5.4
28
NS
39.3 ± 1.1
39.6
99.0 ± 8.1 66.8 ± 10.0 –
3396 ± 241
100 60 0
3300
20–38.5
30.2 ± 4.9
30
22–44
37.3–41.4
35.2 ± 4.1
36.4
26–39.6 50.05
90–120 60–90
148.1 ± 12.1 93.3 ± 4.8
0–0
–
2930–3750 2686 ± 825
140 90 2
2670
140–180 50.05 90–100 50.05 2–3
50.001
780–4190 50.05
Values expressed as mean±SD and median (min-max). PE, preeclampsia; BMI, body mass index; GA, gestational age; NS, not significant. *p50.05 was considered as statistically significant.
Is prohepcidin associated with preeclampsia?
urea and creatine (Table 2). There were also statistically significant differences among gestational age and birth weight of infants (p50.05 all).
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DISCUSSION We found that serum prohepcidin, IL-6, iron and ferritin concentrations were similar in PE and healthy pregnant women. In normal pregnancy, urinary protein excretion increases essentially, due to a combination of increased glomerular filtration rate and increased permeability of the glomerular basement membrane (36). Hence, total protein excretion is considered abnormal in pregnant women when it exceeds 300 mg/ 24 h (37). Although proteinuria is one of the cardinal features of PE, proteinuria may be absent (38). In the recent study, all patients in the PE group had proteinuria 2+ which was detected by dipstick. PE is associated with a substantial elevation in serum indices of iron status. This has been ascribed to increased heme catabolism resulting from mild continuing hemolysis. The increases in serum iron and ferritin are striking and may even have the potential to be used diagnostically to warn of incipient PE (20). It is not known whether the rise in serum iron precedes or contributes to the clinical manifestations of PE, or is solely a result of the disease. There is some evidence to suggest that, through effects on formation of oxygen free radicals and subsequent lipid peroxidation, iron might be a significant etiologic factor in the endothelial cell damage of PE.
Table 2. Laboratory findings. Healthy pregnancy (n = 30) Mean ± SD Prohepcidin 71.9 ± 22.1 (ng/mL) Hemoglobin 12.0 ± 1.1 (g/dl) Serum iron 70.2 ± 43.5 (mg/dl) IBC 468.9 ± 97.5 TIBC 531.5 ± 75.9 Serum ferritin 21.9 ± 12.4 (ng/ml) Transferin 1272.3 ± 1870.9 Transferin sat 13.1 ± 7.4 CRP 26.2 ± 43.7 IL-6 22.5 ± 45.9 Urea 15.1 ± 6.7 Creatinine 0.7 ± 0.6 ALT 7.8 ± 12.3 AST 11.0 ± 11.8 T.Protein 6.3 ± 0.7 Albumine 3.5 ± 0.4
Median Min–Max
PE (n = 30) Mean ± SD
Median Min–Max
p
70.4
34–117
69.4 ± 19.7
66.1
27–105
NS
12.4
9.8–13.5
12.0 ± 0.9
12.1
10–13.7
NS
56
25–214
75.6 ± 40.8
62.5
32–159
NS
523 526.5 20
285–600 368.1 ± 119.5 460–613 424 ± 86.8 7.3–43.4 39.5 ± 43.0
362 435 26.5
135–621 50.05 263–599 50.05 6.6–158 NS
474.5 13.1 9 3.6 13 0.5 5 8 6.2 3.4
339–5334 353.5 ± 84.8 7.9–18.3 20.5 ± 20 1–168 19.1 ± 28.0 1–181 22.0 ± 29.5 3–35 18.6 ± 4.6 0.4–3.2 0.6 ± 0.1 2–57.7 15.7 ± 6.6 6–58.8 2 ± 13.42.5 5–8 5.7 ± 0.9 3–4.6 3.1 ± 0.5
379.5 14.9 7.3 7.8 18 0.6 15 18.9 5.8 3.1
199–469 5.7–84 1–106 1–108 9.9–28 0.4–0.8 6–29 10–71 4.1–7 2.2–3.8
50.05 NS NS NS NS NS 50.05 50.05 50.05 50.05
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Contrary of our study Rayman et al. (20) showed that serum iron concentration, ferritin and percent saturation of transferrin were significantly higher in the PE patients than in control subjects. There were also reported that unsaturated iron-binding capacity and apotransferrin levels were significantly lower. Similarly Siddiqui et al. (39) found that significantly high serum iron and ferritin concentration in the PE group than in the normal pregnant women. No significant differences were observed in other hematological parameters, including hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and MCHC. Same authors suggested that raised serum iron and ferritin may have the potential to be used diagnostically to warn of incipient PE (20,39,40). Another a further possible link between iron status and PE arises from the role that iron plays in the hypoxia-inducible factor transcriptional activation pathway. Several factors regulate hepcidin expression. Its major trigger is the proinflammatory cytokine, IL-6 (30). Casart et al. (41) showed that the serum level of IL-6 was significantly higher in PE women than in normal women as in Greer et al. (42). Elevated IL-6 and higher concentrations of iron are major stimulants of hepcidin expression. Although previous reports (30,39,41,42) represented higher levels of IL-6 and iron, we did not find any difference in PE women which may the reason for normal prohepcidin levels in our study. Laskowska-Klita et al. (43) investigated that maternal serum prohepcidin level in the groups of pregnant women with pregnancy-induced hypertension (PIH) and showed that serum prohepcidin concentrations were similar with healthy pregnant women. Although they investigated serum prohepcidin concentration in the PIH group not in PE women, their results are compatible with our results. Only one study describes the relationship between hepcidin and iron status in PE. This investigation demonstrated that plasma hepcidin, IL-6, iron and ferritin concentrations were increased in PE compared with healthy pregnant women (33). However, these data cannot be compared with our result, as we investigated prohepcidin concentration instead of active hepcidin concentration, this may partly explain why we found no correlations with PE. Our findings indicate that during third trimester pregnancy prohepcidin is detectable in the serum, without any correlation with PE. Prohepcidin may not identify active hepcidin but only the non-functional prohormone. To characterize the role of prohepcidin and active hepcidin in PE further longitudinal studies on larger population are needed.
DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
REFERENCES 1. Roberts JM, Taylor RN, Musci TJ, et al. Preeclampsia: an endothelial cell disorder. Am J Obstet Gynecol 1989;161:1200–4. 2. Brown MA, Lindheimer MD, de Swiet M, et al. The classification and diagnosis of the hypertensive disorders of pregnancy: statement from the International Society for the Study of Hypertension in Pregnancy (ISSHP). Hypertens Pregnancy 2001; 20:IX–XIV.
Hypertens Pregnancy Downloaded from informahealthcare.com by Nyu Medical Center on 05/18/15 For personal use only.
Is prohepcidin associated with preeclampsia?
3. North RA, Taylor RS, Schellenberg JC. Evaluation of a definition of pre-eclampsia. Br J Obstet Gynaecol 1999;106:767–73. 4. Hubel CA. Oxidative stress in the pathogenesis of preeclampsia. Proc Soc Exp Biol Med 1999;222:222–35. 5. Roberts JM, Lain KY. Recent Insights into the pathogenesis of pre-eclampsia. Placenta 2002;23:359–72. 6. Challis JR, Lockwood CJ, Myatt L, et al. Inflammation and pregnancy. Reprod Sci 2009;16:206–15. 7. Webster RP, Roberts VH, Myatt L. Protein nitration in placenta – functional significance. Placenta 2008;29:985–94. 8. Hubel CA, Roberts JM, Taylor RN, et al. Lipid peroxidation in pregnancy: new perspectives on preeclampsia. Am J Obstet Gynecol 1989;161:1025–34. 9. Gutteridge JM, Halliwell B. Comments on review of free radicals in biology and medicine, second edition, by Barry Halliwell and John M. C. Gutteridge. Free Radic Biol Med 1992;12:93–5. 10. Halliwell B, Gutteridge JM. Oxygen free radicals and iron in relation to biology and medicine: some problems and concepts. Arch Biochem Biophys 1986;246:501–14. 11. Burton GJ, Jauniaux E. Placental oxidative stress: from miscarriage to preeclampsia. J Soc Gynecol Investig 2004;11:342–52. 12. Redman CW, Sargent IL. Placental stress and pre-eclampsia: a revised view. Placenta 2009;30:S38–42. 13. Myatt L, Cui X. Oxidative stress in the placenta. Histochem Cell Biol 2004;122: 369–82. 14. Reslan OM, Khalil RA. Molecular and vascular targets in the pathogenesis and management of the hypertension associated with preeclampsia. Cardiovasc Hematol Agents Med Chem 2010;8:204–26. 15. Khalil RA, Granger JP. Vascular mechanisms of increased arterial pressure in preeclampsia: lessons from animal models. Am J Physiol Regul Integr Comp Physiol 2002;283:R29–45. 16. Jauniaux E, Watson AL, Hempstock J, et al. Onset of maternal arterial blood flow and placental oxidative stress. A possible factor in human early pregnancy failure. Am J Pathol 2000;157:2111–22. 17. Balla J, Vercellotti GM, Jeney V, et al. Heme, heme oxygenase and ferritin in vascular endothelial cell injury. Mol Nutr Food Res 2005;49:1030–43. 18. Serdar Z, Gur E, Develioglu O. Serum iron and copper status and oxidative stress in severe and mild preeclampsia. Cell Biochem Funct 2006;24:209–15. 19. Basher K, Deb K. Alteration in iron status in pre eclampsia. Mymensingh Med J 2006;15:22–4. 20. Rayman MP, Barlis J, Evans RW, et al. Abnormal iron parameters in the pregnancy syndrome preeclampsia. Am J Obstet Gynecol 2002;187:412–18. 21. Halliwell B, Gutteridge JM. The antioxidants of human extracellular fluids. Arch Biochem Biophys 1990;280:1–8. 22. Jonsson JJ, Johannesson GM, Sigfusson N, et al. Prevalence of iron deficiency and iron overload in the adult Icelandic population. J Clin Epidemiol 1991;44:1289–97. 23. Balla J, Jeney V, Varga Z, et al. Iron homeostasis in chronic inflammation. Acta Physiol Hung 2007;94:95–106. 24. Hubel CA, Kozlov AV, Kagan VE, et al. Decreased transferrin and increased transferrin saturation in sera of women with preeclampsia: implications for oxidative stress. Am J Obstet Gynecol 1996;175:692–700. 25. Krause A, Neitz S, Magert HJ, et al. LEAP-1, a novel highly disulfide-bonded human peptide, exhibits antimicrobial activity. FEBS Lett 2000;480:147–50. 26. Anderson GJ, Frazer DM, McLaren GD. Iron absorption and metabolism. Curr Opin Gastroenterol 2009;25:129–35. 27. Aisen P, Enns C, Wessling-Resnick M. Chemistry and biology of eukaryotic iron metabolism. Int J Biochem Cell Biol 2001;33:940–59. 28. Singh B, Arora S, Agrawal P, et al. Hepcidin: a novel peptide hormone regulating iron metabolism. Clin Chim Acta 2011;412:823–30.
151
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152
C. I. Duvan et al.
29. Fleming RE. Iron and inflammation: cross-talk between pathways regulating hepcidin. J Mol Med (Berl) 2008;86:491–4. 30. Ganz T. Hepcidin, a key regulator of iron metabolism and mediator of anemia of inflammation. Blood 2003;102:783–8. 31. Ganz T, Nemeth E. Iron sequestration and anemia of inflammation. Semin Hematol 2009;46:387–93. 32. Nemeth E, Valore EV, Territo M, et al. Hepcidin, a putative mediator of anemia of inflammation, is a type II acute-phase protein. Blood 2003;101:2461–3. 33. Nicolas G, Chauvet C, Viatte L, et al. The gene encoding the iron regulatory peptide hepcidin is regulated by anemia, hypoxia, and inflammation. J Clin Invest 2002; 110:1037–44. 34. Yapakci E, Tarcan A, Celik B, et al. Serum pro-hepcidin levels in term and preterm newborns with sepsis. Pediatr Int 2009;51:289–92. 35. Toldi G, Stenczer B, Molvarec A, et al. Hepcidin concentrations and iron homeostasis in preeclampsia. Clin Chem Lab Med 2010;48:1423–6. 36. Roberts M, Lindheimer MD, Davison JM. Altered glomerular permselectivity to neutral dextrans and heteroporous membrane modeling in human pregnancy. Am J Physiol 1996;270:F338–43. 37. Higby K, Suiter CR, Phelps JY, et al. Normal values of urinary albumin and total protein excretion during pregnancy. Am J Obstet Gynecol 1994;171:984–9. 38. Thornton CE, Makris A, Ogle RF, et al. Role of proteinuria in defining preeclampsia: clinical outcomes for women and babies. Clin Exp Pharmacol Physiol 2010;37:466–70. 39. Siddiqui IA, Jaleel A, Kadri HM, et al. Iron status parameters in preeclamptic women. Arch Gynecol Obstet 2011;284:587–91. 40. Entman SS, Richardson LD. Clinical applications of the altered iron kinetics of toxemia of pregnancy. Am J Obstet Gynecol 1983;146:568–74. 41. Casart YC, Tarrazzi K, Camejo MI. Serum levels of interleukin-6, interleukin-1beta and human chorionic gonadotropin in pre-eclamptic and normal pregnancy. Gynecol Endocrinol 2007;23:300–3. 42. Greer IA, Lyall F, Perera T, et al. Increased concentrations of cytokines interleukin6 and interleukin-1 receptor antagonist in plasma of women with preeclampsia: a mechanism for endothelial dysfunction? Obstet Gynecol 1994;84:937–40. 43. Laskowska-Klita T, Chelchowska M, Blumska-Janiak M, et al. Pro-hepcidin concentration in serum of women in uncomplicated pregnancy, pregnancy complicated by premature rupture of membranes and in pregnancy induced hypertension. Med Wieku Rozwoj 2010;14:374–7.
